RNA AND DNA INACTIVATION STRATEGIES TO · PDF fileRNA AND DNA INACTIVATION STRATEGIES TO ON VIA tly in use for HIV/AIDS therapy, a number of gene therapy strategies have been designed

By

Reza Nazari

A thesis submitted in conformity with the requirements for the Degree of Philosophy

Graduate Department of Laboratory Medicine and Pathobiology University of Toronto

© Copyright by Reza Nazari (2008)

RNA AND DNA INACTIVATION

STRATEGIES TO PREVENT OR INHIBIT

HIV-1 REPLICATION VIA GENE THERAPY

RNA AND DNA INACTIVATION STRATEGIES TO

ON VIA

tly in use for

HIV/AIDS therapy, a number of gene therapy strategies have been designed as alternative

therapies. Most of these therapies target HIV RNA/proteins, which are subject to high rate of mutation,

resulting in escape mutants. Viral entry is mediated by CCR5 co-receptor in most routes of transmission. To

downregulate CCR5 as a gene therapy approach, we targeted seven unique sites within the CCR5 mRNA

by a multimeric hammerhead ribozyme, Rz1-7. Hammerhead ribozyme is a small RNA that cleaves a target

RNA upon binding to it. Expressing the Rz1-7 from HIV-1- and MSCV-based vectors in otherwise

susceptible cells inhibited replication of a CCR5-tropic strain of HIV-1 by 99-100%. The Rz1-7 will be tested

for inhibition of HIV-1 replication in the CD4+ T-lymphoid and myeloid progeny of transduced human

CD34+ hematopoietic progenitor stem cells.

It may be preferable to interfere HIV-1 life cycle at the DNA level since a one-time inactivation might

suffice to confer a complete and permanent inhibition of virus replication in the gene modified cells and

their progeny. This is what other strategies that target the HIV-1 RNA/protein can hardly offer. For this

purpose, group II introns, which are able to splice out and get incorporated into a specific DNA sequence,

PREVENT OR INHIBIT HIV-1 REPLICATI

GENE THERAPY

Reza Nazari A thesis submitted in conformity with the requirements for the Degree of Philosophy

Graduate Department of Laboratory Medicine and Pathobiology

University of Toronto

2008

AIDS is caused by a lentivirus, HIV-1. In addition to antiretroviral drugs that are curren

ii

can be designed/modified to gain novel DNA targeting specificities. As a novel approach, we have

examined whether insertion of a modified intron into an infectious HIV-1 clone at two sites within the

integrase domain of HIV-1 pol gene could inhibit virus replication. Intron insertion into the HIV-1 clone

was induced and mammalian cells were transfected with intron-inserted HIV-1 clones. Although similar

amounts of HIV-1 RNA, protein, and progeny virus were produced from the clones as from wild-type HIV-

1 provirus DNA, in the absence of a functional integrase, the HIV-1 reverse-transcribed DNA failed to

integrate and virus replication was aborted. These results demonstrate that modified group II introns can

confer complete inhibition of virus replication at the level of second round of infection. We are now

developing vectors to assess whether intron insertion can take place in mammalian cells.

iii

الَحكيمليم الَعم اِهللابْس

In the name of Allah,

The Knowing and The Wise

iv

Dedications and Acknowledgements

Praise to Allah for all the blessings!

To me, this thesis was more than a process of working on some projects and obtaining results, it

was a part of my life, in which I learned important lessons. I would like to briefly acknowledge

those individuals who have been with me during this challenging endeavor.

I dedicate this work to my wife, Parmis, who has my eternal gratitude for her love and patience. I

also dedicate this thesis to my parents, Mohammad and Mina, and my sister, Bita, who have

always provided me with their unconditional love, prayers, and support.

I am indebted to my supervisor, Dr. Sadhna Joshi, for her mentorship and guidance. I feel very

fortunate to have learned under her guidance, and I will always regard her as a close friend.

I greatly appreciate the advice and support given by my graduate advisory committee members,

Dr. Joe Minta and Dr. Stanley Read. I am also thankful to the department’s graduate

coordinators, Dr. Dittikavi Sarma and Dr. Harry Elsholtz for their kind support and wise advice.

My appreciations can never suffice for my closest friend, Dr. Masoud Ameli’s, innumerous

assistance and selfless sacrifices and dedications. His deep belief in Allah’s merci eased all my

difficulties.

Last but not least, I would like to extend my gratitude to my friends who helped and supported

me during all these years: My close friends: Dr. Sara Arab and Dr. Payman B. Bokaei; my

colleagues: Dr. Alka Arora, Darinka Sakac, Sabah Asad; and our undergraduate students: John

Kraft and Suraj Sharma.

v

Table of Contents

RNA and DNA Inactivation Strategies to Prevent or Inhibit HIV-1 Replication via Gene Therapy Abstract ii Dedications and Acknowledgements v Table of contents vi List of figures ix List of tables x List of abbreviations and short names xi Chapter 1: General Introduction 1 1.1. HIV-1 2

1.1.1. HIV-1 Life Cycle 4 1.1.2. Anti-HIV-1 Therapies 8 1.1.3. Anti-HIV-1 Gene Therapy Strategies 10

1.2. CCR5 as a Target for HIV-1 Therapy 11 1.2.1. HIV-1 Tropism and Co-receptor Utilization 11 1.2.2. Other HIV-1 Co-receptors 15 1.2.3. The Fusion Process 17 1.2.4. Dominance of CCR5 and Co-receptor Switch 17 1.2.5. Importance of CCR5 23 1.2.6. Receptor Downregulation and HIV-1 Therapy 24

1.2.6.1. Ligands and Intrakines 26 1.2.6.2. Anti-CCR5 Monoclonal Antibodies and Intrabodies 29 1.2.6.3. Zinc Finger-nucleases 31 1.2.6.4. RNA Interference 32 1.2.6.5. Antisense RNA 34 1.2.6.6. Ribozymes 35

1.2.7. Possible Consequences of CCR5 Inhibition 37 1.3. HIV-1 Gene Therapy at Pre-integration and Proviral DNA Levels 39

1.3.1. Integration Process 39 1.3.2. Gene Therapy Strategies to Target Components at Pre-integration and 41

Integration Steps 1.3.2.1. Targeting the Released Viral RNA 41 1.3.2.2. Targeting the Reverse Transcriptase 46 1.3.2.3. Targeting the Integrase 49 1.3.2.4. Targeting the Pre-integration Complex 50 1.3.2.5. Targeting the HIV-1 dsDNA or Integrated Proviral DNA 51

1.3.3. Group II Introns 51 1.3.4. Group II Intron as a Therapeutic Agent 57

1.4. Thesis Objectives 61

Chapter 2: Inhibition of HIV-1 Entry Using Vectors Expressing a Multimeric 63 Hammerhead Ribozyme Targeted Against the CCR5 mRNA

2.1. Abstract 64 2.2. Background 65 2.3. Materials and Methods 67

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2.3.1. Bacteria and Virus Strains and Cell Lines 67 2.3.2. Oligonucleotides and Primers 68 2.3.3. Plasmids 68 2.3.4. Construction of a Multimeric Hammerhead Ribozyme Targeted against 70

CCR5 mRNA 2.3.5. In vitro Cleavage Activity of Rz1-3 and Rz4-7 71 2.3.6. Vector Constructions 72 2.3.7. Transduction and Selection of Stable PM1 Transductants 73 2.3.8. PCR Analysis of Genomic DNA from Stable PM1 Transductants 74 2.3.9. RT-PCR Analysis of Total RNA from Stable PM1 Transductants 75 2.3.10.Immunoflowcytometry Analysis of Stable PM1 Transductants 75 2.3.11.HIV-1 Susceptibility of Stable PM1 Transductants 76 2.3.12.Progeny Virus Infectivity 77 2.3.13.PCR Analysis to Detect the HIV-1 Proviral DNA in the HIV-infected PM1 77

Transductants 2.4. Results 78

2.4.1. Design, Construction, and Activity of the Anti-CCR5 Multimeric 78 Hammerhead Ribozyme, Rz1-7

2.4.2. Oncoretroviral and Lentiviral Vectors Expressing Rz1-7 81 2.4.3. Development of Pools of Stable PM1 Transductants Expressing Rz1-7 81 2.4.4. Rz1-7-Mediated Downregulation of CCR5 mRNA 85 2.4.5. Rz1-7-Mediated Downregulation of the Surface CCR5 Co-receptor 85 2.4.6. HIV-1 Susceptibility of Pools of Stable PM1 Transductants Expressing Rz1-7 85 2.4.7. Infectivity of Progeny Viruses 90 2.4.8. PCR Analyses to Detect the Presence of HIV-1 Proviral DNA in Infected 90

PM1 Transductants 2.5. Discussion 92 2.6. Conclusion 95

Chapter 3: Exploring the Potential of Using Group II Introns to Inactivate HIV-1 97 3.1. Abstract 98 3.2. Introduction 99 3.3. Materials and Methods 100

3.3.1. Bacteria and Virus Strains and Cell Lines 100 3.3.2. Oligonucleotides and Primers 101 3.3.3. Plasmid Constructions 103 3.3.4. Intron Insertion Assay 103 3.3.5. Intron Insertion in HIV-1 Proviral DNA Clone and Purification of Intron- 104

inserted HIV-1 Proviral DNA Clones 3.3.6. Transfection of 293T Cells with Group II Intron-inserted HIV-1 Proviral 104

DNA Clones 3.3.7. RT-PCR Analysis to Detect Group II Intron-inserted HIV-1 RNA in 105

Transfected 293T Cells 3.3.8. Intracellular HIV-1 p24 and Progeny Virus Production from Transfected 105

293T Cells 3.3.9. RT-PCR Analysis to Detect Group II Intron-inserted HIV-1 RNA in the 106

Progeny Viruses Produced from Transfected 293T Cells 3.3.10.PM1 Cell Infection with Progeny Viruses Produced from the Transfected 106

293T Cells

vii

3.3.11.PCR Analyses to Detect Proviral DNA and Reverse-transcribed HIV-1 107 dsDNA in Infected PM1 Cells

3.3.12.Progeny Virus Production from Infected PM1 Cells 107 3.4. Results 108

3.4.1. Modified Group II Introns 108 3.4.2. Modified Group II Intron Insertion Frequencies 108 3.4.3. Group II Intron Insertion into an Infectious HIV-1 Proviral DNA Clone 111 3.4.4. Group II Intron-inserted HIV-1 Proviral DNA Replication in Mammalian Cells 115 3.4.5. Group II Intron-inserted Progeny Virus Replication during the Second Round 119

of Infection 3.5. Discussion 123

Chapter 4: General Discussion and Future Directions 126 4.1. Discussion and Thesis Summary 127 4.2. Future Directions 149 References 154

viii

List of Figures Figure 1.1. The HIV-1 schematic structure. 3

Figure 1.2. A schematic diagram of the HIV-1 LTRs and genes. 5

Figure 1.3. A schematic diagram of HIV-1 life cycle. 6

Figure 1.4- Schematic structure of CC chemokine receptor 5, CCR5. 14

Figure 1.5- Model for HIV-1 Entry. 18

Figure 1.6- A schematic diagram of the Ll.LtrB group II intron. 53

Figure 1.7- A schematic diagram of LtrA protein encoded by Ll.LtrB group II intron 54

Figure 1.8- Specifications of intron-insertion site. 56

Figure 1.9- Mechanism of Ll.LtrB intron splicing and the RecA-independent insertion 58 into the target DNA.

Figure 2.1- In vitro cleavage activity of the anti-CCR5 multimeric hammerhead 79 ribozyme, Rz1-7

Figure 2.2- Schematic diagrams of oncoretroviral and lentiviral vectors. 80 Figure 2.3- PCR analyses to determine the presence of vector DNA sequences in the 83

transduced PM1 cells.

Figure 2.4- RT-PCR analyses to determine the production of Rz1-7 RNA in transduced 84 PM1 cells.

Figure 2.5- RT-PCR analyses to determine CCR5 mRNA levels in transduced PM1 cells. 86

Figure 2.6- Immunoflowcytometry analyses to determine CCR5 co-receptor expression 87

level on the surface of untransduced and transduced PM1 cells.

Figure 2.7- Susceptibility of transduced PM1 cells to R5-tropic HIV-1 (Ba-L). 89

Figure 2.8- Assessment of HIV-1 proviral DNA from untransduced PM1 cells, as well 91 as from control or ribozyme vector-transduced PM1 cells challenged with HIV-1.

Figure 3.1- Structure of the group II intron-inserted HIV-1 proviral DNA, its transcripts 109 and the amino acid sequence of C-terminal region of the mutant Gag-Pol

Figure 3.2- Insertion frequencies of introns I4021s, I4021sIN, I4021sN, I4021s∆N, I4069s, I4069sIN, 112 I4069sN, and I4069s∆N.

Figure 3.3- Experimental scheme used to show insertion of I4021sN and I4069sN introns 113 into pHIV and isolation of the intron-inserted HIV-1 proviral DNA.

Figure 3.4- RT-PCR analysis for determining intron insertion. 116

Figure 3.5- HIV-1 p24 antigen detection in cell lysates and culture supernatants. 118

Figure 3.6- PCR analysis for determining intron insertion pHIV. 121

Figure 3.7- Progeny virus production by PM1 cells infected with the progeny virus 122 from pHIV-, pHIV-I4021sN-, or pHIV-I4069sN- transfected 293T cells.

ix

List of Tables Table 2.1- Oligonucleotides and primers sequences. 69

Table 3.1- Oligonucleotides and primers sequences. 102

Table 4.1- Summary of gene therapy approaches to downregulate CCR5 surface expression. 132

Table 4.2- Summary of gene therapy approaches to inhibit HIV-1 replication at pre- integration or integration stages. 141

x

List of Abbreviations and Short Names 3TC: Lamivudine

ABC: Abacavir succinate

ABT-378/r: Lopinavir

AIDS: acquired immunodeficiency syndrome

Ap: Ampicillin

APV: Amprenavir

ATP: adenosine triphosphate

AZT: azidothymidine

BAF: barrier-to-autointegration factor

CXCR4: CXC chemokines receptor 4

CCR5: CC chemokines receptor 5

cDNA: complementary deoxyribonucleic acid

Cm: Chloramphenicol

CMV: cytomegalo virus

CTL: cytotoxic T lymphocyte

CTP: cytidine triphosphate

d4T: Stavudine

dATP: deoxyadenosine 5'-triphosphate

dCTP: deoxycytidine 5'-triphosphate

ddI: Didanosine

ddC: Zalcitabine

dGTP: deoxyguanosine 5'-triphosphate

DMEM: Dulbecco's modified Eagle's medium DMSO: dimethyl sulfoxide

DNA: deoxyribonucleic acid

DNase: deoxyribonuclease

dNTP: deoxynucleoside 5'-triphosphate

DOHH: deoxyhypusine hydroxylase

dsDNA: double-stranded DNA

dTTP: deoxythymidine 5'-triphosphate

dUTP: deoxyuridine 5-triphosphate

DLV: Delaviridine

EBS: exon-binding site

EBV: Epstein Barr virus

xi

EF-1α: human elongation factor-1α

EFZ: Efavirenz

EGFP: enhanced green fluorescent protein

EMV: Emivirine

En: endonuclease

Env: envelop protein

ER: endoplasmic reticulum

E. coli: Escherichia coli

FACS: fluorescence-activated cell sorting

FDA: Food and Drug Administration

FTC: Emtricitabine

Gag: group antigen

GFP: green fluorescent protein

gp: glycoprotein

HAART: highly active anti-retroviral therapy

HIV-1: human immunodeficiency virus type 1

HIV-2: human immunodeficiency virus type 2

HLA-DR: human leukocyte antigen-DR

HMGA1: high-mobility group protein A1

HSA: heat-stable antigen marker

HS/PCs: hematopoietic stem/progenitor cells

IBS: intron-binding site

IDV: Indinavir

IEP: intron-encoded protein

IL: interleukin

imp: Importin

IN: integrase

IPTG: Isopropyl β-D-1-thiogalactopyranoside

IRES: internal ribosome entry site

Kn: Kanamycin

LEDGF: lens epithelium-derived growth factor

LTR: long terminal repeat

M-tropic: macrophage-tropic

MA: matrix protein

xii

MDM: monocyte-derived macrophage

MIP-1α: macrophage inflammatory protein-1α

MIP-1β: macrophage inflammatory protein-1β

MOI: multiplicity of infection

MoMuLV: moloney murine leukemia virus

mRNA: messenger ribonucleic acid

MSCV: mouse stem cell virus

Nef: negative factor

NFV: Nelfinavir mesylate

neo: neomycin phosphotransferase

NLS: nuclear localizing sequence

NNRTIs: non-nucleoside analogue RT inhibitors

NPC: nuclear pore complex

NRTIs: nucleoside/nucleotide analogue RT inhibitors

NSI: non-syncytium inducing

nt(s): nucleotide(s)

NVP: Nevirapine

ORF: open reading frame

PBL: peripheral blood lymphocyte

PBMC: peripheral blood mononuclear cell

PBS: primer binding site

PBS: phosphate buffered saline

PCR: polymerase chain reaction

PIC: pre-integration complex

Pol: polymerase

R: repeat sequence

RANTES: regulated on activation, normal T-cell expressed and secredted

Rev: regulator of expression viral proteins

RH: rapid-high RNA: ribonucleic acid

RNAi: interfering ribonucleic acid

RNase: ribonuclease

RNP: ribonucleoprotein

RPMI medium: Roswell Park Memorial Institute medium

RT: reverse transcriptase

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RT-PCR: reverse transcription polymerase chain reaction

RTV: Ritonavir

Rz: ribozyme

scFv: single-chain variable fragment

SCID: severe combined immunodeficiency

SCID-hu: humanized severe combined immunodeficiency

SDS: sodium dodecyl sulfate

shRNA: short hairpin RNA

SI: syncytium inducing

siRNA: small interfering RNA

SIV: simian immunodeficiency virus

SL: slow-low

SQV: Saqinavir mesylate

+sssDNA: plus-strand strong stop DNA

T-tropic: T-cell-tropic

T-20: enfuvirtide, Fuzeon

TAR : transactivation responsive element

Tat: trans-activator of transcription

TCID50: 50% Tissue Culture Infective Dose

TDF: Tenofovir

Tet: Tetracycline

TPV: Tiparanavir

tRNA: transfer ribonucleic acid

TRTI : template analog RT inhibitor

U5: unique 5'

U3 : unique 3'

UTP: uridine 5'-triphosphate

VH: variable heavy

Vif: viral infectivity factor

VL: variable light

Vpr: viral protein R

Vpu: viral protein U

WHO: World Health Organization

WT: wild-type

xiv

ZDV: Zidovudine

ZFN: Zinc finger nuclease

xv

CHAPTER 1

GENERAL INTRODUCTION

1

The human immunodeficiency virus (HIV) was first identified in 19831,2. The complete

nucleotide sequence of the viral genome and the viral proteins were identified and characterized

soon after3,4. There are two types of HIV: HIV-1 and HIV-2. Both types are known to cause

acquired immunodeficiency syndrome (AIDS), however infection with HIV-1 is more common

worldwide.

Each year more than three million people die from AIDS. More than 25 million people have died

from AIDS since 1981. It is predicted that the number of infected people worldwide can rise to

90 million and 48 million will die by 2010. According to the surveillance report of the Public

Health Agency of Canada in April 2006, until the end of year 2005, 57,780 people were

diagnosed with HIV in Canada. According to the estimates from UNAIDS/WHO AIDS

Epidemic Update (December 2006), over 40 million people are living with HIV-1 worldwide

with the following distribution: Sub-Saharan Africa: 63%; Asia: 21%; North America, Western

and Central Europe: 5%; Eastern Europe and Central Asia: 4%; Latin America: 4%; North Africa

and Middle East: 1%; Caribbean: 0.8%; and Oceania: 0.2%. The overwhelming majority of

people with HIV-1 live in the developing world. The infection rate continues to rise in these

countries due to poverty, poor health care systems, and limited resources for prevention and care.

1.1. HIV-1

HIV-1 is a retrovirus of the lentivirus subfamily. Retroviruses (retro from Latin, `turning back`)

are RNA viruses that replicate via DNA intermediates using the viral reverse transcriptase (RT)

enzyme. The mature virion of HIV-1 has a diameter of approximately 100 nm (Figure 1.1). The

outer envelope, which is formed from the host cell membrane, is a lipid bilayer that contains host

cell proteins and spikes of the viral envelope glycoproteins (gp120 and gp41). Inside the lipid

2

Figure 1.1. The HIV-1 schematic structure. The virus is enveloped by a cell-originated lipid

bilayer membrane, containing HIV-1 surface and transmembrane glycoproteins. Matrix proteins

are arranged under the envelope. Capsid proteins enclose the virus core, which includes two plus

RNA strand genome, associated with nucleocapsid proteins. A tRNA3Lys and a reverse-

transcriptase are also bound to each of the RNA strands. The core also includes PR and IN

enzymes. Vpr, Vif, and p6 proteins are located in the space between matrix and the core. (From

Joshi & Joshi, 1996)5

3

bilayer are the internal structural capsid and core proteins, p17, p24, p7, and p6. These proteins

enclose two copies of the single-stranded 9-kb RNA genome and multiple RT molecules in the

center of the virus particle, which is called “core”. The virions also carry viral integrase (IN) and

protease (PR) enzymes inside the core. This core structure is often visible in thin sections viewed

by electron microscopy.

The HIV-1 genome contains at least nine different genes that encode 15 different proteins. Three

main genomic regions are: gag (coding for structural proteins), pol (coding for the viral enzymes

PR, RT and IN) and env (coding for the envelope glycoproteins) (Figure 1.2). The other HIV-1

gene products, tat, rev, vpr, and nef regulate the virus life cycle. The viral genome contains long

terminal repeat (LTRs) sequences at each end. The LTRs contain the viral promoter, as well as

binding sites for cellular proteins that are able to activate transcription. The HIV-1 genomic

RNA also contains signal and structural elements, which are involved in different stages of viral

life cycle, such as activation, transportation, packaging, and reverse-transcription5.

1.1.1. HIV-1 Life Cycle

HIV-1 begins the infection of a susceptible human host cell by binding to the CD4 receptor on

the cell surface. CD4+ is expressed on T lymphocytes, macrophages, microglial, dendritic and

Langerhans cells and makes them targets for HIV-1 infection. The CD4 transmembrane protein

along with one of two chemokine receptors, CCR5 and CXCR4, are necessary for viral

attachment and fusion to occur (Figure 1.3). During fusion, viral membranes fuse with target cell

membranes and HIV-1 matrix and capsid proteins eventually disassemble.

4

U3 U5R

5’ LTRgag

PR RT

pol

vif

vpr

tat vpu

rev

rev

tat

env

nef

U3 U5R

3’ LTRIIII IIIN

MA CA NC P6

gp120 gp41

Figure 1.2. A schematic diagram of the HIV-1 LTRs and genes. Like all retroviruses, HIV-1

genome encodes for gag, pol, and env. However, HIV-1 also contains six accessory gene

products (tat, rev, vif, vpu, vpr, and nef), some of which are essential for HIV replication and

reproduction.

5

Figure 1.3. A schematic diagram of HIV-1 life cycle. After binding and fusing to the surface of

the host cell, the HIV-1 core enters the cell. RT synthesizes dsDNA, which is transported to the

nucleus. Integration occurs and the resulting provirus expresses viral RNA that will synthesize

viral proteins using the host's ribosomes. New virions are created by assembly and budding

through the infected cell membrane and subsequent maturation due to the actions of PR. Adapted

from www.medspace.com

6

http://www.medspace.com/

When the virus enters the cell, the genomic RNAs are released and undergo reverse transcription

into DNA by viral RT enzyme. Synthesis of proviral DNA includes several steps: binding of the

primer tRNA, synthesis of DNA from RNA by RT, degradation of viral RNA by the RNase H

domain of RT, and second strand DNA synthesis by RT.

The proviral DNA then enters the host cell nucleus, where it can be integrated into the genetic

material of the cell. In order for integration to occur in non-replicating cells, the HIV-1 pre-

integration complex (PIC) must be actively transported into the nucleus. A nuclear localization

signal (NLS) on the HIV-1 matrix protein, as well as other interactions with some cellular

proteins, have been reported to facilitate this transportation6. Viral protein R (VPR), an accessory

protein that appears to be incorporated in the virion, has also been reported to facilitate targeting

of HIV-1 PIC to the nucleus7.

After entry into the nucleus, HIV-1 double-stranded DNA (dsDNA) of the PIC undergoes

specific cleavages at the 5' and 3' termini and is integrated into the host DNA through the action

of the HIV-1 IN.

Although HIV-1 replicates extensively throughout all stages of infection8,9, latently infected cells

do exist10, which serve as HIV-1 reservoir in the body. The vast majority of virion production is

from the newly infected cells and not the activation of latently infected cells8,9.

In the cells where the virus is replicating, newly synthesized viral RNA is transported out of the

nucleus and translated (in the case of mRNA) or incorporated into new virions (in the case of

genomic RNA). The gag and pol genes are encoded out of frame on a single mRNA. Frame-

7

shifting during the translation of the viral gag-pol messenger RNA, therefore, is essential for the

production of pol gene products (PR, RT, and IN)11.

The viral proteins and genomic RNA then migrate to and accumulate on the cell membrane of

the releasing virions. The progeny virus buds from the cell membrane, and after release, undergo

the maturation process in which the pol-encoded PR cleaves the gag and gag-pol precursor into

matrix (MA, p17), capsid (CA, p24) and nucleocapsid (NC, p7) proteins12,13. Assembly of

infectious virions is also dependent on the action of cellular N-protein myristoyl transferase

(NMT), which adds myristic acid to the N-terminus of gag, gag-pol and nef precursors before the

virions bud14.

1.1.2. Anti-HIV-1 Therapies

To date, 22 FDA-approved antiretroviral agents are in use for HIV/AIDS therapy and several

others are currently in different stages of basic development and clinical trials. The year 2006

was the 10th anniversary of advent of highly active anti-retroviral therapy (HAART), which

significantly decreased the AIDS morbidity and mortality, and provided great hopes for the HIV-

positive individuals. The drugs that are used in HAART are developed to target viral proteins

RT, PR, IN and viral fusion compartments.

Anti-RT drugs include nucleoside/nucleotide analogue RT inhibitors (NRTIs) such as zidovudine

(ZDV, also called azidothymidine or AZT), didanosine (ddI), zalcitabine (ddC), stavudine (d4T),

lamivudine (3TC), abacavir succinate (ABC), emtricitabine (FTC), and tenofovir (TDF), and

non-nucleoside analogue RT inhibitors (NNRTIs) such as nevirapine (NVP), delaviridine (DLV),

efavirenz (EFZ), and emivirine (EMV). Anti-PR drugs include saquinavir mesylate (SQV),

ritonavir (RTV), indinavir (IDV), nelfinavir mesylate (NFV), amprenavir (APV), lopinavir

8

(ABT-378/r), tipranavir, and atazanavir. Anti-IN drugs consist of oligonucleotides, dinucleotides

and different kinds of chemical agents, such as pdCpI dicaffeoylquinic acids and 2,4-

dioxobutanoic acid analogous. The drugs that inhibit HIV-1 binding and fusion are T-20

(enfuvirtide, Fuzeon) and T-1249. These drugs are currently used as components of a multi-drug

cocktail in HAART15.

Other drugs are also being tested currently in different clinical trial phases, including four RT

inhibitors such as racivir (phase II), etravirine (phase III), rilpivirine (phase II), and BILR-355

BS (phase IIa), two PR inhibitors such as tiparanavir (TPV- phase II) and BMS 232,632 (phase

II), one IN inhibitor, MK-0518 (phase III), and three fusion inhibitors BMS-488043 (phase II),

NBD-556 and NBD-557 (both phase I)16. The drugs that target cellular factors include a

ribonucleotide reductase inhibitor (hydroxycarbamide (Hydrea), previously known as

hydroxyurea), cyclophilin A inhibitor (ciclosporin), deoxyhypusine hydroxylase (DOHH)

inhibitor (deferiprone), deoxyhypusine synthase inhibitor (CNI-1493, phase II), anti-CD4

monoclonal antibody (TNX-355), CCR5 inhibitors (maraviroc and vicriviroc, phase II), CXCR4

inhibitor (PRO 140, phase I)16.

In general, low intracellular permeability, drug toxicity, poor patient adherence to complicated

drug regimens, high mutation rate resulting in the emergence of drug-resistant isolates, and

persistence of viral reservoirs are the major obstacles facing current drug therapy. These

problems have led researchers to develop new drugs with novel mechanisms of action, as well as

alternative therapies such as gene therapy16,17.

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http://www.aidsmap.com/cms1032256.asp

1.1.3. Anti-HIV-1 Gene Therapy Strategies

A number of gene therapy strategies have been designed to inhibit HIV-1 replication. The

`therapeutic gene(s)` against identified key target(s) in HIV-1 or host cell were delivered to the

cell by a variety of vectors to interfere with various steps of HIV-1 life cycle15,18,19. Those

strategies that use interfering molecules against HIV-1 genes/RNAs/proteins, target highly

conserved regions in the virus. However, there is always a chance of escape mutations. So far,

many anti-HIV-1 genes that targeted HIV-1 proteins failed at different stages of trials. However,

it has been shown that by a combinational strategy that targets different stages of HIV-1 life

cycle, the effectiveness can be increased and the chances of generating escape mutants may be

minimized. Many examples of such combination therapies are described in reviews by Lamothe

and Joshi (2000)18 and Nielsen et al. (2005)20.

The anti-HIV-1 gene therapy strategies are categorized into two groups: protein-based and RNA-

based. The protein-based strategy includes intrakines, single-chain antibodies (intrabodies),

transdominant mutants, targeted/packageable nucleases, and Zinc finger-nucleases.

Transdominant RevM1021, gp41-disruptive peptide22, and anit-HIV-1 antibodies23 are among the

protein-based strategies that are currently at different stages of clinical trials. The RNA-based

approaches consist of antisense24,25, ribozymes26-28, RNA aptamers and decoys29-33, and RNAi

(shRNA and siRNA)34-37. Ribozyme against HIV tat and antisense RNA against HIV env are two

of the anti-HIV-1 RNA-bases therapeutic molecules that are currently in clinical trials23.

Another approach will be targeting a cellular factor that undergoes less diversity. CCR5 co-

receptor has such characteristic, because, as will be explained later, it is the only naturally

dispensable cellular molecule involved in HIV-1 infection. Downregulation of CCR5 expression

via gene therapy strategies has been attempted by several groups. Anti-CCR5 zinc-finger and

10

nucleases (protein-based strategies), and ribozyme and RNAi against CCR5 (RNA-bases

strategies) are now being tested in animals23.

The gene therapy strategies that interfere with HIV-1 RNA or proteins, mostly target the virus

life cycle at post-integration stages. While proviral DNA in infected cells continues producing

viral RNA and proteins, the therapeutic molecules must be expressed in the cells with the pace of

HIV-1 gene expression to be able to inhibit virus replication. Even if the virus replication is

inhibited, the originally infected cells are unable to function normally in the immune system.

Therefore, we believe that the best approach would be preventing viral entry to the cells via

downregulation of CCR5 expression or inactivating the proviral DNA in the infected cells by

insertion of a modified group II intron into specific sites of HIV-1 proviral DNA. In this

introduction, I will provide an in-depth review of the anti-HIV-1 gene therapy strategies that are

designed to downregulate or block CCR5 expression, and those that are designed to interfere

with pre-integration and integration process or to inactivate the proviral DNA.

1.2. CCR5 as a Target for HIV-1 Therapy

1.2.1. HIV-1 Tropism and Co-receptor Utilization

HIV-1 can infect various CD4+ human target cell types38. The viral isolates obtained from

peripheral blood of individuals, shortly after infection and during the asymptomatic phase, are

predominantly macrophage-tropic (M-tropic). As the infection progresses to AIDS, T-cell-tropic

(T-tropic) viruses can be isolated from many, but not all, patients.

The HIV-1 entry into the target cells starts by fusion between the viral envelope and the cell

membranes, which is initiated by a high-affinity binding of the viral envelope glycoprotein (Env)

11

to CD4 on the cell surface. However, CD4 is not the only cellular molecule involved in fusion, as

evidenced by studies showing that its expression alone on non-human cells does not render them

permissive to infection39.

Research focused on identification of the co-factors led to the discovery of the first HIV-1 co-

receptor by Feng et al. (1996)40. They showed that this co-receptor belongs to the superfamily of

the seven transmembrane G protein–coupled receptors. The co-receptor was called “Fusin” due

to its activity in HIV-1 Env-mediated fusion40. When Fusin and CD4 were co-expressed in non-

human cells, the cells could be infected by some of HIV-1 strains. In addition, it had been found

that anti-Fusin antibodies could inhibit infection of primary human CD4+ T lymphocytes.

However, Fusin could play roles in fusions and infections only when T-tropic HIV-1 strains were

used, not the M-tropic strains. Thus, Fusin was considered as the T-tropic HIV-1 co-receptor.

Also, Bleul et al. (1996) reported that infections of CD4+ T lymphocytes by the T-tropic HIV-1

strains were inhibited with use of stromal cell-derived factor-1 (SDF-1 or CXCL-12)41. Fusin

was later shown to be a receptor responding to SDF-141-43 and it was renamed CXCR4 as it

represented the fourth receptor for CXC chemokines. Chemokines (the abbreviation for

chemoattractant cytokines) are small proteins (92-125-amino acid long)44 produced during

immune activation, inflammation, and autoimmune diseases to induce a chemotactic migration

signal so that the cells migrate to the source of chemokine43.

The CC chemokines regulated on activation, normal T-cell expressed and secreted (RANTES or

CCL-5), macrophage inflammatory protein-1α (MIP-1α or CCL-4), and MIP-1β (CCL-4)45,

which are released mainly by CD8+ T lymphocytes, suppressed infection by M-tropic HIV-1

strains46. A receptor corresponding to these chemokines was identified by Samson et al. (1996),

Combadiere et al. (1996) and Raport et al. (1996)47-49. It was first designated CC CKR5 and was

12

later called CCR5 (Figure 1.4). CCR5 was shown to be the major co-receptor for M-tropic HIV-

1 strains50-54. It was also shown to be the only co-receptor used by the simian immunodeficiency

virus (SIV)55.

The identification of the chemokine receptors CCR5 and CXCR4 as HIV-1 co-receptors shed

light on the molecular basis of viral tropism and pathogenesis. CCR5 is expressed on the surface

of macrophages and CD4+ T-lymphocytes51, and is the common receptor for the M-tropic strains

that predominate during transmission56. Generally, strains that use this co- receptor cause the

majority of new infections47,57. In fact, the viruses that are transferred by infected persons can

replicate in both macrophages and primary CD4+ T-cells, but, neither will be able to infect T-

cell lines58-61, nor will form syncytia in T-cell lines. Therefore, the M-tropic strains were named

non-syncytium inducing (NSI) viruses. Considering the slow replication of the M-tropic viruses

in cell cultures, they were also called slow-low (SL) strains59. In the recent nomenclature, the M-

tropic strains are named R5-tropic, as they use CCR5 co-receptor.

Generally in about 50% individuals, about 4-5 years after the initial infection, viral strains evolve

to be able to infect T-cell lines, too40,62. Viral strains that utilize the CXCR4 co-receptor are

called X4-tropic. The virus evolution from R5-tropic to X4-tropic strain is correlated with

accelerated CD4+ T-cell decline and progression to AIDS63. Although sometimes the X4-tropic

strains loose their ability to replicate in macrophages, most of the time the primary isolates will

still be able to use both CCR5 and CXCR4 co-receptors and, therefore, they are called dual-

tropic or R5X4-tropic strains64.

13

HOOC

NH2

Extracellular Loops

Transmembrane Domains

Intracellular Loops

Figure 1.4. A schematic structure of CC chemokine receptor 5, CCR5, a 352-amino acid protein

encoded from chromosome 3p2149. Adapted from Lederman et al. (2006)65.

14

Viruses that can use CXCR4 and infect the T-cell lines have also been referred to as T-tropic,

syncytium-inducing (SI), or rapid-high (RH), respectively based on their ability to infect T-cells,

form syncytia in cultured T-cell lines, and rapid replication kinetics60.

The described nomenclature systems are sometimes confusing. For example, naming a virus

strain as macrophage (M)-tropic or T-cell (T)-tropic may be implied that the stain cannot infect

the other type of cells. Calling a strain as NSI may mean that the virus Env protein is deficient in

binding to the co-receptor. It may also be incorrectly understood that SI viruses are more

cytopathic and fusogenic than the NSI viruses, which we now know that this is not correct in

primary CD4+ T-cell cultures. It seems that this effect is more related to the type of cells than the

strain of virus. It has been shown that NSI viruses are able to form syncytia when infect the

CCR5-expressing cells lines. Therefore, for many instances, it is not appropriate to use terms

NSI, SL and M-tropic as synonyms; likewise for the terms SI, RH and T-tropic66.

To evolve to X4-tropic strains, R5-tropic viruses undergo mutations in the Env glycoprotein 120

(gp120), which are usually located in the V3-loop. It has been shown that the V3-loop of X4-

tropic HIV-1 Env protein is more basic than that of R5-tropic strains67,68. The X4-tropic strains

use the first and second extracellular loops of the CXCR4, which are noticeably more anionic

than the same domains of CCR569,70, therefore, an opposite-charge interaction may be involved

in the interaction of gp120 and CXCR4. In contrast, gp120 from the R5-tropic strains interacts

with the N-terminus of CCR5 co-receptor. Although the extracellular domains of CCR5 and

CXCR4 are less than 20% identical in amino acid sequence, R5X4-tropic strains are able to use

both co-receptors to efficiently enter the cells. The Env from the dual-tropic strains can interact

with the first and second extracellular loops of CXCR4 as well as the N-terminus of CCR570. It is

not clearly understood if the appearance of dual-tropic strains is an evolutionary step between

15

R5- to X4-tropic convergence or a final result of such phenomenon. However, it has been shown

that although R5X4-tropic HIV-1 strains are able to use both co-receptors, they prefer to use

CXCR4 to enter primary T-cells71.

1.2.2. Other HIV-1 Co-receptors

All HIV-1 strains examined so far use one or both of CCR5 and CXCR4 co-receptors66,72. In

addition to CCR5 and CXCR4, at least twelve other chemokine or chemokine receptor–like

orphan receptors have been detected to be involved in the cellular entry of one or more viral

strains. These include CCR2b53, CCR351,53,73, CCR874-76, CCR977, CXCR672, CX3CR1 (formerly

named CMKBRL1 or V28)78, GPR155, GPR15/BOB55,79, Apj77,80, US2881, ChemR2382, and

STRL33/Bonzo79,83.

There are discrepancies about the use of additional co-receptors. The most variable factor in

determining whether a given chemokine or orphan receptor can serve as an HIV-1 co-receptor is

the level of its expression. For example, when CCR3 is highly expressed in cells, most of the

tested X4-, R5-, or X4R5-tropic HIV-1 strains are able to infect the cells. However, when the

level of CCR3 expression is lower, only a few viral strains can infect the cells via this co-

receptor74. Another important factor is relative expression of the co-receptor compared to CD484.

It has been shown that low levels of CCR5 or CXCR4 can suffice virus entry when high level of

CD4 receptor is expressed on cell surface. In contrast, when the CD4 receptor level is low, high

levels of CCR5 or CXCR4 are required to allow virus entry84. However, viral entry via some

chemokine receptors is only shown to occur in vitro and there is not enough evidence of their

usage in vivo85.

16

It is known that the HIV-1 strains are very dependent on CD4 receptor to infect the target cell.

Only a few strains are found to be able to infect CD4-negative cells. However, presence of CD4

is not really crucial for some HIV-2 and SIV strains as they are able to use CCR5 or CXCR4 to

enter the target cells without need to CD466.

1.2.3. The Fusion Process

HIV-1 entry into target cells begins with interactions between viral Env gp120 and cell’s CD4

receptor. The HIV-1 envelope glycoproteins gp120 and gp41 trimers are non-covalently bound

together on the virus envelope (Figure 1.5)86,87. Gp120 consists of five variable loops (V1-V5)

and five constant domains (C1-C5), making an inner and an outer domain. A four-stranded

antiparallel β-sheet (bridging sheet) connects the domains to each other86. A highly conserved

groove at the boundary of the inner and outer domains and the bridging sheet of gp120 binds to

the first extracellular domain of the CD4 receptor88,89. Formation of the gp120-CD4 complex

causes conformational changes to the gp120 core structure that exposes a co-receptor-binding

site on gp120. This site contains a core of hydrophobic amino acids that is surrounded by

positively charged residues. Since the co-receptor is anchored in the host membrane, binding

moves the gp120 bridging sheet close to the target membrane89. The gp120-co-receptor

interaction causes additional conformational changes in the gp120-gp41 trimer that forces the

hydrophobic, glycine-rich fusion peptide region of gp41 to insert into the target cell’s plasma

membrane90.

1.2.4. Dominance of CCR5 and Co-receptor Switch

As mentioned before, CCR5 is the common co-receptor for R5-tropic HIV-1 strains that are

associated with transmission56,66,91. It is expressed on the surface of effector cells (e.g. T-cells,

17

Target cell

HIV virion

CD4

CCR5

b HIV virion a

gp41

gp120

Target cell

HIV virion

CCR5

CD4

c d

Target cell

HIV virion

Figure 1.5. Model for HIV-1 Entry. (a) the HIV-1 Env protein as a heterodimer consisting of

three gp120/gp41; (b) upon binding of cellular CD4 to gp120, the gp120 undergoes a

conformational change; (c) the altered gp120 now is capable of binding to the co-receptor, here

CCR5. The gp120-CCR5 interaction causes a conformational change in gp41, which enables it to

insert the hydrophobic parts into target cell membrane; (d) folding of gp41 trimer on itself brings

the membranes of virus and cell close together. Adapted from Lederman et al. (2006)65.

18

natural killer cells, and natural killer T-cells that produce inflammatory cytokines or destroy

infected cells)49,92,93, antigen presenting cells (e.g. monocytes, macrophages, and dendritic cells

that initiate immune responses)48,65,94-96, as well as the Langerhans cells97 and the mucosa of

rectum, colon, vagina, and cervix96,98. Viruses that use the CCR5 co-receptor cause the vast

majority of new infections57,99, are more frequently found in asymptomatic infected individuals,

and are involved in person-to-person and mother-to-child transmission100.

The co-receptor switch is not necessary for disease progression since X4-tropic strains are not

detected in 50% of individuals who develop AIDS101. However, the co-receptor switch is an

important factor contributing to HIV-1 pathogenesis since individuals, in which X4-tropic strains

dominate during the disease, show a faster decline in CD4+ T-cells and progress significantly

faster to AIDS than individuals who only bear the R5-tropic HIV-163.

There are three theories that explain the predominance of the R5-tropic virus in transmission.

Based on the “transmission-mutation hypothesis” the reasons for the dominance of R5-tropic

virus during or soon after transmission could be selection in favor of R5-tropic virus either in the

donor or the recipient101. The selection in donor can occur due to deferential distribution of R5-

and X4-tropic viruses in organs (such as the male genital tract) that are involved in transmission.

However, dominance of R5-tropic virus in semen could not be demonstrated102. CCR5 is also

thought to play an important role in selection in the recipient during early stages of transmission.

For instance, CCR5 is mainly expressed on the surface of intestinal epithelial cells. These cells

play an important role in infections via oral–genital contact (uncommon) and mother-to-child

transmissions. This may explain the reason of predominance of R5-tropic viruses when the

infection is transmitted through the above venues103,104. It has also been shown that among the

two types of viruses, mainly R5-tropics bind to dendritic cells and can be transported from the

19

mucosal tissues to lymph nodes by these cells105. On the other hand, mucosal surfaces express

high levels of SDF-1, causing the CXCR4 to be selectively downregulated on intestinal

lymphocytes103. However, we know that R5-tropic viruses form the majority of virus population

in patients that are infected through intravenous drug injection, blood transfusion, or sexual

intercourse91. Therefore, the predominance of R5-tropic virus occurs regardless of transmission

route106,107.

The “transmission-mutation hypothesis” also suggests that evolution in the virus population

during the infection results in co-receptor switch from CCR5 to CXCR4101. Neither selection of a

specific transmission route in the epithelial or mucosal tissue nor an advantage for predominance

of R5-tropic viruses in the competition for infecting the target cells is supported by other

observations. If competitive advantage is the reason of early-stage predominance of R5-tropic

strains, X4-tropic strains should predominate in infection of CCR5-deficient individuals. In

contrast, it has been shown that homozygous ccr5∆32/∆32 individuals are highly resistant to

HIV-infection108. Therefore, it was suggested that the predominance of R5-tropic strains may

occur because of replication of virus in some tissues, such as mucosa of the gastrointestinal tract,

in which higher levels of CCR5, but not CXCR4, are expressed63,109. However, late appearance

of X4-tropic virus strains is in contrast with this hypothesis110.

It is shown that, in most cases, for an R5-tropic strain to evolve to an X4-tropic type, only two

mutations are required. Considering the high mutation rate of HIV111 and the high production of

virus progeny (~1010–1012 virions every two days)112, appearance of X4-tropic strains must occur

earlier during infection68. It is known that switch to X4-tropic virus occurs only in ~50% of

individuals and at various times after infection. The reason Pastore et al. (2004)113 suggested for

20

these facts is that the intermediate mutants between R5-tropic and X4-tropic viruses have fitness

disadvantage and X4-tropic virus is fitter than R5-tropic virus.

Another hypothesis, the “immune-control hypothesis”, suggests that the patient’s immune

system recognizes the X4-tropic virus better than the R5-tropic virus and removes them at the

early stages of infection. By the time virus replication results in immune-system deterioration,

the selection against the X4-tropic virus is decreased101. Although the mathematical models

support this hypothesis, there is relatively little evidence to show such selective pressure

exists101. A specific immune mechanism that could lead to stronger selection pressure against

X4-tropic virus by the immune system is yet to be identified114.

The other hypothesis that tries to explain the predominance of R5-tropic virus at early stages of

infection and switch to X4-tropic virus later is called “target cell-based hypothesis”. We know

that CD4+ T-cells are the major HIV-1 target cells in vivo115. While high proportion of naïve

CD4+ T-cells expresses CXCR4, smaller fractions of memory CD4+ T-cells express both CCR5

and CXCR4116. This causes both R5- and X4-tropic viruses to have different target cell ranges,

which, however, may overlap117. During the infection, the CD4+ T-cell population changes in

several related manners: 1) in the peripheral blood, the number of memory CD4+ T-cells

increases and the number of naïve CD4+ T-cells decreases118. If we assume that the same

changes occur in the lymphatic system, this will cause the selection pressure to increase in the

favor of R5-tropic virus. 2) the number of proliferating naïve CD4+ T-cells at the early stages of

infection is lower than the number of proliferating memory CD4+ T-cells. This ratio will convert

at the later stage of infection in a way that the number of proliferating naïve CD4+ T-cells

increases to reach the level where the memory CD4+ T-cells used to be119. Such conversion

would increase the selection pressure in favor of X4-tropic virus.

21

In addition, when the immune system is activated upon infection, the expression of human

leukocyte antigen-DR (HLA-DR) increases, which results in proliferation of CCR5-expressing

CD4+ T-cells120. Higher number of CCR5-expressing CD4+ T-cells will increase the selection

pressure in favor of the R5-tropic virus. This may explain why in almost all cases of infection,

appearance of X4-tropic virus is delayed by years. More knowledge about the pathways of CD4+

T-cells differentiation and the CD4+ T-cell progeny’s susceptibility to R5- and X4-tropic virus is

required to approve this hypothesis114.

Other investigators focused on the kinetics and life cycle of HIV-1 in the infected target cells.

Rodrigo suggested that, during primary infection, there is a competition between R5- and X4-

tropic viruses121. This model hypothesizes that R5- and X4-tropic viruses would infect the same

cells. However, because X4-tropic virus causes syncytia and, therefore, has a higher

cytopathicity and shortens the lifespan of infected cells, it has a replicative disadvantage and R5-

tropic virus predominates.

Sometimes, actual findings support one or more of the above hypotheses. For instance, both

transmission-mutation hypothesis and the target-cell-based hypothesis are supported by the fact

that the X4-tropic viruses are subject to higher diversifying selection pressure than R5-tropic

viruses. However, other times, the findings can criticize the basis of a hypothesis. For example,

the fact that CCR5∆32 homozygous individuals are highly resistant to infection is in contrast to

the statement of the transmission-mutation hypothesis suggesting that predominance of R5-tropic

viruses at the early stages of infection is because of their competitive advantages over X4-tropic

viruses108.

22

1.2.5. Importance of CCR5

There are two reasons why CCR5 is an attractive target for HIV-1 therapy: first, the R5-tropic

HIV-1, which utilizes the CCR5 co-receptor, predominates during early infection. Second,

approximately 1% of Caucasians, who lack CCR5, are highly resistant to HIV-1 infection52,57,122.

In individuals heterozygous for the mutant allele, the infection is delayed123,124 and the plasma

viremia is lower52,122-124. The mutant allele of CCR5, which is present at a relatively high

frequency (allele frequency, 0.092 or ~10%) in Caucasians, but absent in black and Asian

populations, contains a 32-bp deletion (CCR5∆32) within the coding region57,99. This deletion

results in a frame-shift and generates a non-functional receptor that is truncated and cannot be

exported to the cell surface57. The membrane fusion or infection by R5- and R5X4-tropic HIV-1

strains does not occur in individuals who are homozygous for CCR5∆3299,108.

The reason progression to AIDS is delayed in CCR5 +/∆32 heterozygotes, is that the amount of

CCR5 protein on the cell surface of such individuals is lower than that expected from a simple

gene dosage effect92. This can be because of the transdominant effect of the ∆32 truncated

protein, which results in production of a deficient heterodimer that remains in the endoplasmic

reticulum99,125.

Rare cases of infection with X4-tropic strains have been reported in CCR5∆32

homozygotes56,126,127. This strongly indicates that CCR5 is the major co-receptor for HIV-1

transmission in vivo and it is not known why X4-tropic strains cannot inefficiently initiate

infection in a new host66. The fact that the R5-tropic strains are not able to infect stimulated

peripheral blood mononuclear cells (PBMCs) from CCR5∆32 homozygotes, also indicates that

receptors such as CCR2b and CCR3 do not play an important role in the process of the in vivo

virus entry into the primary target cells128.

23

1.2.6. Receptor Downregulation and HIV-1 Therapy

Interference with viral entry seems to be an optimistic approach to treat or prevent HIV-1

infection. Interfering molecules used for this purpose may target either HIV-1 or host cell

elements.

A small number of human monoclonal antibodies against the gp120 and gp41 are able to

neutralize HIV-1 infectivity and prevent entry129-131. However, using them in patients did not

show a prolonged inhibition132. Small molecules that block the CD4-binding site on gp120 have

also been developed. One of them, called BMS 378806 (Bristol-Myers Squibb, New York, NY)

was shown to have antiviral activity in vitro and is being developed to prevent mucosal HIV-1

transmission133. The first entry inhibitor approved for clinical use is a parenterally administered

peptide containing sequences of HIV-1 gp41. This peptide, called enfuvirtide or “Fuzeon”

(Roche/Trimeris, Basel, Switzerland/Durham, NC), blocks the zippering of gp41, which

normally brings the viral and cell membranes together to promote their fusion134.

Compared to the high variability of the viral Env protein, lack of variability in receptors and co-

receptors makes them better targets for therapeutic intervention. However, since there are several

receptors involved in the process of viral entry, it is likely that several different inhibitors will

have to be used in order to cover all possible means by which HIV-1 can enter.

Interference with the binding of CD4 and gp120 by using soluble CD4 was ineffective in early

studies135,136, but a polyvalent CD4-IgG fusion protein (PRO 542, Progenics Pharmaceuticals,

Tarrytown, NY) showed some antiviral activity in vivo137.

24

Complete and broad downregulation of CD4 or CXCR4 is possibly harmful to immune system

and immune cells maturation and homing. CXCR4 deficiency is lethal for mice embryos.

CXCR4-/- mice embryos were shown to have severe cardiac, neural, and hematopoietic

developmental defects. In adult mice, expression of CXCR4 and its interaction with SDF-1 is

required during homing and migration of hematopoietic progenitor cells, as well as cellular

positioning during thymic differentiation and immigration to the periphery43,138,139. On the

contrary, individuals carrying a non-functional CCR5 do not show any deficiency in their

immune system, have less susceptibility to HIV-1 infection, and the AIDS progression is delayed

in them140. Also, one study suggests that CCR5 downregulation in an HIV-2-infected cohort of

Senegalese women protected them from HIV-1 superinfection141. In addition, binding of β-

chemokines to CCR5 results in intracellular signal transduction and internalization of the co-

receptor, which prevents subsequent infection by HIV-1. For these reasons, CCR5 is an attractive

antiviral target and different approaches have been developed either to block the co-receptor

function or to decrease its expression on the cell surface.

To block the co-receptor function, ligands41,50,72,142-144 and anti-CCR5 monoclonal

antibodies92,145-147 were developed and reported with properties that may be attractive for anti-

HIV therapy, including reduced agonist activity, enhanced blocking of fusion/entry, and

improved selectivity for the desired co-receptor.

Among different CCR5-blocking approaches, CCR5 antagonists are the most advanced in their

clinical development and trials. However, investigation of some anti-CCR5 drugs, such as

aplaviroc and vicriviroc, was stopped at different stages of phase IIb and III trials due to

hepatotoxicity and inferior efficiency. For these reasons, many researchers, including ourselves,

have focused on gene therapy strategies to downregulate CCR5 expression.

25

Gene therapy strategies have been developed to inhibit either co-receptor synthesis or surface

expression. The anti-HIV-1 genes used to prevent surface expression include intrakines148-150 and

single-chain antibodies (or intrabodies)151 and those used to decrease co-receptor synthesis

include antisense RNAs152,153, ribozymes153-157, interfering RNAs (RNAi)35-37,158-163, and zinc

finger-nuclease164.

1.2.6.1. Ligands and Intrakines

The co-receptor blocking agents that seem useful are the natural chemokines that bind to and

inhibit fusion, entry and infection mediated by the corresponding co-receptors. Many

researchers, including Cocchi et al. (1995), Alkhatib et al. (1996), Dragic et al. (1996), Oravecz

(1996), Bleul et al. (1996), and Oberlin (1996) demonstrated inhibition of HIV-1 entry into

CD4+ T-cells and PBMCs, as well as monocytic and CD4+ T-cell lines by using ligands for

CCR5 (RANTES, MIP-1α, and MIP-1β)46,50,143,165 and CXCR4 (SDF-1)41,42. Therefore, it seems

that increasing expression of these ligands is part of host immune response to the infection46.

However, Yang et al. (1997) reported that a CCR5+/CD4+ human lymphoid cell line (PM1)

expressing RANTES and MIP1-α under the control of a cytomegalovirus (CMV) promoter failed

to grow166. In addition, there are conflicting reports by Schmidtmayerova et al. (1996), Coffey et

al. (1997), Simmons et al. (1997) and Kinter (1998) about the effects (enhancement, inhibition,

or no effect) of these ligands on HIV-1 replication in macrophages167-170. For example, in sharp

contrast to observed antiviral effects in T-cells, Schmidtmayerova et al. (1996) showed β-

chemokines stimulated the replication of primary HIV-1 strains in macrophages167. In the

presence of RANTES, Oravecz (1996) observed inhibition only in M-tropic isolates, and

RANTES did not inhibit virus replication in chronically infected PM1 cells or did not reduce

virus attachment to the cell membrane143. Tedla et al. (1996) demonstrated that β-chemokine

expression was strongly enhanced in lymph nodes of patients with HIV-1 disease171. Thus,

26

knowing that the primary physiologic role of β-chemokines is to direct the trafficking of

mononuclear cells to lymph nodes and sites of inflammation, cells recruited to lymph nodes are

likely to interact with β-chemokines before exposure to the virus171. However, studies to date

have examined the influence of β-chemokines only when added simultaneously with and/or after

HIV-1 infection. Accordingly, the effects of timing of exposure to β-chemokines on HIV-1

replication in monocytes and monocyte-derived macrophages (MDMs) were studied using an in

vitro system144. Kelly et al. (1998) reported that RANTES, MIP-1α and MIP-1β exposure

produced dichotomous effects on HIV-1 replication. When both monocytes and MDMs were

exposed to β-chemokines before infection, the HIV-1 replications were enhanced. By contrast,

addition of β-chemokines either simultaneously with or after HIV-1 infection inhibited

subsequent viral replication144. Acceleration of HIV-1 replication in the monocytes and MDMs

that are exposed to β-chemokines before infection suggests that mononuclear cells that are

exposed to β-chemokines during being recruited to the lymph nodes may be more susceptible to

HIV-1 infection. Also, note that high concentrations of β-chemokines used in some of these

studies may be well above those present in vivo46,165. The enhancing effects of β-chemokines on

HIV-1 replication observed in monocytes and MDMs were also observed in CD4+ T

lymphocytes. Furthermore, primary clinical isolates have also demonstrated similar results144.

Since interaction of β-chemokines with G protein-linked receptors stimulates the cell’s signaling

pathways, it may result in increased HIV-1 replication. The receptor signaling may upregulate a

nuclear transcription factor, such as κB, that may boost HIV-1 transcription172. Therefore,

developing an HIV-1/AIDS treatment based on using ligands cannot be successful unless the

entire role of β-chemokines during the disease is completely understood.

The chemokines can be converted to “intrakines”, which are intracellular chemokines that bind

to the chemokine receptors to block and decrease their surface expression. Intrakines can be

27

designed to contain KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum (ER) retention signal to

trap the bound protein in the ER during translation and/or recycling166.

Yang et al. (1997) expressed two intrakines, RANTES and MIP1-α containing KDEL, under the

control of a CMV promoter from a pCMV plasmid and a retroviral LNCX vector. The intrakines

decreased cell surface expression of CCR5 and syncytia formation, as well as R5-tropic HIV-1

replication in a T-lymphoid cell line (PM1)166. Since leakage of expressed intracellular

chemokines may induce signal transduction or inflammatory responses, Bai et al. (1998)

developed a deletion mutant (∆2-8) RANTES, which lacked amino acids 2-8. This intrakine, and

a KDEL-tagged derivative of it, ∆RANTES-KDEL, were cloned under the control of CMV

promoters in pRc/CMV plasmid and LNCX retroviral vector. ∆RANTES-KDEL demonstrated

the same efficiency as its ancestor in downregulating surface expression of CCR5, inhibiting

syncytia formation, R5-tropic HIV-1 replication, and desensitizing to chemotaxis in transduced

PM1 cells and peripheral blood lymphocytes (PBLs)148. In addition, to show the antigen-

stimulated function of ∆RANTES-KDEL-expressing lymphocytes, IL2 production and DNA

synthesis rate in these cells were assayed after exposure to tetanus toxoid as an antigen. These

cells demonstrated the same response as the untransduced control cells, suggesting that

∆RANTES-KDEL-expressing cells retained the basic biological functions in response to antigen

stimulation148.

Schroers et al. (2002) designed another intrakine, RANTES-SK, by adding a six amino acid ER

retaining sequence (Ser-Glu-Lys-Asp-Glu-Leu or SEKDEL) to the C-terminus. This intrakine

was cloned in LOX lentiviral vectors, expressing GFP (green fluorescent protein), under the

control of the human elongation factor-1α (EF-1α) promoter149. When PM1 cells were

transduced with LOX RANTES-SK vector particles, the surface expression of CCR5 was

28

reduced. Since RANTES binds to CCR1, CCR3, and CCR5, surface expression of CCR1 and

CCR3 were also downregulated. Cells were then challenged with R5-tropic HIV-1 (ADA,

SF162, and JRCSF) and X4-tropic HIV-1 (IIIB) at a multiplicity of infection (MOI) of 0.01 or

0.1. The HIV-1 p24 values obtained from infected RANTES-SK-expressing cells were much

lower than from control cells. Incomplete inhibition of virus infection might be due to residual

amounts of CCR5 molecules that were still expressed on cells surface. Using quantitative PCR

method, 44 copies of HIV-1 proviral DNA/ng genomic DNA were detected after 3 days in

infected RANTES-SK-expressing cells, compared to 743 copies/ng genomic DNA in control

cells. The HIV-1 proviral DNA copy number in RANTES-SK-expressing cells remained the

same during three weeks, while in control cells this number increased as high as 5277 copies/ng

genomic DNA149.

The disadvantages of using intrakines include off-target cellular effects and induction of an

inflammatory response. Besides, RANTES-intrakines disrupt expression of other RANTES

receptors, CCR1 and CCR3, whose normal expression during allergic reactions and

inflammatory responses are crucial for proper lymphocyte functions149.

1.2.6.2. Anti-CCR5 Monoclonal Antibodies and Intrabodies

Many anti-CCR5 monoclonal antibodies have also been made and tested, among which, some

were more successful at inhibiting HIV-1 entry. For example, the humanized monoclonal

antibody HGS004 developed and tested by Roschke et al. (2004)146 is in a phase I clinical trial93.

Another anti-CCR5 antibody, Pro-140, is a humanized monoclonal antibody developed by

Castagna et al. (2005)147 and results from a phase 1 clinical trial that was recently completed

showed a dose-dependent binding of Pro-140 to CCR5-expressing cells. At the highest

concentration tested, Pro-140 was shown to coat these cells for at least 60 days. A phase II study

29

in HIV-1 infected individuals is underway16. Binding of these agents to CCR5 initiates its

internalization, which is considered as an advantage: by intracellular arresting of the co-receptor,

there will be less chance for emergence of the drug-resistant HIV-1 isolates that are able to use

other regions of CCR5 to enter the cell. The only disadvantage is that the efficiency of the agent

is dependent on the extent and duration of receptor internalization.

An “intrabody” is an intracellularly expressed single-chain variable fragment (scFv) of antibody

against a specific protein173,174. Similar to intrakines, intrabodies can be designed to contain

KDEL signal to retain the target protein in ER173. Because of their high affinity, target

specificity, and stability in cellular environments173, intrabodies were also used to inhibit surface

CCR5 expression. Steinberger et al. (2000) produced an intrabody against N-terminal

extracellular domain of CCR5 for downregulation of CCR5 expression and inhibition of R5-

tropic HIV-1 infection151. To this end, the scFv was dimerized using a linker and tagged with

KDEL at the C-terminus. The resulting intrabody, ST6, was expected to be more efficient

because it had two CCR5 binding sites and two ER-retention signals. Steinberger et al. (2000)

expressed ST6 intrabody and RANTES-KDEL intrakine148 separately from pIB6 and pRAN

plasmids, respectively. ST6 was also expressed from Babe-Puro retroviral vector. To test the

efficiency of ST6 and RANTES-KDEL in downregulation of CCR5 surface expression, 293T

cells were first transfected with a CCR5-encoding plasmid (to produce high levels of CCR5) and

then with pIB6 or pRAN. Intracellular staining revealed that both intrakine and intrabody were

expressed equally. While ST6 resulted in a complete inhibition, RANTES-KDEL led to only a

slight reduction of CCR5 surface expression. Syncytia formation was also shown to be

completely inhibited by ST6, but only slightly by RANTES-KDEL. PM1 cells transduced with

the retroviral vector expressing ST6 showed a complete reduction of surface expression of CCR5

and inhibition of syncytia formation. These cells were also resistant to R5-tropic HIV-1 (SF162

30

and JR-CSF) infection at an MOI of 0.01 over the 10-day period of experiment151. Swan et al.

(2006) also showed that expression of ST6 intrabody from a lentiviral vector efficiently

disrupted surface expression of CCR5 in transduced primary CD4+ T-cells and macrophages

derived from transduced CD34+ cells175.

Cagnon et al. (2000) modified the scFv of an anti-CCR5 antibody (2C7) to contain KDEL and

then cloned it into an SV40-based vector under the control of CMV promoter to produce

pSV(2C7)176. Transduced PM1 cells and primary monocytes expressed 2C7 scFv for two months

and two weeks, respectively. When the monocytes were induced to differentiate into

macrophages, over 90% of the differentiated MDMs expressed 2C7 scFv. Surface CCR5

receptor was reduced 50-60% in transduced SupT1/CCR5 cells, PM1 cells, and MDMs. When

transduced SupT1/CCR5 and PM1 cells were challenged with 0.05-0.1 ng p24 equivalents of

HIV-1 (BaL), the infection was inhibited, but not completely. With 1 ng p24 equivalents of

virus, the infection was ~90% inhibited only when the cells were sequentially transduced with

SV(2C7) and SV(VCKA1), a vector expressing single hammerhead ribozyme against CCR5177.

SV(2C7)-transduced MDMs and microglial cells were partially resistant (20-50%) to 0.3 and 1

ng p24 equivalents of HIV-1 (BaL), respectively. The combination strategy showed a better

inhibition of virus infection. However, when SV(2C7)- and combined SV(2C7)/SV(VCKA1)-

transduced human monocytes were induced to differentiate to macrophages, only partial

inhibition of virus replication was observed at 1.5 ng p24 equivalent of HIV-1 (BaL).

1.2.6.3. Zinc Finger-nuclease

Proteins containing a Zinc finger (ZF) domain can bind with high affinities to specific DNA

sequences. Zinc finger nucleases (ZFNs) have been developed by fusing the non-specific

cleavage domain (N) of Fok I restriction enzyme to the ZF proteins, which can specifically bind

31

to an 18-bp target sequence within plant and mammalian genome. Upon binding the ZF domain

to target site, the nuclease cleaves the dsDNA in vitro73,178,179. Mani et al. (2005) combined and

fused the three ZF domains to the C-terminal 196 amino acids of Fok I restriction enzyme, which

constitute the Fok I cleavage domain, to develop a CCR5-specific ZFN to disrupt the ccr5 gene

at the DNA level164. A target site close to the start codon was chosen to target, so that only a

small peptide would be produced. The efficiency of this ZFN in downregulation of surface

expression of CCR5 is not reported yet.

1.2.6.4. RNA Interference

The endogenous or exogenous micro-RNAs and small interfering RNAs (siRNAs) control gene

expression, mRNA degradation and translation, as well as chromatin structure in eukaryotic

cells. All these pathways are referred to as RNAi180. The siRNAs, ranging in size from 19-24

nucleotides, can be targeted to any gene of interest. Silencing is performed by an inherent RNase

III-like endonuclease that uses specific siRNAs as triggers in cleaving target mRNAs181.

Martinez et al. (2002) showed that an siRNA, RNAR53i, corresponding to nucleotides +554 to

+572 of CCR5 relative to the start codon, conferred a 48% reduction of surface CCR5 expression

in transfected U87 cells that express CD4, CCR5, and CXCR4 receptors36. It also displayed a

33% inhibition of HIV-1 (BaL; MOIs between 0.03-0.24) entry as evaluated by intracellular p24

antigen detection 24 hrs post-infection, and a 79% inhibition of progeny virus production 48 hrs

post-infection. Therefore, the CCR5 mRNA cleavage was incomplete.

In order to further improve this strategy, Qin et al. (2003) designed a lentiviral FG12 vector to

express a CCR5 mRNA-specific siRNA, CCR5-siRNA (186), targeting nucleotides 186-204

within the CCR5 open reading frame37. CD4+ PBLs transduced with this vector showed >90%

32

reduction of surface CCR5 expression. By challenging the transduced CD4+ PBLs with a R5-

tropic HIV-1 reporter virus (expressing murine heat-stable antigen marker, HSA, instead of HIV-

1 Vpr gene) that could only undergo a single round of infection, more than 98% inhibition of

progeny virus production was observed. As expected, these cells were still susceptible to X4-

tropic HIV-137.

Anderson et al. (2003), Butticaz et al. (2003), Anderson et al. (2005) and Morris et al. (2006)

also investigated application of siRNAs in CCR5 downregulation159,160,163,182. Anderson et al.

(2003) designed a bispecific siRNA (with an 8-nucleotide spacer) to target both CCR5 and

CXCR4 mRNAs. The MAGI-CCR5 cells were transfected with the in vitro-transcribed

bispecific siRNA, which was shown to be processed in the cell to give rise to two 20-nt. long

monospecific products159. A 53% reduction of CCR5 expression was observed in the transfected

MAGI-CCR5 cells. Two days later, the cells were challenged with HIV-1 (BaL; MOI of 0.001),

and it was shown that there was inhibition of HIV-1 progeny virus production by ~95% at day 5

post-infection. However, when R5/X4 siRNA-transfected PBMCs were challenged with the

same virus (MOI of 0.001), only 32% inhibition of infection was observed on day 5 post-

infection. These results indicate that siRNA can be designed and assembled as multiple effector

motifs.

Anderson et al. (2005) co-expressed an anti-CXCR4 short hairpin183 that was expressed with the

anti-CCR5 siRNAs159 from a lentiviral vector, HIV-7-GFP-XHR, under the control of two

separate Pol-III promoters, U6 and H1, respectively163. In comparison to control cells, the

surface expression of CXCR4 and CCR5 co-receptors in transduced MAGI-CXCR4 and Ghost

R5 cells was reduced by 73% and 72%, respectively; however, the CCR5 mRNA was not

completely eliminated. When transduced cells were challenged with X4-tropic (NL4-3) or R5-

33

tropic (BaL) strains of HIV-1 (MOI of 0.01), over 90% reduction in progeny virus production

was observed with both cells on day 5 post-infection, as compared to untransduced or empty

vector-transduced cells. However, increased progeny virus production was detected on day 5-7

post-infection, probably because of the presence of cells that are untransduced and/or produce

low levels of siRNA. When PBMCs transduced with this vector were challenged with the same

HIV-1 strains, 33% reduction of p24 Ag inhibition was observed on days 3-7 post-infection163.

Lower levels of protection in PBMCs could have been due to the lower transduction efficiency.

Besides incomplete inhibition of HIV-1 replication, disadvantages of siRNA approach include

possibility of an interferon response and off-target gene regulation184-186.

1.2.6.5. Antisense RNA

Li et al. (2006) designed a 653-nt. long antisense RNA against the CCR5 mRNA (nts. 187-839

within the coding region) and cloned it into an adenovirus-based vector, to make pAd-

antisenseR5152. Inhibition of surface expression of CCR5 on U937 cells transduced with this

vector was 98.1%, compared to 13.8% from transduced cells expressing a sense RNA

corresponding to the same region. The amount of CCR5 mRNA in U937/Ad-antiR5 cells was

less than in control cells. No difference was seen in chemotactic activities responding to

RANTES in any of the cells. Compared to controls, when U937/Ad-antiR5 cells were challenged

with R5-tropic HIV-1 (CN97001; MOI of 0.01), ~55% inhibition of progeny virus production

was observed in 12 days post-infection. However, this antisense RNA possesses an ~87%

sequence homology to the CCR2a and CCR2b mRNAs, therefore, it may also inhibit the

function of these mRNAs, which may not be desired.

34

1.2.6.6. Ribozymes

Ribozymes are enzymatic RNA molecules that can be engineered to site-specifically cleave the

target RNAs187,188. The advantage of ribozymes over siRNA is that ribozymes have minimal

cellular toxicity and do not induce an interferon immune response154,189.

Qureshi et al. (2006)153 designed an anti-CCR5 ribozyme (CCR5Rz) against 13 nucleotides

flanking the second GUC (nts. 262-264) within the CCR5 mRNA. A 13-nt. long control

antisense RNA was also designed to bind to the same region153. These RNAs were expressed

from separate plasmids under control of a T7 promoter. When PBMCs were co-transfected with

either pCCR5Rz or pCCR5As, along with plasmids expressing the T7 RNA polymerase, CCR5

mRNA production was reduced by 95% in CCR5Rz-expressing cells, and by 80% in CCR5As-

expressing cells. The inhibition of surface CCR5 expression in PBMCs from different sources

varied from 50-90% between day 3-7 post-transfection. On day 5 post-transfection, the CCR5Rz-

or CCR5As-expressing PBMCs were challenged by 3 ng p24 equivalent of R5-tropic HIV-1

(SF162). On day 7 post-infection, progeny virus production was shown to be inhibited by 68%.

Another monomeric ribozyme targeted against nucleotide 23 within the CCR5 open reading

frame (ORF) was extensively studied. Cagnon et al. (2000)177 transfected HOS-CD4.CCR5 cell

line with a plasmid expressing this ribozyme and showed a 70% decrease in surface CCR5

expression, compared to a 50% decrease from a mutant ribozyme. However, both the active and

the mutant ribozymes conferred only a 1-3 days delay in R5-tropic HIV-1 (BaL; MOI of 0.001)

replication177. PM1 cells transduced with pBabe-Puro oncoretroviral vector expressing this

ribozyme conferred 70% (active ribozyme) vs 50% (mutant ribozyme) inhibition of BaL virus

replication (MOI of 0.02) on day 7 post-infection177. Bai et al. (2000) also used pG1Na

oncoretroviral vector expressing this ribozyme transduce CD34+ hematopoietic stem cells. The

35

differentiated macrophages showed inhibition of BaL virus replication (MOI of 0.02) on day 17

post-infection. However this inhibition was only slightly better than with the mutant ribozyme155.

Li M et al. (2005) also used an HIV-1-based vector, pHIV-7-GFP, expressing this ribozyme to

transduce primary T-cells and CD34+ stem cells190. The transduced primary T cells and the

monocytes differentiated in vitro from the transduced CD34+ cells, were challenged with the R5-

tropic HIV-1 (JR-FL; MOIs 0.01 and 0.05). A certain level of selective survival was observed,

compared to the control vector-expressing cells. Viral replication was reduced by 99% compared

to cells expressing the pHIV-7-GFP control vector, but still high amounts of progeny virus (1-10

ng/ml on day 7 and 500 ng/ml on day 28 post-infection) were produced190.

Ramezani et al. 199726 demonstrated that a multimeric ribozyme functions more efficiently than

a single (monomeric) ribozyme. Multimeric hammerhead ribozymes have an increased

probability of recognizing and cleaving at least one of the multiple target sites within the CCR5

mRNA. Therefore, Bai et al. (2001) designed a trimeric ribozyme against nucleotides 17, 153,

and 249 within the CCR5 ORF157. Oncoretroviral vectors, LN and MND, expressing this trimeric

ribozyme decreased CCR5 expression by 10-15% and conferred ~30% inhibition of R5 Env-

psuedotyped HIV-1 NL4-3 replication on day 4 post-infection157. Similar results were obtained

for inhibition of replication of HIV-1 (BaL; MOI of 0.001) in macrophages derived from

transduced CD34+ stem cells157.

The partial inhibition of HIV-1 replication observed using anti-CCR5 monomeric and trimeric

ribozymes could have been due to incomplete downregulation of surface CCR5 expression.

Therefore, to further improve this strategy, we designed a multimeric hammerhead ribozyme, Rz1-7,

which targets seven unique sites within the CCR5 mRNA. Rz1-7 was designed against nucleotides 17,

380, 390, 520, 556, 811, and 824 within the CCR5 ORF. These ribozymes were shown to effectively

36

cleave the CCR5 RNA in vitro191. An oncoretroviral vector (MGIN) and an HIV-1-based lentiviral

vector (HEG1) were used for the delivery and expression of Rz1-7. Rz1-7 expressed in the

transduced PM1 cells was shown to be active, since the CCR5 mRNA levels were found to be

decreased. As expected, high-levels of progeny virus were produced when the untransduced

cells, as well as MGIN-, MGIN-Rz1-7-, HEG1-, or HEG1-Rz1-7-transduced PM1 cells, were

challenged with the NL4-3 strain, suggesting that the cells were permissive to X4-tropic HIV-1

replication. Control untransduced PM1 cells and MGIN- or HEG1-transduced cells could be

infected by the R5-tropic HIV-1 (BaL; MOIs of 0.225, 0.675, and 2.025). However, when the

MGIN-Rz1-7-transduced cells were challenged with the BaL strain at all three MOIs, 99-100%

inhibition of progeny virus production was observed for the duration of the experiment (2-3

months post-infection). Inhibition of replication of the BaL strain was more prominent when the

multimeric ribozyme was expressed from the MGIN-Rz1-7 vector than from the HEG1-Rz1-7

vector. When the HEG1-Rz1-7-transduced cells were challenged with the BaL strain (MOIs of

0.225, 0.675, and 2.025), 80-99% inhibition of virus replication was observed for the duration of

the experiment (2 months post-infection). PCR analyses at different time intervals confirmed that

the inhibition of BaL virus replication in MGIN-Rz1-7- and HEG1-Rz1-7-transduced cells is at the

level of entry, as no or very little proviral DNA was detected by PCR.

1.2.7. Possible Consequences of CCR5 Inhibition

Although congenital deletion of CCR5 is well-tolerated, probably due to functions of other co-

receptors, use of mutated chemokines or antagonists to block CCR5 in a normal individual might

still result in induction of inflammatory responses or might block the trafficking and cellular

activation mediated by CCR5 in response to chemokines65. Also, as mentioned before, some

CCR5 inhibitors may increase the risk of opportunistic infections and malignancies in persons

who are already infected with HIV-1192,193. Recently, Glass et al. 2005194 showed that ∆32

37

homozygote individuals have a higher rate of mortality from a symptomatic infection with West

Nile virus.

38

1.3. HIV-1 Gene Therapy at Pre-integration and Proviral DNA

Levels

1.3.1. Integration Process

After fusion of HIV-1 envelop with cell membrane, the viral core enters the cytoplasm. Like all

known retroviruses, HIV-1 expresses RT, an enzyme that catalyzes the synthesis of proviral

linear DNA using the viral RNA as a template and a cellular tRNA3lys that binds to the primer

binding site (PBS) region located immediately 3' to the U5 region to serve as a primer. This

process consists of three catalytic steps: 1) synthesis of complementary DNA strand of the RNA

genome, resulting in a RNA-DNA hybrid; 2) removal of the RNA strand by ribonuclease H

(RNase H); and 3) simultaneous synthesis of a second DNA strand, complementary to the first

one. RT is an error-prone enzyme, introducing 3.4 × 10-5 mutations per base pair per cycle into

the HIV-1 genome20.

Following reverse-transcription, the PIC has to be quickly transported to the host cell’s nucleus,

but because the size of the PIC of most retroviruses is bigger than the nuclear pore complex

(NPC), the PIC cannot enter the nucleus of nondividing cells, such as macrophages and

microglia. To overcome this problem, HIV-1 and related lentiviruses, use a nuclear transport

machinery in a cell division-independent, ATP-dependent process195. Vpr, containing a nuclear

localization signal, is one of the viral proteins involved in transporting the PIC through NPC by

interacting directly with proteins in it196. A functional IN protein is not required for nuclear

import of the PIC197. The HIV-1 MA protein, is a component of the PIC, as well202. It facilitates

both nuclear import of the PIC (especially in non-dividing cells) and viral particle assembly with

the help of its two subcellular localization signals6,204.

39

Importin (imp) 7, a receptor for ribosomal proteins and histone H1, was found to be involved in

the process of importing the PIC198. A host transcriptional co-activator called lens epithelium-

derived growth factor (LEDGF/p75, also known as PSIP1), was shown to interact directly with

HIV-1 and other lentiviral IN protein in the PIC to localize it to the nucleus198, specifically into

the genes whose expression is regulated by LEDGF/p75199. This might be because LEDGF/p75

and, perhaps, other transcriptional factors make cellular DNA more accessible to the PIC200. It is

also shown that LEDGF/p75 protects IN from proteosomal degradation198. Another cellular

protein that directly and specifically binds to HIV-1 IN is the transcription factor Integrase

Interactor 1 (INI1/hSNF5), which is suggested to be required for the integration process and acts

as an ATP-dependent chromatin-remodeling protein20. It is shown that INI1/hSNF5 also

incorporates into the HIV-1 particles201. Two other cellular proteins were also identified in the

HIV-1 PIC: high-mobility group protein A1 (HMGA1) and barrier-to-autointegration factor

(BAF)202,203.

The imported dsDNA is integrated into the host chromosomal DNA by IN, which catalyzes

multiple steps, including DNA cutting and joining reactions: 1) the IN protein recognizes short

inverted repeats (ATT sites) at both ends of the proviral DNA, removes two nucleotides (AT)

from each 3’ end of the viral DNA, leaving recessed CA-OH’s at the 3’ end; 2) the IN cuts both

strands of target chromosomal DNA at a non-specific location with a 5-base distance, followed

by ligation between the viral processed 3’ end and host 5’ end; 3) IN fills the gap resulting in

production of a 5-bp duplication of host DNA at both ends on proviral DNA integration

site205,206.

IN is a protein encoded by the gag-pol polycistronic gene and is produced when the virion-

assembled gag-pol polyprotein is cleaved into individual components during viral maturation. IN

40

consists of three domains, a relatively highly conserved C-terminal domain, a central catalytic

core domain with a highly conserved D,D(35)E motif, which is required for the catalytic activity,

and an N-terminal zinc-binding domain (HHCC)201,206. In addition to its function in nuclear

localization of the PIC using its nuclear localization signals, IN plays roles in viral morphology,

particle formation, particle release, particle-associated RT activity, and infectivity201.

1.3.2. Gene Therapy Strategies to Target Components at Pre-Integration and

Integration Steps

As mentioned before, many drugs have been developed to target RT and IN, however, low

intracellular permeability, drug toxicity and high mutation rate resulting in emergence of drug-

resistant isolates, persistence of viral reservoirs, and poor patient adherence to complicated drug

regimens are the major obstacles faced with current drug therapy. These problems have led

researchers to develop new drugs with novel mechanisms of action, as well as alternative

therapies such as gene therapy15-17. A number of strategies have been developed to inhibit HIV-1

replication. Strategies designed to target the released viral RNA, RT, IN, PIC, and the integrated

proviral DNA are summarized below.

1.3.2.1. Targeting the Released Viral RNA

One of the potential targets for HIV-1 gene therapy is the viral RNA right after the viral core

enters the cytoplasm and before it has a chance to be reverse-transcribed into DNA. In a study by

Sarver et al. (1990), HeLa CD4+ cells allowing transient expression of a hammerhead ribozyme

against HIV-1 gag-coding region were >97% resistant to in-coming HIV-1 for 7 days post-

infection and the synthesized proviral DNA was 100-fold less than in control cells207. The

41

decreased amount of proviral DNA could be due to ribozyme-mediated cleavage of viral RNA

prior to reverse-transcription.

Ojwang et al. (1992) designed a hairpin ribozyme against a conserved site at position +111/112

relative to the transcription initiation site, within the U5 region of LTR of HIV-1 strain HXB2

mRNA208. HeLa cells allowing transient expression of ribozymes showed reductions in p24

protein (by 75%) and Tat activity (by 80%), suggesting the ribozyme efficiency in inhibition of

HIV-1 infection. This ribozyme was then expressed from a Moloney murine leukemia virus

(MoMuLV)-based LNL6 oncoretroviral vector, under the control of tRNAval, adenovirus VA1,

or β-actin promoters. Again, transient transfection of HeLa cells with these vectors and HIV-1

clones resulted in reduction of viral p24 protein. The best results were obtained when the

ribozyme was expressed under control of tRNAval promoter. Yu et al. (1993) reported that HeLa

cells allowing transient expression of this ribozyme inhibited progeny virus production by 95%

on day 2 post-transfection209. When Yamada et al. (1994) transduced a human T lymphoid Jurkat

cell line with the LNL6 oncoretroviral vector expressing the hairpin ribozyme under control of a

tRNAval promoter, virus replication was shown to be inhibited for up to 35 days post-infection

with HIV-1 (HXB2 strain). The efficiency of incoming virus to synthesize viral DNA was also

decreased by the ribozyme by approximately 50- to 100-fold210. Leavitt et al. (1994)

demonstrated that human PBLs transduced with this vector were also resistant for 10 days post-

infection to laboratory and clinical isolates of HIV-1211. Yu et al. (1995a) showed that

macrophages differentiated from transduced human fetal cord blood CD34+ cells expressed the

ribozyme and were resistant to infection by R5-tropic HIV-1 (BaL strain). The ribozyme

expressed by the promoter VA1 was more efficient (>95% inhibition for 10 days) than expressed

by the tRNAVal promoter (>90% inhibition for 6 days)212. Yu et al. (1995b) then designed

another hairpin ribozyme against the HIV-1 pol-coding region. This ribozyme was also

42

expressed from the LNL6 vector under the control of adenovirus VA1 promoter. This ribozyme

inhibited viral replication in stable CD34+-derived macrophage transductants till day 25 post-

infection. Inhibition by this ribozyme was slightly better than with the U5-ribozyme213. In a

preclinical study, Li et al. (1998) enriched and stimulated CD34+ cells from placental and

umbilical cord blood from ten newborns of HIV-1-positive mothers, and transduced them with

LNL6 vector expressing the U5-ribozyme under the control of the tRNAVal promoter. These cells

were then induced to differentiate and the progeny macrophage cells were challenged with HIV-

1 (BaL strain), or the HIV-1 isolate from the infant’s mother. Virus replication was significantly

inhibited for 20 to 35 days post-infection214. This study progressed to a phase-I clinical trial, in

which Wong-Staal et al. (1998) used MFT retroviral vector expressing the U5-ribozyme under

the control of the tRNAVal promoter215.

Westaway et al. (1995) fused an anti-HIV-1 hammerhead ribozyme targeted against the PBS

region of HIV-1 RNA to the 3'-terminus of tRNALys3 as it can be co-packaged with viral

genomes in newly synthesized virions216. This tRNA-ribozyme reduced the infectivity of

progeny virus by about 83%, probably by cleaving the viral genomic RNA. During the

subsequent round of infection, this tRNA-ribozyme also interrupted reverse-transcription by

competing with the host tRNALys3 for binding to RT/PBS and failed to prime the cDNA

synthesis due to presence of ribozyme at its 3’ end.

In a continuous study, Cohli et al. (1994) and Ding et al. (1998) demonstrated that production of

two antisense RNAs against Psi-gag-coding regions or U3-5’ untranslated region-gag-env-

coding regions in transduced MT4 cell line inhibited virus replication till day 30 and 78 post-

infection, respectively. These antisense RNAs were also able to inhibit HIV-1 replication in

PBLs for 21 days post-infection. Experiments revealed that both antisense RNAs co-packaged

43

into progeny virus and could interrupt virion RNA encapsidation and reverse-transcription

during the subsequent round of infection24,217.

Peng et al. (1997) also designed sense and antisense RNAs to hybridize to HIV-1 RNA or DNA

to inhibit reverse transcription. These include anti-RTn 1, a plus-sense RNA containing the RU5

sequence; anti-RTn 2, a plus-sense RNA containing the full U3RU5 sequence; anti-RTn 3, same

as anti-RTn 2 but with Sp1 and TATA box deleted, resulting in a defective HIV-1 promoter;

anti-RTn 4, a minus-sense RNA complementary to the plus-strand strong-stop DNA; and anti-

RTn 5, same as anti-RTn 4 along with a sequence complementary to the HIV-1 packaging

sequence218. The sense and antisense RNAs were expressed from a double-copy LSNP vector

under the control of the human tRNAmet promoter. Stably transduced Jurkat cells were

challenged with HIV-1 (NL4-3 strain; MOI of 0.01). While anti-RTn 1 enhanced viral infection

(for some unknown reason), no virus replication was observed in Jurkat cells expressing anti-

RTn 2, 3, 4, and 5 for 140 days post-infection.

Jacque et al. (2002) used siRNAs to destroy virion RNA before it has a chance to be reverse-

transcribed into cDNA34. Several 21-nt. long siRNA duplexes were directed against different

regions of the HIV-1 genome, including two siRNAs against the viral LTR, 5 siRNAs against the

vif-coding region, and 3 siRNAs against the nef-coding region. The siRNAs were co-transfected

with pHIVNL-GFP (containing a green fluorescent protein gene at the beginning of nef region) into

CD4+ MAGI cells. Compared to control cells that were only transfected with pHIVNL-GFP,

progeny virus production was reduced 30- to 50-fold in 24 h post-transfection. To investigate

whether siRNAs were able to specifically find and degrade the virion RNA, MAGI cells were

transfected with various siRNAs and infected with HIVNL-GFP 20 h later. No viral RNA could be

detected 1 h post-infection and no viral cDNA or integrated proviral DNA could be detected 36 h

44

post-infection. These results showed that HIV replication could be inhibited by degradation of

genomic HIV-1 RNA at the level of entry prior to reverse-transcription and provirus integration.

SiRNAs were also shown to be stable in cells because virus replication was inhibited when the

cells were challenged with HIVNL-GFP 20 h or 4 days after siRNA transfection.

SiRNAs were shown to inhibit HIV-1 replication not only before reverse-transcription but also

after transcription219. Capodici et al. (2002) designed two siRNAs, one targeting the gag-coding

region and the other targeting the 3’ LTR region. Infection of U87-CD4+-CXCR4+ and U87-

CD4+-CCR5+ cell lines with HIV-1 strains IIIB or BaL, respectively, and transfection with

either one of the two siRNAs on day 3 post-infection showed a 75 to 96% decrease in progeny

virus production for 3 days. Similar results were obtained using primary CD4+ T cells. Virus

replication was shown to be inhibited upon siRNA addition between day 2 and 5 post-infection.

Transfection of the infected cells with both gag and 3’-LTR siRNAs at one-half the

concentration of each resulted in better inhibition of viral replication, suggesting a synergistic

effect of targeting multiple sites. SiRNA can be protected against RNase A degradation by

incorporating 2’-F-dCTP and 2’-F-dUTP instead of CTP and UTP during transcription. Such

fluorine-derivatized RNA can be recognized by RTs, has the expected weight and structure by

mass spectroscopy, and can be delivered to cell by being directly added to serum without

complexing to lipofectin. Adding the gag-specific fluorine-derivatized siRNA to HIV-1-infected

primary CD4+ T cells culture on day 3 post-infection resulted in inhibition of HIV-1 replication

after 3 days. To determine whether viral infection could be inhibited after viral entry and before

reverse-transcription U87-CD4+-CXCR4+ and U87-CD4+-CCR5+ cells were transfected with

gag- or 3’-LTR-specific siRNA. One day later, cells were infected with HIV-1 strains IIIB and

BaL, respectively, and real-time quantitative PCR analysis for gag DNA of 24-hour cultured

cells demonstrated less gag DNA per cell. These results indicated that inhibition occurred before

45

the completion of reverse-transcription. The same results were obtained when primary CD4+ T

cells were treated with gag- or 3’-LTR-specific siRNA. Analysis with primers for 5’-negative

strand strong stop DNA gave similar results, suggesting that RNAi inhibited early and late stages

of reverse-transcription.

1.3.2.2. Targeting the Reverse Transcriptase

Maciejewski et al. (1995) cloned variable heavy (VH) and variable light (VL) gene fragments of

an anti-RT monoclonal antibody into an Epstein Barr virus (EBV)-based episomal eukaryotic

expression vector, pMEP4, to yield pMEP/VH and pMEP/VL220. This vector produces high levels

of protein in mammalian cells in the presence of cadmium. A lymphoid cell line MOLT-3 was

transfected with either of pMEP/VH and pMEP/VL, or with both of the vectors to express anti-RT

Fab. Stably transfected Fab-expressing cells were challenged by laboratory strains of HIV-1 IIIB

and RF, the clinical isolate HIV-1 571, and a low-passage isolate HIV-1 MN at MOI’s of 2. In

all cases, the infection was completely inhibited for 35 days post-infection. Quantitative PCR

analysis revealed a decrease of viral DNA in cells expressing anti-RT Fab to 1% of controls.

Shaheen et al. (1996) expressed an anti-RT intrabody (scFv fragment) from a MoMuLV-based

vector, SLXCMV, under the control of a CMV promoter221. Transduced SupT1-RT-SFv3 cells

were challenged with HIV-1 strains NL4-3 (MOI’s of 0.012 and 0.006) and R7-HXB2 (MOI’s of

0.01 and 0.001). Compared to controls, ~80 to 97% inhibition of progeny virus production was

observed 15 to 22 days post-infection. However, when cells were challenged with MOI of 1.0 of

viruses, no inhibition took place221. This study showed that intrabodies were able to reach the

target viral protein in the PIC.

46

Wu et al. (1993) produced an anti-RT monoclonal antibody, 1E8, that inhibited both RNA- and

DNA-dependent DNA polymerase, but had no effect on RNase H activity222. Strayer et al.

(2002) cloned the scFv of this and another anti-RT monoclonal antibody (RT#3)221 into an

SV40-based vector. SupT1 cells were individually or sequentially transduce with SV(1E8) and

SV(RT3) vectors223. When individually transduced cells were infected with 40 and 100 TCID50

(50% Tissue Culture Infective Dose) HIV-1 strain NL4-3, the progeny virus production was

highly inhibited. At 800 TCID50 HIV-1, both 1E8 and RT#3 failed to protect the individually

transduced cells, but in the cells, which were expressing both scFv’s, the infection was delayed

for about a week. In another effort, they transduced the cells with both SV(Aw) (an anti-IN scFv

cloned in SV40)224 and SV(RevM10) (a dominant-negative mutant of HIV-1 Rev21 cloned in

SV40) and showed that the cells were resistant to 800 TCID50 HIV-1. Transducing SupT1 cells

with SV(1E8) and SV(Aw) completely protected them from 2500 TCID50 HIV-1, made it the

best combination of scFv’s in one cell line.

Joshi et al. (2002) used anti-RT aptamers to inhibit HIV-1 replication. The anti-RT aptamers,

also referred to as template analog RT inhibitors (TRTI), are small RNA molecules that have

high affinity and specificity for HIV-1 RT and competitively inhibit its enzymatic activity in

vitro225. Ten aptamers were designed, out of which six (70.8,13, 70.15, 80.55,65, 70.28,

70.28t34, and 1.1) were chosen based on their binding constants and levels of RT inhibition in

vitro. Double-stranded fragments encoding different aptamer sequences were cloned into

pcDNA3.1 vector between two self-cleaving ribozymes under the control of the CMV promoter.

The flanking ribozymes were required to cleave and release the aptamers. Jurkat and 293T cells

were transfected by aptamer-expressing vectors separately and stable transfectants were selected.

When untransfected and transfected 293T cells were transfected with an infectious molecular

clone of HIV-1 strain R3B, they all produced the same amount of progeny virus. Immunoblot

47

analysis of viral proteins revealed that the assembly and maturation of the virus particles were

not affected. The authors assumed that the TRTI aptamers were co-packaged with the RT in the

progeny viruses. The infectivity of these viruses was reduced from 90 to 99% compared to the

control virus obtained from non-aptamer-expressing 293T cells. Out of six aptamers, the aptamer

70.8, which was the strongest RT-inhibitor in vitro, displayed the best (99%) reduction of HIV-1

infectivity. To determine the interference step during reverse-transcription, Jurkat cells were

infected with the TRTI-containing virion particles and the cells’ total genomic DNA were

analyzed 12 h post-infection. Synthesis of the earliest intermediate, minus-strand strong-stop

DNA, was not affected, but minus-strand transfer product and the formation of completed

proviral DNA was blocked by the aptamers 70.8,13 and 70.15. When stably transfected aptamer-

expressing Jurkat cells were infected with HIV-1 strain R3B at a low MOI (0.1), 23 to 96%

reduction of viral replication was observed for 22 days tested, 70.8,13 being the most effective

aptamer. Infection of the same Jurkat cells with HIV-1 R3B (MOI of 50) was significantly

inhibited with the three most effective aptamers, 70.8,13, 70.15, and 80.55,65. These three

aptamers also strongly suppressed the replication of a number of viral strains (MOI of 0.1)

resistant to NRTIs, NNRTIs or PR inhibitors225.

Surabhi et al. (2002) designed two other siRNAs (RT1 and RT2) against HIV-1 RT226. MAGI

cells were transfected with either of RT1 or RT2 siRNAs and 24 h later infected with DNase I-

treated HIV-1. Progeny virus production was inhibited by 90% on day 6 post-infection,

compared to the control cells. The inhibition was observed for at least 13 days. Western blot

analysis of whole-cell extracts from these cells demonstrated a specific decrease in the p51/p66

RT proteins following transfection of either of these RT siRNAs. Similar results were obtained

when Jurkat cells transfected with either of RT1 or RT2 siRNAs at a 20-time higher

concentration were infected with HIV-1.

48

1.3.2.3. Targeting the Integrase

Levy-Mintz et al. (1996) designed five anti-HIV-1 IN scFv antibodies against the zinc-finger-

like domain, core (catalytic) domain, and C-terminal nonspecific DNA binding domain227. The

scFvs were cloned under the control of a CMV promoter in the MoMuLV-based oncoretroviral

vector, pSLXCMV. The resulting vectors were used to transduce SupT1 cells. Stable

transductants were then challenged with HIV-1 strain NL4-3 at MOI’s of 0.04 and 0.06.

Compared to control vector-transduced and untransduced cells, low levels of HIV-1 p24 antigen

were detected in SupT1 IN scFv33 and scFv4 cells in a 22-day long experiment, while SupT1 IN

scFv12, scFv17 and scFv21 cells were susceptible to infection. The cytopathic effect (syncytia

formation) was also low and delayed in SupT1 IN scFv33 and scFv4 cells. It was also shown that

when two populations of SupT1 IN scFv33 and SupT1 IN scFv4 cells were mixed, infection by

HIV-1 (NL4-3) was approximately 95 to 98% inhibited. ScFv33/NU, a derivative of scFv33 that

contained the NLS of HIV-1 Tat protein between its VH and VL chains, was also made. When

SupT1 IN scFv33 and scFv33/NU cells were infected with HIV-1 (NL4-3), similar resistance

was observed, although the scFv33 seemed to be more efficient. The anti-IN scFv efficiency was

also tested in stimulated human PBMCs transduced with the retroviral vector expressing scFv33

or scFv33/NU. When these cells were challenged with HIV-1 strain NL4-3 at an MOI of 0.08,

about 92% inhibition was observed for 25 days post-infection.

BouHamdan et al. (1999) cloned the scFv33 in an SV40-based vector to obtain SV(Aw)224.

SupT1 cells transduced with this vector were challenged with two different doses of HIV-1

NL4–3 (0.05 pg and 0.5 pg p24 antigen equivalents). When lower amount of virus was used for

challenge, only very low levels of HIV-1 p24 antigen were detected in the supernatants. With

higher amount of virus, the syncytium formation was delayed for up to 18 days.

49

Goldstein et al. (2002) also injected an SV40-derived vector encoding IN#33 into the human

thymic grafts of thy/liv-SCID-hu mice228. Expression of IN#33 was shown to inhibit HIV-1 (800

TCID50) infection up to about 85% in two weeks after infection.

1.3.2.4. Targeting the Pre-integration Complex

As mentioned before, one of the cellular factors in importing the PIC is imp7. Fassati et al.

(2003) showed that applying an siRNA homologous to nucleotides 1392–1414 of human imp7

mRNA depleted the imp7 mRNA and resulted in 80-90% inhibition of HIV-1 replication198.

Inhibition of virus replication was only observed when HIV-1 infection occurred at an MOI of

0.01, and not when an MOI of >1 was used. The effect of depletion of imp9 on inhibition of

HIV-1 infection was also assessed. In cells transfected with an siRNA homologous to

nucleotides 527–547 of human imp9 mRNA, HIV-1 infection was partially reduced from 2% to

38% in three independent experiments.

Another cellular factor that can be potentially targeted is LEDGF/p75. RNAi-mediated depletion

of LEDGF/p75 resulted in instability and severe reduction of both cytoplasmic and nuclear HIV-

1 IN229. When Ciuffi et al. (2005) knocked down the LEDGF/p75 in Jurkat cells using short

hairpin (sh)RNA, the frequency of proviral DNA integration into genes regulated by

LEDGF/p75, as well as other transcription units, was partially reduced (6-10%), compared to

wild-type (WT) cells199. However, the frequency of integration into GC-rich DNA regions was

slightly increased.

Yung et al. (2004) showed that a transdominant mutant of INI1/hSNF5 (S6) that competes with

the WT protein for binding to IN, inhibited HIV-1 replication by 105 to 106 folds201.

50

To target HIV-1 MA protein, Levin et al. (1997) developed an intracellular Fab antibody against

a C-terminal epitope of MA from the Clade B HIV-1 Levin et al. (1997)230. Expression of

intrabody in the dividing CD4+ T-cells could inhibit HIV-1 infection. The progeny virions’

infectivity was also shown to be substantially reduced.

1.3.2.5. Targeting the HIV-1 dsDNA or Integrated Proviral DNA

To date, there are no reports of successful inactivation of HIV-1 dsDNA or integrated DNA. Our

attempt to test proviral DNA inactivation by targeting it with a mobile, retrotransposing RNA

element, called group II intron, is the first one of its type.

1.3.3. Group II Introns

Group II introns are found in both gram-negative and gram-positive bacteria231, the

mitochondrial and chloroplast genomes of lower eukaryotes and higher plants, but not in the

mitochondrial DNAs of metazoan animals nor in eukaryotic nuclear genes232.

Group II introns are mobile genetic elements that can insert themselves into the intronless allele

via a process called retrohoming. Mobility occurs when the spliced intron RNA reverse-splices

into a DNA site and is reverse-transcribed into dsDNA233. The initial study of group II intron by

Cech234 also showed that a self-splicing intron RNA could reverse-splice into an ectopic site

(similar to the original homing site) in dsDNA, resulting in retrotransposition.

A group II intron consists of a catalytic RNA and a multifunctional, intron-encoded protein (IEP)

with multiple structural and enzymatic activities. The intron RNA performs RNA splicing and

reverse-splicing (integration) reactions by its ribozyme activity and specific sequences, and IEP

facilitates these reactions by stabilizing the catalytically active RNA structure, DNA recognition

51

property, and its own enzymatic activities. Efficiency of this complex lets group II introns

retrohome (at the natural site) at a frequency of about 100%231 and retrotranspose (at an ectopic

site) at low frequencies (typically 10-4 to 10-5)235,236.

The intron of LtrB gene of Lactococcus lactis, also called Ll.LtrB intron, is a very well-studied

model group II intron, which unlike most of other bacterial group II introns, is mobile and can be

expressed at high levels in Escherichia coli. The Ll.LtrB intron RNA has a conserved secondary

structure consisting of six double-helical domains (DI-DVI) (Figure 1.6). Interaction of DI and

DV (containing the ribozyme motif) forms the catalytic core; DII and DIII are responsible for

RNA folding and catalytic efficiency; DIV encodes the intron’s IEP ORF, which can be provided

in trans for ribozyme activity; DVI contains a bulged A, which is necessary for intron

splicing238,239. In order for the splice junctions at the intron’s active site to be close together for

RNA splicing and reverse splicing reactions, the flanking 5’- and 3’-exon sequences base-pair

with three short sequences on intron RNA: are exon-binding site (EBS)1 and EBS2 in DI, which

base-pair with the 5’-exon sequences intron-binding site (IBS)1 and IBS2, and the sequence δ

adjacent to EBS1, which base pairs with δ’ made of the first three nucleotides of the 3’

exon232,240.

In vivo intron splicing requires RNA folding into a catalytically active structure that is facilitated

by the IEP, which binds specifically to the intron RNA to stabilize the active structure

(“maturase” activity)240,241.

The L. lactis IEP, named LtrA, consists of four domains: N-terminal reverse-transcriptase (RT),

maturase (X), DNA-binding (D), and DNA endonuclease (En) (Figure 1.7)242,243. The RT domain

binds the intron RNA, which will serve as a template for reverse transcription242, 243. Domain X

52

Figure 1.6. A schematic diagram of the Ll.LtrB group II intron, showing secondary structure of

double-helical domains (DI to DVI) and complementary intron and exon sequences. The ORF in

domain IV encodes the LtrA protein. EBS and IBS refer to exon- and intron-binding sites,

respectively. EBS1-IBS1, EBS2-IBS2, δ-δ’ interactions facilitates introns tertiary structure for

splicing and target DNA hybridization during insertion. Adapted from Guo et al. (2000)237.

53

N C

Z RT X D En

I II III IV V VI VII1 36 70 361 383 487 543 599

Figure 1.7. A schematic diagram of LtrA protein encoded by Ll.LtrB group II intron. Region Z

is characteristic of the RTs of non-LTR retroelements. The RT domain contains conserved motifs

I–VII. Domain X is an RNA binding domain with maturase activity, which is required for RNA

splicing. Other domains are D (DNA-binding) and En (DNA endonuclease). Key amino acids

flanking various domains are numbered. Adapted from San Filippo & Lambowitz (2002)244.

54

is an RNA binding domain with maturase activity that is required for RNA splicing242,243.

Although RNA splicing and synthesis of single-stranded DNA substrates occur independently

from the C-terminal D and En domains, they are required for intron mobility and prompting the

synthesis of dsDNA244. The En domain cleaves the second-strand DNA244.

The IEP is translated from unspliced precursor RNA. The interaction between the IEP and intron

RNA is crucial for all steps of intron splicing and mobility. It has been shown using the Ll.LtrB

that specific, high affinity binding of LtrA protein dimer to the DIV of intron RNA245 is

sufficient to initiate RNA splicing in vitro at physiologic Mg2+ concentrations241,246. LtrA also

interacts with intron’s catalytic core regions DI, DII, and DVI to fold the intron RNA into the

active structure241,245. This interaction is also crucial for (i) positioning the RT to initiate reverse-

transcription245, (ii) downregulation of translation by covering ribosome attachment site247,

which, in turn, prevents the accumulation of excess LtrA, and (iii) keeping ribosomes away from

the intron, which might, otherwise, prevent RNA splicing.

For reverse-splicing, first the ribonucleoprotein (RNP) complex binds nonspecifically to DNA

and then searches for a specific binding site. The binding site is located in the distal 5’ exon at

positions –24 to –13 corresponding to insertion site, where T–23, G–21 and A–20 seem to be the

most critical nucleotides. When the LtrA protein recognizes and binds to this location via major

groove interaction, it unwinds the dsDNA, enabling the EBS2, EBS1 and δ regions of the intron

RNA to base-pair to the IBS2, IBS1 and δ’ sequences of top-strand at positions –12 to +3

relative to the insertion site (Figure 1.8)231,233,248. While the protein is still associated with RNA,

it cleaves the bottom-strand of DNA (at +9) through interaction with a small number of bases in

the 3’ exon, especially T+5, and uses it as a primer to initiate reverse-transcription233,241,244.

55

Figure 1.8. Specifications of intron-insertion site. The most critical positions recognized by the

LtrA in the 5’ exon (T–23, G–21 and A–20) and 3’ exon (T+5) are indicated by squares. The

IBS2, IBS1 and δ’ sequences (positions –12 to +3) base-paired to intron RNA’s EBS2, EBS1

and δ sequences are shown. The black triangles indicate the positions where the dsDNA breaks.

The intron RNA inserts directly into the top-strand of DNA at the insertion site (IS) via reverse

splicing. Bottom-strand cleavage (CS) requires additional interaction between the LtrA and the 3'

exon. Adapted from Singh & Lambowitz (2001)233.

56

LtrA has very low efficiency DNA-dependent DNA polymerase activity. Therefore, second-

strand DNA synthesis might be performed by cellular machinery, suggesting that intron insertion

would require actively DNA replicating249.

Both retrohoming and retrotransposition of Ll.LtrB intron are Recombinase (Rec)A-independent

processes (Figure 1.9)231,250.

1.3.4. Group II Intron as a Therapeutic Agent

Since intron insertion into the DNA target site initiates by IEP recognition and the intron RNA

base-pairing, different DNA sites can be targeted for gene therapy purposes by modifying the

intron’s sequence237,248. Now, the target sequence can be scanned by computer programs to find

the best matches to the positions recognized by the IEP. These programs can be used to design

primers for modifying the intron’s EBS and δ sequences for insertion in the target sites251. An

advantage of using introns is that each gene can be targeted with multiple introns for each of its

available IEP-recognition sites. Efficient targeted gene disruptions have been shown already in

both gram-negative and gram-positive bacteria using Ll.LtrB intron251-254. In addition, the group

II introns can carry a gene, cloned in domain IV, into a specific chromosomal location252. This

property of Ll.LtrB intron was even more highlighted when it was shown that a drug-resistance

marker could be cloned in DIV in order to develop an intron-based vector243.

If group II introns can be modified and used to target genes in eukaryotes as efficiently as they

now do in bacteria, they would have potentially a broad range of gene therapy applications,

including gene disruption, gene repair/substitution, and gene insertion. To investigate the group

II abilities in gene repair, Jones III et al. used a modified Ll.LtrB group II intron to insert the WT

β-globin exons 2 and 3 into the second intron of a mutated β-globin gene in an E. coli system.

57

Second-strand synthesisand DNA repair

LtrA

Nicking of the DNA Strand by LtrA protein

Nicking of the DNA Strand by intron

Target recognition and hybridization

RNP Intron

LtrAIntronless allele

E1 E2

E2E1

LtrA

LtrA

LtrA

Intron

Intron releaseLtrA

Translation

Transcription and Translation of precursor

E1 E2 Ll.LtrB Intron

LtrA

Figure 1.9

Reverse transcription

Complete insertion

58

Figure 1.9. Mechanism of Ll.LtrB intron splicing and the RecA-independent insertion into the

target DNA. L1.LtrB (thick black line) is found in the LtrB gene (gray) between exons E1 and

E2. The intron encodes the LtrA protein (white), which facilitates self-splicing of the Ll.LtrB

intron (thin black line). The excised intron is in the form of a lariat that is bound to the LtrA

protein. This RNP complex targets an intronless target DNA. The RNA component recognizes

the IBS1, IBS2, and δ’ regions within the target DNA, nicks the DNA, and inserts itself (thin

black line) into the top strand of target dsDNA (gray). The LtrA protein (white) then cleaves the

opposite DNA strand and reverse transcribes the inserted intron RNA. Second-strand DNA

synthesis and DNA repair then complete the intron insertion into the intronless target DNA.

Adopted from Long et al. (2003)255.

59

Results revealed that approximately 2% of the mutant genes were repaired by this insertion and

could express RNAs that correspond to the WT β-globin transcript. The repaired gene also

expressed the full length β-globin protein compared to the truncated one from the mutant gene256.

This study demonstrated for the first time that a mutated mammalian gene can be repaired by

insertion of an intron carrying the correct exons in a bacterial model system and result in

expression of WT transcripts and proteins.

Another study to investigate intron insertion into a human gene and HIV-1 sequence has been

done by Dr. A. Lambowitz and his team237. They scanned CCR5 and HIV-1LA1 DNA sequences

and found 5 and 6 positions, respectively, that could be recognized by L. lactis IEP on sense and

antisense strands. The EBS-IBS and δ-δ‘ sequences of intron RNA were then modified to enable

it to base-pair with either of those positions. Each intron was cloned in a pACYC184 plasmid

while the LtrA ORF in domain IV was replaced by a T7 promoter and LtrA ORF was cloned

downstream of the intron, to make intron-donor pACD plasmids. To determine the introns

mobility, different CCR5 and HIV-1 DNA fragments were separately cloned in a recipient

plasmid immediately upstream of an intron-less TetR gene. Intron and LtrA expressions in E. coli

cells by a donor and a relevant recipient plasmid were induced by isopropyl β-D-1-

thiogalactopyranoside (IPTG). TetR cells were considered as cells in which the intron expression,

mobility and insertion had occurred. With this method, the insertion frequencies were measured,

which varied from 0.16-53% for CCR5-targeting introns and 3-66% for HIV-targeting introns.

To investigate whether the group II intron RNPs could function in human cells environment,

293T or CEM-T cells were co-transfected using liposome containing either of CCR5 nt. 332-

targeting or HIV-1 nt. 4069-targeting intron RNPs and their related recipient plasmids using

transfection. Note that the intron and LtrA protein were pre-expressed and bound to form RNP

prior to packing in the liposomes. PCR analysis on DNA of co-transfected cells revealed

60

occurrence of intron insertion. Note that the liposomes may have fused outside the cells before

delivering the material to the cell. Therefore, the insertion could have happened in a non-cellular

environment. Another question that one can ask about the study is that, while the ccr5 gene

exists in 293T cells, why did the researcher use a CCR5-expressing plasmid. Intron insertion into

the genomic ccr5 gene could be investigated by PCR analysis using Alu sequence-specific

primers.

1.4. Thesis Objectives

As mentioned before, one of the major reasons why most of the post-infection anti-HIV-1 gene

therapy strategies have not been successful to date is that they must inhibit and interfere with the

viral RNA/protein production and functions. Therefore, it is thought that the strategies to block

viral entry to the cell or to inactivate proviral DNA in the cells’ genome may be better

approaches. Since the viral genes undergo a high rate of mutation, human genes are more

constant targets for gene therapy. It is believed that among the human cells’ receptors, which

facilitate the virus entry, CCR5 is dispensable. The 10% of Caucasians, who carry this ∆32

allele, have a functional immune system and are resistant to HIV-1 infections. So far, the

ribozymes that are designed to downregulate CCR5 expression had better outcomes compared to

other therapeutic molecules. Therefore, I studied the inhibition of infection of an R5-tropic HIV-

1 strain by an anti-CCR5 multimeric hammerhead ribozyme.

The approach of inactivation of proviral DNA has not been well studied. Since Guo et al.

showed that 5 sites on HIV-1 proviral DNA can be targeted by modified group II introns,237 we

planned to investigate the feasibility of HIV-1 proviral DNA inactivation by further modified

group II introns and its effects on the virus infectivity.

61

Therefore, two objectives are pursued in this thesis:

1) Development and testing retroviral vectors expressing a 7-meric hammerhead ribozyme (Rz1-

7) against CCR5 mRNA. I will investigate whether transduction of mammalian cell lines with the

vectors expressing the Rz1-7 would inhibit ccr5 gene expression in these cells and their progeny,

rendering them non-permissive to HIV-1 infection and syncytia formation compared with the

untransduced HIV-1-infected cells.

2) Development a gene therapy strategy using a modified group II intron, which inhibits HIV-1

replication upon insertion within the HIV-1 provirus DNA and interfers with viral RNA/protein

production. I will develop a modified group II intron to insert into HIV-1 provirus DNA in the

IN domain of pol gene in order to inactivate the IN protein. Then I will investigate whether a) the

intron could target the full-length HIV-1 clone, b) further modifications to the intron to have a

selectable marker might alter the intron’s function and mobility, and c) the intron insertion would

inhibit HIV-1 replication.

62

CHAPTER 2

Inhibition of HIV-1 Entry Using Vectors Expressing a Multimeric Hammerhead Ribozyme Targeted against the

CCR5 mRNA

Reza Nazari1, Xue Zhong Ma1, Sadhna Joshi1,2,§

1Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 3E2, Canada 2Department of Medical Genetics and Microbiology, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 3E2, Canada

§ Corresponding author

Email address:

SJ: [email protected]

RN: [email protected]

This chapter has been modified from a published article in Journal of General Virology in 2008.

63

mailto:[email protected]

mailto:[email protected]

2.1. Abstract

HIV-1 entry is mediated by the specific interaction of the viral envelope glycoproteins with a cell

surface molecule, CD4, which serves as the primary receptor, and a chemokine receptor, CCR5

or CXCR4, which serves as a co-receptor. CCR5 appears to be required for all routes of

transmission. Therefore, research has been focused on downregulating CCR5 expression and/or

function.

Rz1-7 is a multimeric hammerhead ribozyme targeted against seven unique sites within the human

CCR5 mRNA. This multimeric ribozyme was shown to be active in vitro. It was inserted into a

mouse stem cell virus-based vector, as well as into an HIV-1-based vector so as to express the

multimeric ribozyme in a human CD4+ T lymphoid PM1 cell line. Stable transductants

expressed the virus-based ribozyme, which was further shown to be active since the CCR5

mRNA level decreased. High-levels of progeny virus were produced when the cells were

challenged with an X4-tropic HIV-1 strain, suggesting that cells were permissive to X4-tropic virus

replication. Replication of an R5-tropic HIV-1 strain was inhibited. Inhibition was more

prominent in cells transduced with the mouse stem cell virus-based vector than with the HIV-1-

based vector. When the PM1 cells transduced with the mouse virus-based vector were challenged

with the R5-tropic HIV-1 strain, 99-100% inhibition of progeny virus production was observed

for the duration of the experiment. Inhibition occurred at the level of viral entry, as no HIV-1

proviral DNA could be detected.

Our results demonstrate that the multimeric ribozyme can efficiently cleave the CCR5 mRNA and

prevent R5-tropic HIV-1 infection at the level of entry. Since this ribozyme confers excellent

64

inhibition of R5-tropic HIV-1 replication, we anticipate that it will be beneficial for HIV-1 gene

therapy.

2.2. Background

The anti-HIV-1 genes developed to date encode RNAs or proteins that interfere with the

functions of viral or cellular RNAs/proteins15,18,257. However, most of the strategies to prevent

infection, including several that are currently being evaluated in clinical trials, confer incomplete

inhibition. This would only delay disease progression and would not lead to the reconstitution of

an HIV-1 resistant immune system. Therefore, strategies are being developed to confer complete

inhibition of HIV-1 replication in the gene-modified cells15,190.

The best interference site to protect healthy cells from infection is at the level of entry. Hence,

strategies that inhibit viral entry are of particular interest. HIV-1 entry is mediated by the specific

interaction of the viral envelope glycoproteins with a cell surface molecule, CD4, which serves

as the primary receptor, and a chemokine receptor, CCR5 or CXCR4, which serves as a co-

receptor258. HIV-1 strains that utilize the CCR5 co-receptor are called R5-tropic strains and those

utilizing the CXCR4 co-receptor are called X4-tropic strains. To inhibit viral entry, either the

receptor or the co-receptor may be targeted. CD4 and CXCR4 cannot be downregulated because

of their critical roles in the immune function or cell maturation and homing138,259,260. However,

CCR5 appears not to play a major role in immune function. The ccr5 gene is polymorphic and a

mutant phenotype has been reported in the Caucasian population at a frequency of 1-2%57. The

defective ccr5 gene (∆32ccr5) in this population contains a 32-bp deletion; it gives rise to a

truncated ∆32CCR5 protein, which is not expressed on the cell surface. Individuals homozygous

for the defective ccr5 gene are resistant to HIV-1. Disease progression in heterozygotes is also

65

slower than in individuals with normal ccr5 alleles52,122,261. Resistance in ∆32CCR5

homozygotes implies that other co-receptors do not substitute CCR5 for infection by R5-tropic

HIV-1, which initiates transmission. Furthermore, CCR5 appears to be required for all routes of

transmission since ∆32CCR5 homozygous individuals, among hemophiliacs and intravenous

drug-users, are also protected from HIV-1 transmission57. ∆32CCR5 homozygotes that lack

CCR5 appear to be completely normal, implying that either CCR5 is not functionally critical or

other co-receptors can substitute for normal cell functions261. Therefore, research has been

focused on downregulating CCR5 expression and/or function15,18,262. A concern in using this

strategy to block HIV-1 entry was the potential of selective pressure on HIV-1 to use other co-

receptors, i.e. CCR1, CCR2b or CCR3, or to convert more rapidly towards X4-tropic HIV-1.

However, in vitro replication of two R5-tropic HIV-1 isolates in the presence of an anti-CCR5

monoclonal antibody did not convert the two isolates to use CCR1, CCR3, or CXCR4263.

Monomeric155,176,177,190,264 and trimeric157 hammerhead ribozymes were designed to target CCR5

mRNA and were tested for their ability to inhibit viral entry. A decrease in surface CCR5

expression was observed with ~70% inhibition of virus replication in the ribozyme-expressing

cells. Incomplete inhibition of HIV-1 replication may be due to the fact that only a portion of the

CCR5 mRNA was cleaved.

Since anti-HIV-1 multimeric hammerhead ribozymes targeted against HIV-1 RNA inhibited

HIV-1 replication better than monomeric ribozymes did18,26,28, we have developed and tested a

multimeric hammerhead ribozyme that targets seven unique sites within the CCR5 mRNA.

Oncoretroviral and lentiviral vectors were developed and used to express this multimeric

ribozyme. Stably transduced cells were then tested for CCR5 mRNA downregulation,

susceptibility to X4- and R5-tropic HIV-1, and absence of HIV-1 proviral DNA.

66

2.3. Materials and Methods

2.3.1. Bacteria and Virus Strains and Cell Lines

E. coli DH5α cells were obtained from GIBCO-BRL.

NL4-3 (Cat.# 2479) and BaL (Cat#. 510) were obtained from NIH AIDS Research and

Reference Reagent Program.

PA317, an adherent mouse packaging cell line (expressing MoMuLV gag, pol, and env genes)265

was obtained form ATCC (Cat.# CRL-9078) and used to obtain amphotropic retroviral vector

particles.

293T, an adherent human embryonic kidney cell line266, was obtained form Stanford Reference

(Cat.# S97-079) and used to produce lentiviral vector particles.

PM1267, a suspension human CD4+/CCR5+/CXCR4+-espressing T-lymphoid cell line, was

obtained from NIH AIDS Research and Reference Reagent Program and used for expression of

Rz1-7, producing stocks for HIV-1 strains NL4-3 and BaL, and challenged with the same HIV-1

strains.

The adherent 293T and PA317 cells were cultured in complete Dulbecco's modified Eagle's

medium (DMEM), and the PM1 cells in complete Roswell Park Memorial Institute medium

(RPMI-1640). Both media were supplemented with 10% cosmic calf serum (HyClone, USA),

1% antibiotic-antimycotic (Gibco-BRL, USA), and 1% L-Glutamine 200 mM (Gibco-BRL,

USA). Cells were incubated in a 5% CO2 incubator at 37°C. For freezing, media were removed

67

from cells by centrifugation at 600 × g for 5 min. Cell pellet was resuspended in cosmic calf serum

containing 10% dimethyl sulfoxide (DMSO), followed by 2 hr at -20°C and overnight at -70°C,

and stored in liquid nitrogen. To revive, frozen cells were melted at room temperature and mixed

with 3 ml fresh medium, followed by centrifugation as above. Cell pellet was resuspended in the

appropriate amount of fresh medium and transferred to flask or plate.

2.3.2. Oligonucleotides and Primers

All oligonucleotides and primers were synthesized by ACGT Corporation (Toronto, Canada). Long

oligonucleotides were cartridge-purified. Primers were received in powder form (0.2 µmole). See

table 2.1 for sequences of oligonucleotides and primers.

2.3.3. Plasmids

pGEM-7Zf(+) was purchased from Promega, USA. Mouse stem cell virus (MSCV)-based vector,

MGIN, containing the internal ribosome entry site (IRES) from encephalomyocarditis virus,

enhanced green fluorescent protein (egfp), and neomycin phosphotransferase (neo) genes was

obtained from Dr. R.G. Hawley268. pMD.G, expressing vesicular stomatitis virus membrane

glycoprotein (VSV-G) was obtained from Dr. J. C. Burns269. pCMV∆8.9, expressing HIV-1 gag,

gag-pol, tat and rev proteins, was obtained from Dr. L. Naldini270.

68

Table 2.1- Oligonucleotides and primers sequences

Name Sequence

β-actin-R 5’-caa-aca-tga-tct-ggg-tca-tct-tct-c-3’

β-actin-F 5’-gct-cgt-cgt-cga-caa-cgg-ctc-3’

EGFP-F 5’-tgg-tga-gca-agg-gcg-agg-a-3’

HEG1-R 5’-cta-aga-tct-aca-gct-gcc-3’

MGIN-R 5’-cag-tcg-act-acg-tag-cgg-3’

R5-Ti-F or R5-Ti-5’* 5’-ata-ggt-acc-tgg-ctg-tcg-tcc-atg -3’

R5-Ti-R or R5-Ti-3’* 5’-ggt-cca-acc-tgt-tag-agc-tac-tgc-3’

T7-Tat-F or Tat-5’* 5’-ata-tca-tat-gta-ata-cga-ctc-act-ata-ggg-cga-ata-ctt-ggg-cag-gag-tgg-aag-c-3’

Tat-R or Tat-3’* 5’-gat-cta-tgc-atg-agc-cag-3’

* Either name has been used in the text.

69

2.3.4. Construction of a Multimeric Hammerhead Ribozyme Targeted against CCR5 mRNA

Multimeric hammerhead ribozymes were designed to target CCR5 mRNA at nts. 17, 380, 390, 520,

556, 811, and 824. The ribozyme (Rz) target sites within CCR5 mRNA are as follows: Rz1 target

site: 5’-TCAAGTGTC17 AAGTCCAA-3’, Rz2 target site: 5’-CGATAGGTA380 -CCTGGCTG-

3’, Rz3 target site: 5’-CTGGCTGTC390 GTCCATGC-3’, Rz4 target site: 5’-AAGAAGGTC520 T

TCATTAC-3’, Rz5 target site: 5’-CATACAGTC556 AGTATCAA-3’, Rz6 target site: 5’-ATTG

CAGTA811 GCTCTAAC-3’, and Rz7 target site: 5’-TAACAGGT-T824 GGACCAAG-3’;

cleavage sites are indicated by arrows (Figure 2.1A).

The following oligonucleotides were designed to construct the multimeric ribozymes: Rz1F: 5’-

GCAGATCTAATCGCAAGGATCCTCAAGTGTTTCGTCCTCACGGACTCATCAGAAGTC

CAA-3’ (containing a BamH I site), Rz2R: 5’-CAGCCAGGCTGATGAGTCCGTGAGGACGAA

ACCTATCGTTGGACTTCTGATGAG-3’, Rz3R: 5’-ATCCATCTTGTTCCACCCGGATCGATG

CATGGACCTGATGAGTCCGTGAGGACGAAACAGCCAGGCTGATGAG 3’ (containing a

Cla I site), Rz4F: 5’-GCAGATCTAATCGCAAGGATCCAAGAAGGTTTCGTCCTCACGGAC

TCATCAGTTCATTAC-3’ (containing a BamH I site), Rz5R: 5’-TTGATACTCTGATGAGTCC

GTGAGGACGAAACTGTATGGTAATGAACTGATGAG-3’, Rz6R: 5’-GTTAGAGCCTGATG

AGTCCGTGAGGACGAAACTGCAATTTGATACTCT GATGAG-3’, and Rz7R: 5’-ATCCAT

CTTGTTCCACCCGGATCGATCTTGGTCCCTGATGAGTCCGTGAGGACGAAACCTGTTA

GAGCCTGATGAG-3’ (containing a Cla I site); the restriction sites are shown in italics, the

ribozyme flanking sequences that hybridize to the target sites are underlined, and the ribozyme

catalytic domains are in bold.

The oligonucleotides Rz1F (containing a BamH I site), Rz2R, and Rz3R (containing a Cla I site) were

joined by overlapping PCR271 to yield a 189-bp product, Rz1-3. This PCR product was digested with

70

BamH I and Cla I and cloned into the same sites in pGEM-7Zf(+) to yield pGEM-Rz1-3. Similarly,

oligonucleotides Rz4F (containing a BamH I site), Rz5R, Rz6R, and Rz7R (containing a Cla I site)

were joined by overlapping PCR271 to yield a 230-bp product, Rz4-7. This PCR product was digested

with BamH I and Cla I and cloned into the same sites in pGEM-7Zf(+) to yield pGEM-Rz4-7. Rz1-3

and Rz4-7 do not contain any nucleotides between individual ribozymes.

To obtain Rz1-7, pGEM-Rz4-7 was digested with BamH I and the 3’-CTAG-5’ overhang was partially

filled with the DNA polymerase I Klenow fragment (New England BioLabs, Canada) in the presence

of dGTP, dATP, and dTTP. This resulted in a 3’-G-5’ overhang. The DNA was then digested with

EcoR I, and the modified BamH I-EcoR I fragment containing the Rz4-7 was gel-purified. The

pGEM-Rz1-3 was digested with Cla I and the 5’-CG-3’ overhang was partially filled with the DNA

polymerase I Klenow fragment in the presence of dCTP to yield a 5’-C-3’overhang. This plasmid

was digested with EcoR I and used as the backbone to insert the modified BamH I-EcoR I fragment

containing Rz4-7. Note that the modified Cla I and BamH I sticky ends are compatible with each

other. The resulting plasmid was designated pGEM-Rz1-7.

2.3.5. In Vitro Cleavage Activity of Rz1-3 and Rz4-7

Rz1-3 and Rz4-7 RNAs were transcribed in vitro from pGEM-Rz1-3 and pGEM-Rz4-7, and the 32P-

labeled target CCR5 RNA was transcribed in vitro from a plasmid designated pc.CCR5, as

described27. Briefly, the pc.CCR5 plasmid was digested by Xba I. The linearized plasmid containing

the ccr5 gene was used as a transcription template for in vitro transcription reaction using T7 RNA

Polymerase kit (Invitrogen, USA) for 3 hr at 37°C. 20 µCi [α-32P]UTP was also added to the mixture

when labeled RNA was required. The produced RNAs were purified by running on a 5%

polyacrylamide, 7 M urea gel, followed by incubating the gel fragment containing the RNA in an

elution buffer, containing 2 M NH4Ac, 1% sodium dodecyl sulfate (SDS), and 25 µg tRNA for

71

overnight at 37°C. The eluted RNAs were precipitated using 1 ml 100% Ethanol, incubation on

ice for 15 min, centrifugation at maximum 15000 × g for 15 min, followed by air-drying and

dissolving in RNase-free water. The cleavage reactions were performed by mixing the ribozymes

and labeled CCR5 RNA at a 1:1 molar ratio followed by gel electrophoresis, as described27.

Briefly, Rz1-3 or Rz4-7 was mixed with the labeled CCR5 RNA in a buffer containing 40 mM Tris-

HCl (pH 8.0) and 10 mM NaCl. After 5 min of incubation at 65°C, the mixture was cooled stepwise

to 37°C, 13.3 mM MgCl2 was added, and the incubation continued for 30 min at 37°C. The cleavage

products were analyzed on a 5% polyacrylamide, 7 M urea gel.

2.3.6. Vector Constructions

The MGIN vector268 was previously modified in our laboratory to contain unique Csp45 I and

Bgl II sites downstream of the egfp gene28. The Csp45 I-BamH I 274-bp fragment of pGEM-Rz1-

7 was used to clone Rz1-7 into the modified MGIN vector at the Csp45 I and Bgl II sites, to obtain

MGIN-Rz1-7. The correct clone was identified and characterized by PCR and restriction enzyme

analyses (Figure 2.2).

To construct the HIV-1-based vectors, HEG1 and HEG1-Rz1-7, the Sfi I-EcoR I 5360-bp fragment of

SIN-EF-EGFP (containing the plasmid sequence, the HIV-1 5’ LTR, and the human EF-1α

promoter)272, the EcoR I-Not I fragment of MGIN (756 bp containing the egfp gene) or the same

digestion fragment of MGIN-Rz1-7 (1030 bp containing the egfp and Rz1-7 genes), and the Not I-

Sfi I 4100-bp fragment of the pHR’CMVLacZ vector (containing the HIV-1 3’ LTR and the

downstream sequences)270 were ligated using T4 DNA ligase (Invitrogen, USA). The correct clones

were identified and characterized by PCR and restriction enzyme analyses (Figure 2.2).

72

2.3.7. Transduction and Selection of Stable PM1 Transductants

PA317 was transfected with the MGIN or MGIN-Rz1-7 vector, as described27. Briefly, the

adherent cells were trypsinized and 4 × 106 of them were transferred to each 10 cm cell culture

plate containing 10 ml complete DMEM medium. Cells were incubated in a 37°C CO2 incubator

for 5-6 h until attached to the bottom of the plate. Alternatively, 2-3 × 106 cells were seeded in

late evening and incubated for 18 h. 15 µg of vector DNA (here MGIN or MGIN-Rz1-7), as well

as packaging constructs (where applicable), were mixed in up to 450 µl H2O by vortexing. 50 µl

ice-cold 2.5 M CaCl2 was added and vortexed briefly. 500 µl of ice-cold 2× HeBS (containing

0.283 M NaCl, 1.5 mM Na2HPO4.7H2O, 0.023 M HEPES, pH 7.05; filter-sterilized) was added

drop-wise while mixing with bubbles made by a transfer pipette and then mixed more by finger

tapping. The mixture was incubated at room temperature for 20 min and then added drop-wise to

the cell culture while rotating the plate in all directions. Cells were incubated in a 37°C CO2

incubator for over night. The medium was changed next morning. The transfected cells were

cultured for three weeks in medium containing 400 µg/ml G418 to select for stable transfectants.

These cells were used to collect vector particles, which were filtered through a 0.42 µm

Millipore filter and stored at -70°C until used.

To obtain lentiviral vector particles, the 293T cells were cotransfected with the following

plasmids: pCMV∆8.9 (5 µg), pMD.G (10 µg), and the HEG1 (15 µg) or HEG1-Rz1-7 (15 µg)

vectors, as described above. The vector particles were collected on day 3 post-transfection,

filtered and stored at -70°C until used.

The MGIN, MGIN-Rz1-7, HEG1, and HEG1-Rz1-7 vector particles were used to transduce PM1

cells as described previously28. Briefly, depending on the concentration of vector particles

73

determined by p24 Ag assays, 5 × 104 to 1 × 106 suspension cells were transduced. The cells were

mixed with appropriate amount of vector particles in the presence of 6-8 µg/ml of polybrene in a well

of a 12-well plate and left for 7-8 h in a 37°C CO2 incubator. Then 100-200 µl of fresh complete

RPMI-1640 medium was added to each well and cells were cultured for overnight. Next

morning, the supernatant was removed completely and fresh medium was added. Alternatively,

cells were mixed with vector particles in the presence of 6-8 µg/ml of polybrene in a 5-ml tube and

centrifuged at 450 × g for 2 h at 32°C. Then, cells were resuspended in the same mixture and

transferred to a well of a 12-well plate, where 100-200 µl fresh complete RPMI-1640 was added,

and cultured in a 37°C CO2 incubator for overnight. Next morning, the culture was centrifuged at

450 × g for 5 min and the cells’ pellet was resuspended in the same mixture of vector particles. On

the fourth day, the cells were resuspended in fresh media and cultured. The pools of green

fluorescent stable PM1 transductants were sorted twice by fluorescence-activated cell sorting

(FACS) machine at 2-3 month intervals and used in subsequent experiments.

2.3.8. PCR Analysis of Genomic DNA from Stable PM1 Transductants

Genomic DNA was extracted from the individual pools of stable PM1 transductants by phenol-

chloroform-isoamyl alcohol extraction protocol273. The EGFP-F and MGIN-R primer pair was

used to amplify the Rz1-7 gene from genomic DNA of the MGIN-Rz1-7-transduced cells. Another

primer pair, EGFP-F and HEG1-R, was used to amplify this gene from genomic DNA of the

HEG1-Rz1-7-transduced cells. Endogenous β-actin gene was amplified as control using the β-

actin-F and β-actin-R primer pair. PCRs were performed for 40 cycles (95°C for 1 min, 56°C for

1 min, and 72°C for 1.5 min), in a 5 µl reaction mixture containing 0.4 µM of each primer, 1×

PCR buffer, 100 µM of each dNTP, 0.5 µg DNA, and 2.5 units Taq DNA polymerase. The PCR

74

products were analyzed by 2% agarose gel electrophoresis along with the λ–Hind III DNA

marker (Gibco-BRL, USA).

2.3.9. RT-PCR Analysis of Total RNA from Stable PM1 Transductants

Total RNA was extracted from stable PM1 transductants using RNeasy Mini Kit (QIAGEN,

Canada). The RNA was incubated with RQI RNase-free DNase (Promega, USA) for 5 min at

37°C to degrade any residual DNA. To ensure that the DNase treatment was complete, the RNA

samples were analyzed by PCR. Reverse transcriptions were performed for 1 h at 37°C using the

MGIN-R, HEG1-R, β-actin-R, or R5-Ti-R primers and the RT-PCR kit (Invitrogen, USA). The

cDNAs were then PCR-amplified, as described above, using the EGFP-F/MGIN-R or EGFP-

F/HEG1-R primer pair. β-actin-F/β-actin-R or R5-Ti-F/R5-Ti-R primer pairs were used to

amplify a 353-bp or 456-pb long region within the β-actin or CCR5 ORF as endogenous PCR

controls. The RT-PCR products were analyzed on a 2% agarose gel along with the λ–Hind III

DNA marker (Gibco-BRL, USA).

2.3.10. Immunoflowcytometry analysis of stable PM1 transductants

Transduced and untransduced PM1 cells (2 × 106) were washed twice with 10 ml phosphate

buffered saline (PBS) and resuspended in 250 µl PBS containing 2% fetal calf serum (FCS)

(Hyclone, USA). Anti-CCR5 mouse IgG2aκ 2D7 monoclonal antibody (mAb; 2.5 µl)

(PharMingen, Canada) was added and the cells were incubated on ice in the dark for 30 min. The

cells were then washed twice with PBS and incubated in a similar manner with 2.5 µl

biotinylated goat anti-mouse IgG2a antibody (Southern Biotechnology Associates Inc., USA) and

then with 2.5 µl allophycocyanin (APC)-streptavidin conjugate. Finally, the cells were washed

twice with PBS, resuspended in 2 ml PBS containing 2 mM EDTA and 2% FCS, and analyzed

75

by flowcytometry. To remove background staining during flowcytometry, PM1 cells, which

were incubated in the presence of only one of the two antibodies or APC-streptavidin, were also

analyzed and the parameters were set to ignore these responses.

2.3.11. HIV-1 Susceptibility of Stable PM1 Transductants

Stable transductants were challenged with the BaL274 and NL4-3275 strains of HIV-1. To obtain

virus stocks, untransduced PM1 cells were infected with either the BaL or the NL4-3 strain and

the supernatants from day 10 to 14 were collected, filtered through a 0.42 µm Millipore filter,

and stored at -70°C.

To determine the MOI, the TCID50 was calculated as follows: 600,000 PM1 cells in 100 µl were

seeded in a 96-well plate in four repeats. BaL or NL4-3 viruses were diluted in series of 1:10,

1:100, 1:1000, 1:10,000, 1:100,000, 1:1,000,000, and added to each well. Cells were incubated

for 7 days and at day 7, samples were assayed for p24 Ag values. TCID50 was calculated by the

Karber, Reed and Muench method276 using the following formula: TCID50=L-d(S-0.5), where

L=log10 of last dilution that makes 100% infection, S=sum of positive wells that includes the

last row with 100% infection plus the following well, d=log10 of dilution factor. The TCID50

gives the number of infectious viral particles in the volume of inoculation. The MOI was

calculated using this formula: MOI=(V × NVP) / (1000 × NC), where V=volume of inoculated

virus, NVP=number of viral particles in that volume, and NC=number of cells initially used for

infection.

The pools of actively dividing stable PM1 transductants (6 × 105 cells in 1 ml) were each

inoculated with the BaL or NL4-3 strain at an MOI of 0.225, 0.675, or 2.025 for 3 h at room

temperature, with gentle shaking. The cells were then washed three times with phosphate

76

buffered saline (PBS), suspended in 2 ml complete RPMI-1640 medium, and cultured at 37°C. ~1

ml of each cell culture was collected every 3-4 days and replaced with 1 ml of fresh medium.

These aliquots were centrifuged at 450 × g for 5 min, and the cell pellets and supernatants were

both stored at -70°C. Experiments were stopped 2-3 months post-infection. The supernatants of

the frozen cultures were diluted as appropriate, and the amount of HIV-1 p24 antigen was

measured by the enzyme linked immunosorbent assay (ELISA) p24 Antigen EIA kit (Beckman

Coulter, USA).

2.3.12. Progeny Virus Infectivity

Progeny viruses produced from BaL virus-infected untransduced, as well as HEG1 or HEG1-

Rz1-7-transduced stable PM1 cells (MOI of 2.025) were collected from the supernatants from day

17 post-infection, filtered through a 0.42 µm Millipore filter, and stored at -70°C. The progeny

viruses (13 ng of HIV-1 p24 equivalent) were then used to inoculate actively dividing

untransduced PM1 cells (6 × 105 cells in 1 ml) for 3 h at room temperature, with gentle shaking.

The cells were then washed three times with PBS, suspended in 2 ml complete RPMI-1640

medium, and cultured at 37°C. ~1 ml of each cell culture was collected every 3-4 days and

replaced with 1 ml of fresh medium. These aliquots were centrifuged at 450 × g for 5 min, and

the supernatants were stored at -70°C. The amount of HIV-1 p24 antigen was measured by the

ELISA p24 Antigen EIA kit (Beckman Coulter, USA).

2.3.13. PCR Analysis to Detect the HIV-1 Proviral DNA in the HIV-infected PM1

Transductants

Genomic DNA was extracted from MGIN- and MGIN-Rz1-7-transduced frozen PM1 cell pellets

from day 4 and 43 post-infection. Genomic DNA was also extracted from HEG1- and HEG1-

Rz1-7-transduced frozen PM1 cell pellets on day 4 and day 29 post-infection. The T7-Tat-F

77

primer and the Tat-R primer were used for PCR to amplify a 424-bp region of the HIV-1 tat

gene. The R5-Ti-F/R5-Ti-R primer pair was used for PCR to amplify a 465-bp region of the

endogenous ccr5 gene. The PCRs were performed for 40 cycles (95°C for 15 sec, 53°C for 1

min, and 72°C for 45 sec) and the products analyzed by a 2% agarose gel electrophoresis.

2.4. Results

2.4.1. Design, Construction, and Activity of the Anti-CCR5 Multimeric Hammerhead

Ribozyme, Rz1-7

The multimeric hammerhead ribozyme was designed against seven unique sites at nucleotides 17,

380, 390, 520, 556, 811, and 824 within the human CCR5 open reading frame (ORF) (Figure

2.1A). These sites were not found anywhere else within the human genome. Overlapping PCRs

were performed using synthetic oligodeoxynucleotides to assemble Rz1-3 and Rz4-7, which were

cloned in pGEM-7Zf(+).

In vitro cleavage reactions were performed to determine the activity of cloned ribozymes. The

expected lengths of the products resulting from cleavage of a 989-nt. long 32P-labelled CCR5

target RNA by Rz1-3 or Rz4-7 are shown in Figure 2.1B. The in vitro cleavage products are shown

in Figure 2.1C. Rz1-3 cleaved the CCR5 RNA at three sites. Cleavage by Rz1 gave rise to 61- and

928-nt. long products, while cleavage by Rz2 or Rz3 resulted into 424- and 565-, or 434- and

555-nt. long products, respectively. Cleavage by Rz1 and Rz2 or Rz3 produced 363- and 565-, or

373- and 555-nt. long products, respectively, from the 928-nt. long fragment. Rz4-7 cleaved the

CCR5 target RNA at four sites. Cleavage by Rz4 should have given rise to 564- and 425-nt. long

products, which were not clearly detectable. Cleavage by Rz5 gave rise to 580- and 409-nt. long

products. Cleavage of the 409-nt. long product by Rz6 or Rz7 gave rise to 275 and 134, or to 288

78

Rz117 380

Rz2380

Rz3390 520

Rz4520 556

Rz5556

Rz6811 824

Rz7824

CCR5 ORF

a.

b.

121-134 b

275-288 b409 b580 b855-868 b

c.

928 b

363-373 b424-434 b555-565 b

61 b

CCR5 + Rz4 -7

CCR5+ Rz1 -3

CCR5

61 b

Rz2 Rz3

363 b 555 b

CCR5 RNA (989 b)

CCR5 RNA (989 b)

Rz1

Rz4 Rz5

Rz1-3 cleavage products:

10 b

Rz6 Rz7


424 b 565 b

555 b434 b

61 b 928 bRz1 cleavage products

Rz2 cleavage products


Rz1, Rz2, Rz3cleavage products

564 b 121 b16 b 13 b275 b

564 b 425 b

580 b 409 b

855 b 134 b

868 b 121 b

Rz4, Rz5, Rz6, Rz7cleavage products





989 b

Rz117 380

Rz2380

Rz3390 520

Rz4520 556

Rz5556

Rz6811 824

Rz7824

CCR5 ORFCCR5 ORF

a.

b.

121-134 b

275-288 b409 b580 b855-868 b

c.

928 b

363-373 b424-434 b555-565 b

61 b

CCR5 + Rz4 -7

CCR5+ Rz1 -3

CCR5

61 b

Rz2 Rz3

363 b 555 b

CCR5 RNA (989 b)

CCR5 RNA (989 b)

Rz1

Rz4 Rz5


10 b

Rz6 Rz7


424 b 565 b

555 b434 b

61 b 928 bRz1 cleavage products



Rz1, Rz2, Rz3cleavage products

564 b 121 b16 b 13 b275 b564 b 121 b16 b 13 b275 b

564 b 425 b

580 b 409 b

855 b 134 b

868 b 121 b

Rz4, Rz5, Rz6, Rz7cleavage products





989 b

C B

A

Figure 2.1- In vitro cleavage activity of the anti-CCR5 multimeric hammerhead ribozyme,

Rz1-7: A) Rz1 to Rz7 cleavage sites are shown within the 1058-nt. long CCR5 ORF. B) The

expected lengths of the CCR5 RNA products upon cleavage by Rz1, Rz2, and/or Rz3 and Rz4,

Rz5, Rz6, and/or Rz7. C) In vitro cleavage activities of Rz1-3 and Rz4-7. The CCR5 target RNA

was incubated with the Rz1-3 or Rz4-7, which were transcribed in vitro from pGEM-Rz1-3 or

pGEM-Rz4-7. Rz1-3 cleavage products (lane 1), CCR5 RNA (lane 2), and Rz4-7 cleavage products

(lane 3) were analyzed on a 5% polyacrylamide, 7 M urea gel.

79

Cap (A)n

Cap (A)n

Cap (A)n

Cap(A)nCap(A)n

Cap (A)n

Cap(A)nCap(A)n

Cap (A)n

Cap (A)n

Cap (A)n

Cap(A)nCap(A)n

Cap (A)n

Cap(A)nCap(A)n

Figure 2.2- Schematic diagrams of oncoretroviral and lentiviral vectors: Schematic

diagrams of MGIN, MGIN-Rz1-7, HEG1, and HEG1-Rz1-7 vectors and their transcripts. MGIN

contains the egfp gene, an IRES element, and the neo gene. HEG1 contains the egfp gene under

control of the EF-1α promoter. The Rz1-7 gene in MGIN-Rz1-7 and HEG1-Rz1-7 vectors is cloned

downstream of the egfp gene.

80

and 121-nt. long products, respectively. These results show that Rz1-3 and Rz4-7 are active. These

ribozymes were then combined to yield Rz1-7. Rz1-7 contains the seven ribozymes with no

intercalated nucleotides, except for eight nucleotides between Rz3 and Rz4.

2.4.2. Oncoretroviral and Lentiviral Vectors Expressing Rz1-7

The mouse stem cell virus (MSCV)-based oncoretroviral vector, MGIN268, contains the

enhanced green fluorescence protein (egfp) gene, an internal ribosome entry site (IRES), and the

neomycin phosphotransferase (neo) gene (Figure 2.2). MGIN-Rz1-7 was engineered to express the

Rz1-7 gene, which was cloned between the egfp gene and the IRES element. In the MGIN and

MGIN-Rz1-7 vectors, the MSCV 5’ long terminal repeat (LTR) promoter allows constitutive

expression of a bicistronic vector RNA, which permits translation of the two ORFs, EFGP and

NEO.

An HIV-1-based HEG1 vector was designed to express the egfp gene under the control of an

internal human elongation factor-1α (EF-1α) promoter. The Rz1-7 gene was cloned downstream

of the egfp gene in the HEG1 vector to yield HEG1-Rz1-7 (Figure 2.2). In the HEG1 and HEG1-

Rz1-7 vectors, the EF-1α promoter allows constitutive expression of EGFP mRNA, whereas the

HIV-1 5’ LTR promoter allows inducible expression of vector RNA, which can also be spliced.

Rz1-7 is present on all the transcripts.

2.4.3. Development of Pools of Stable PM1 Transductants Expressing Rz1-7

Amphotropic MGIN, MGIN-Rz1-7, HEG1, and HEG1-Rz1-7 vector particles were each used to

transduce a PM1 cell line, which is a human CD4+ T lymphoid cell line. Pools of stable PM1

transductants were sorted twice at 2-3 month intervals by fluorescence activated cell sorter

81

(FACS) and used in subsequent experiments. The growth rates of transduced PM1 cells were

comparable to those of untransduced PM1 cells.

The presence of the Rz1-7 gene was confirmed by PCR analysis of genomic DNAs isolated from

various PM1 transductants (Figure 2.3) using the EGFP-F/MGIN-R and EGFP-F/HEG1-R

primer pairs. These forward and reverse primers were designed to hybridize to sequences

upstream and downstream of the ribozyme-cloning sites, respectively. No PCR product was

detected in the untransduced samples (Figure 2.3A and B, lane 1). As expected, by PCR using

the EGFP-F/MGIN-R primer pair, a 771-bp product was detected in the MGIN-transduced

sample, whereas a 1083-bp product was obtained from the MGIN-Rz1-7-transduced sample when

using the same primer pair (Figure 2.3A, lanes 2 and 3). Likewise, 834- and 1099-bp products

were detected in the HEG1- and HEG1-Rz1-7-transduced cells respectively, when the EGFP-

F/HEG1-R primer pair was used (Figure 2.3B, lanes 2 and 3). Genomic DNAs from the

untransduced and transduced cells were also analyzed using the β-actin-F/β-actin-R primer pair

to amplify a 474-bp region of the cellular β-actin gene (Figure 2.3A and B, lower panels).

Rz1-7 production was confirmed by RT-PCR analysis of total cellular RNA from various PM1

transductants. The RNA samples were treated with DNase and analyzed by PCR to ensure

absence of DNA contamination (results not shown). No RT-PCR product was obtained from the

untransduced cells (Figure 2.4A and B, lane 1). RT-PCR using the EGFP-F/MGIN-R primer pair

resulted in the amplification of a 771-bp product from the MGIN- transduced cells and a 1083-bp

product from the MGIN-Rz1-7-transduced cells (Figure 2.4A, lanes 2 and 3). RT-PCR using the

EGFP-F/HEG1-R primer pair resulted in the amplification of an 834-bp product from the HEG1-

transduced cells and a 1099-bp product from the HEG1-Rz1-7-transduced cells (Figure 2.4B,

lanes 2 and 3). RT-PCR analysis using the β-actin-F/β-actin-R primer pair (as a control) yielded

82

a.

b.1099 bp

834 bp

474 bp

1083 bp771 bp

474 bp

MGIN-Rz1-7 DNAMGIN DNA

β-actin gene

HEG1-Rz1-7 DNAHEG1 DNA

β-actin gene

1 2 3 4 5

1 2 3 4 5

a.

b.1099 bp

834 bp

474 bp

1083 bp771 bp

474 bp

MGIN-Rz1-7 DNAMGIN DNA

β-actin gene

HEG1-Rz1-7 DNAHEG1 DNA

β-actin gene

1 2 3 4 5

1 2 3 4 5

A

B

Figure 2.3- PCR analyses to determine the presence of vector DNA sequences in the

transduced PM1 cells: Genomic DNA was extracted from untransduced or transduced PM1

cells. A) PCR products obtained from the untransduced PM1 cells (lane 1), or MGIN (lane 2) or

MGIN-Rz1-7 (lane 3) vector-transduced PM1 cells using the EGFP-F/MGIN-R primer pair. PCR

products from MGIN (lane 4) and MGIN-Rz1-7 (lane 5) vectors were analyzed as controls. B)

PCR products obtained from untransduced PM1 cells (lane 1), or HEG1 (lane 2) or HEG1-Rz1-7

(lane 3) vector-transduced PM1 cells using the EGFP-F/HEG1-R primer pair. PCR products

from HEG1 (lane 4) and HEG1-Rz1-7 (lane 5) vectors were analyzed as controls. The

endogenous β-actin gene was PCR-amplified (lower panels) as a control using the β-actin-F/β-

actin-R primer pair. The PCR products were analyzed on a 2% agarose gel. The product sizes are

indicated on the left of the gels.

83

b.

a.1083 bp

771 bp

353 bp

1099 bp

834 bp

353 bp

MGIN-Rz1-7 RNAMGIN RNA

β-actin mRNA

HEG1-Rz1-7 RNA

HEG1 RNA

β-actin mRNA

1 2 3 4 5

1 2 3 4 5b.

a.1083 bp

771 bp

353 bp

1099 bp

834 bp

353 bp

MGIN-Rz1-7 RNAMGIN RNA

β-actin mRNA

HEG1-Rz1-7 RNA

HEG1 RNA

β-actin mRNA

1 2 3 4 5

1 2 3 4 5

A

B

Figure 2.4- RT-PCR analyses to determine the production of Rz1-7 RNA in transduced PM1

cells: Total RNA was extracted from untransduced or transduced PM1 cells. A) RT-PCR

products obtained from the untransduced PM1 cells (lane 1), or MGIN (lane 2) or MGIN-Rz1-7

(lane 3) vector-transduced PM1 cells using the EGFP-F/MGIN-R primer pair. PCR products

from MGIN (lane 4) and MGIN-Rz1-7 (lane 5) vectors were analyzed as controls. B) RT-PCR

products obtained from untransduced PM1 cells (lane 1), or HEG1 (lane 2) or HEG1-Rz1-7 (lane

3) vector-transduced PM1 cells using the EGFP-F/HEG1-R primer pair. PCR products from

HEG1 (lane 4) and HEG1-Rz1-7 (lane 5) vectors were analyzed as controls. The endogenous β-

actin mRNA was RT-PCR-amplified (lower panels) using the β-actin-F/β-actin-R primer pair.

The RT-PCR products were analyzed on a 2% agarose gel. The product sizes are indicated on the

left of the gels.

84

the expected 353-bp product corresponding to spliced β-actin mRNA in all untransduced and

transduced samples (Figure 2.4A and B, lower panels).

2.4.4. Rz1-7-mediated Downregulation of CCR5 mRNA

Downregulation of CCR5 mRNA in MGIN-Rz1-7- and HEG1-Rz1-7-transduced cells was

assessed by RT-PCR analysis. A 456-bp product, resulting from RT-PCR amplification of CCR5

mRNA, was detected in MGIN- and HEG1-transduced cells (Figure 2.5A and B, lane 1).

Absence of this band in MGIN-Rz1-7- and HEG1-Rz1-7-transduced cells (Figure 2.5A and B, lane

2) reveals that CCR5 mRNA was cleaved by the multimeric ribozyme.

Similar amounts of RNA were present in all samples as shown by RT-PCR amplification of

endogenous β-actin mRNA (Figure 2.5, lower panels).

2.4.5. Rz1-7-mediated Downregulation of the Surface CCR5 Co-receptor

To determine the level of expression of the CCR5 co-receptor on the surface of stable PM1

transductants lacking or expressing Rz1-7, cells were incubated with an anti-CCR5 mouse IgG2aκ

2D7 mAb, followed by a biotinylated goat anti-mouse IgG2a antibody and an APC-streptavidin

conjugate. The immunoflowcytometry results showed 90% and 99.6% downregulation of surface

CCR5 expression on PM1 cells expressing MGIN-Rz1-7 and HEG1-Rz1-7, respectively (Figure

2.6).

2.4.6. HIV-1 Susceptibility of Pools of Stable PM1 Transductants Expressing Rz1-7

Stable PM1 transductants lacking or expressing Rz1-7 were challenged with the BaL and NL4-3

strains of HIV-1. Progeny virus production was measured by determining the amount of HIV-1

85

a.

b.

CCR5 mRNA

1 2

353 bp

456 bp

353 bp

456 bp

β-actin mRNA

β-actin mRNA

CCR5 mRNA

1 2.

.

CCR5 mRNA

a

b1 2

353 bp

456 bp

353 bp

456 bp

β-actin mRNA

β-actin mRNA

CCR5 mRNA

1 2

B

A

Figure 2.5- RT-PCR analyses to determine CCR5 mRNA levels in transduced PM1 cells:

Total RNA was extracted from transduced PM1 cells. A) RT-PCR products obtained from the

MGIN (lane 1) or MGIN-Rz1-7 (lane 2) vector-transduced PM1 cells using the R5-Ti-F/R5-Ti-R

primer pair. B) RT-PCR products obtained from HEG1 (lane 1) or HEG1-Rz1-7 (lane 2) vector-

transduced PM1 cells using the R5-Ti-F/R5-Ti-R primer pair. The endogenous β-actin mRNA

was RT-PCR-amplified (lower panels) using the β-actin-F/β-actin-R primer pair. The RT-PCR

products were analyzed on a 2% agarose gel. The product sizes are indicated on the left of the

gels.

86

GIN PM1-MGIN-Rz1-7PM1-MPM1

PM1 PM1-HEG1 PM1-HEG1-Rz1-7

Figure 2.6- Immunoflowcytometry analyses to determine CCR5 co-receptor expression

level on the surface of untransduced and transduced PM1 cells: Untransduced PM1 cells as

well as MGIN, MGIN-Rz1-7, HEG1, and HEG1-Rz1-7 vector-transduced PM1 cells lacking or

expressing Rz1-7 were analyzed by immunoflowcytometry using a combined antibody system

containing anti-CCR5 mouse IgG2aκ 2D7 mAb, biotinylated goat anti-mouse IgG2a antibody, and

APC-streptavidin conjugate.

87

p24 present in the supernatants of infected cell cultures. Untransduced cells, as well as MGIN-,

MGIN-Rz1-7-, HEG1-, or HEG1-Rz1-7-transduced cells, challenged by the NL4-3 strain at a

multiplicity of infection (MOI) of 0.225 produced high amounts (>460 ng/ml) of HIV-1 p24 on

day 10 post-infection (results not shown). Likewise, both the untransduced cells and the cells

transduced with the MGIN or HEG1 vectors produced high amounts of progeny virus when

challenged by the BaL strain at an MOI of 0.225, 0.675, or 2.025 (Figure 2.7). Complete (100%)

inhibition of progeny virus production was observed up to day 87 (Figure 2.7A) or 66 (Figure

2.7B) post-infection in two independent experiments performed with the MGIN-Rz1-7-transduced

cells challenged by the BaL strain at an MOI of 0.225. Nearly total (~99%) inhibition of progeny

virus production was observed up to day 66 post-infection when the MGIN-Rz1-7-transduced

cells were challenged by the BaL strain at higher MOIs (0.675 and 2.025) (Figure 2.7B). When

HEG1-Rz1-7-transduced PM1 cells were cultured for a long period of time and then challenged

with the BaL strain at an MOI of 0.225, progeny virus production was delayed (up to day 29

post-infection) and strongly diminished (80% inhibition) up to day 60 post-infection (Figure

2.7C). However, nearly total (~99%) inhibition of progeny virus production was observed up to

day 66 post-infection when a fresh batch of HEG1-Rz1-7-transduced cells was challenged by the

BaL strain at three different MOIs (0.225, 0.675, and 2.025) (Figure 2.7D). The purpose of this

experiment was showing the resistance of Rz1-7-expressing cells in comparison with the cells,

which do not express it. The reproductivity of this experiment was tested by different

concentrations of virus rather than simple repeating with the same viral MOI.

2.4.7. Infectivity of Progeny Viruses

The infectivity of the progeny viruses from HEG1- or HEG1-Rz1-7-transduced cells, challenged

by the BaL strain at an MOI of 2.025, was assessed by infecting the untransduced PM1 cells. The

amount of progeny viruses produced on various days post-infection was measured by

88

a. b.

d.c.

0

100

200

300

400

500

600

0 10 20 30 40 50 60 70 80 90

Days post-infection

HIV

-1 p

24 (n

g/m

l)

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1000

0 10 20 30 40 50 60

Days post-infection

HIV

-1 p

24 (n

g/m

l)

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1250

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1750

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HIV

-1 p

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g/m

l)

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500

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HIV

-1 p

24 (n

g/m

l)

a. b.

d.c.

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100

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Days post-infection

HIV

-1 p

24 (n

g/m

l)

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HIV

-1 p

24 (n

g/m

l)

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-1 p

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Days post-infection

HIV

-1 p

24 (n

g/m

l)

D C

B A

Figure 2.7- Susceptibility of transduced PM1 cells to R5-tropic HIV-1 (BaL): Cells were

challenged with HIV-1 BaL at an MOI of 0.225, 0.675, or 2.025. HIV-1 p24 released in the

culture supernatant was determined (Y axis) at different time intervals (X axis). A) Progeny virus

production following HIV-1 infection (MOI of 0.225) of untransduced PM1 cells (×), PM1 cells

transduced with MGIN (open circles), or PM1 cells transduced with MGIN-Rz1-7 (closed

circles). B) Progeny virus production following HIV-1 infection of PM1 cells transduced with

MGIN (open symbols) or MGIN-Rz1-7 (closed symbols) at an MOI of 0.225 (circles), 0.675

(triangles), or 2.025 (rectangles). C) Progeny virus production following HIV-1 infection (MOI

of 0.225) of untransduced PM1 cells (×), PM1 cells transduced with HEG1 (open circles), or

PM1 cells transduced with HEG1-Rz1-7 (closed circles). D) Progeny virus production following

HIV-1 infection of PM1 cells transduced with HEG1 (open symbols) or HEG1-Rz1-7 (closed

symbols) at an MOI of 0.225 (circles), 0.675 (triangles), or 2.025 (rectangles).

89

determining the amount of HIV-1 p24 present in the supernatants of infected cell cultures.

Similar amounts of progeny viruses were produced from PM1 cells that were challenged with the

progeny viruses produced from the HEG1- (199 pg/ml) or HEG1-Rz1-7- (196 pg/ml) transduced

PM1 cells on day 14 post-infection.

2.4.8. PCR Analyses to Detect the Presence of HIV-1 Proviral DNA in Infected PM1

Transductants

PCR analyses of the genomic DNA of BaL and NL4-3 virus-infected untransduced and

transduced PM1 cells revealed the presence of HIV-1 proviral DNA in all cells that were

infected with the NL4-3 strain (Figure 2.8A and B, lanes 1, 3, and 5), as well as the untransduced

and the MGIN- or HEG1-transduced PM1 cells infected with the BaL strain (Figure 2.8A and B,

lanes 2 and 6). No HIV-1 proviral DNA could be detected at the two time points tested (day 4

and 43 post-infection) in the MGIN-Rz1-7-transduced PM1 cells (Figure 2.8A, lane 4), whereas a

faint band corresponding to this DNA was detectable by day 29 post-infection in the HEG1-Rz1-

7-transduced cells (Figure 2.8B, lane 4).

2.5. Discussion

CCR5 is an attractive candidate for HIV-1 gene therapy. A monomeric ribozyme targeted against

nt. 23 within the CCR5 ORF was extensively studied by Cagnon et al. (2000), Bai et al. (2000),

and Li et al. (2003). A cell line transfected with a plasmid expressing this ribozyme was shown

to lead to a 70% decrease in surface CCR5 expression, compared to a 50% decrease for a mutant

ribozyme. However, both the active and the mutant ribozymes conferred only a 1-3 day delay in

BaL virus replication177. PM1 cells transduced with an oncoretroviral vector expressing this

ribozyme conferred 70% (active ribozyme) vs 50% (mutant ribozyme) inhibition of BaL virus

replication on day 7 post-infection177. Another oncoretroviral vector expressing this ribozyme

90

a.

b.

Day 29

Day 4HIV-1 tat gene

424 bp

ccr5 gene Day 29 456 bp

Day 43

Day 4HIV-1 tat gene

424 bp

Day 43 456 bp

1 2 3 4 5 61 2 3 4 5 6

ccr5 gene

1 2 3 4 5 61 2 3 4 5 6

a.

b.

Day 29

Day 4HIV-1 tat gene

424 bp

ccr5 gene Day 29 456 bp

Day 43

Day 4HIV-1 tat gene

424 bp

Day 43 456 bp

1 2 3 4 5 61 2 3 4 5 6

ccr5 gene

1 2 3 4 5 61 2 3 4 5 6

B

A

Figure 2.8- Assessment of HIV-1 provirus DNA from untransduced PM1 cells, as well as

from control or ribozyme vector-transduced PM1 cells challenged with HIV-1: A)

Assessment of HIV-1 proviral DNA from untransduced PM1 cells (lanes 1 and 2), as well as

from MGIN-Rz1-7-transduced (lanes 3 and 4), and MGIN-transduced (lanes 5 and 6) PM1 cells

challenged with HIV-1 strains NL4-3 (lanes 1, 3, and 5) or BaL (lanes 2, 4, and 6). Genomic

DNA samples from day 4 and 43 post-infection (from the experiment described in Figure 2.7a)

were analyzed by PCR using the T7-Tat-F/Tat-R primer pair. B) Assessment of HIV-1 proviral

DNA from untransduced PM1 cells (lanes 1 and 2), as well as from HEG1-Rz1-7-transduced

(lanes 3 and 4) and HEG1-transduced (lanes 5 and 6) PM1 cells challenged with HIV-1 strains

NL4-3 (lanes 1, 3, and 5) or BaL (lanes 2, 4, and 6). Genomic DNA samples from day 4 and 29

post-infection (from the experiment described in Figure 2.7c) were analyzed by PCR using the

T7-Tat-F/Tat-R primer pair. As a control, a 456-bp region of the cellular ccr5 gene was PCR-

amplified (lower panels) using the R5-Ti-F/R5-Ti-R primer pair. The PCR products were

analyzed on a 2% agarose gel. The product sizes are indicated on the left of the gels.

91

was also used to transduce CD34+ hematopoietic stem cells. The differentiated macrophages

showed inhibition of BaL virus replication on day 17 post-infection. However this inhibition was

only slightly better than with the mutant ribozyme155. An HIV-1 based vector expressing this

ribozyme was also used to transduce primary T cells and CD34+ stem cells264. The transduced

primary T cells and the monocytes differentiated in vitro from the transduced CD34+ cells, were

challenged with the JR-FL strain. A certain level of selective survival was observed compared to

the control vector-expressing cells; inhibition of virus replication was not assessed264.

Multimeric hammerhead ribozymes have an increased probability of recognizing and cleaving at

least one of the multiple target sites within the CCR5 mRNA. Therefore, a trimeric ribozyme

was designed against nts. 17, 153, and 249 within the CCR5 ORF157. Oncoretroviral vectors

expressing this trimeric ribozyme decreased CCR5 expression by 10-15% and conferred ~30%

inhibition of R5-tropic HIV-1 replication on day 4 post-infection157. Similar results were

obtained for inhibition of HIV-1 replication in macrophages derived from transduced CD34+

stem cells157.

The partial inhibition of HIV-1 replication observed using anti-CCR5 monomeric and trimeric

ribozymes could have been due to incomplete downregulation of surface CCR5 expression.

Therefore, to further improve this strategy, we designed a multimeric hammerhead ribozyme, which

targets seven unique sites within the CCR5 mRNA. Rz1-7 was designed against nts. 17, 380, 390,

520, 556, 811, and 824 within the CCR5 ORF. These ribozymes were shown to effectively cleave

the CCR5 RNA in vitro (Figure 2.1).

Unlike oncoretroviral vectors, lentiviral vectors are able to pass through the nuclear envelope

into the cell nucleus, thereby allowing transduction of non-dividing cells. A lentiviral vector is

92

therefore more suitable for anti-HIV-1 gene delivery into human hematopoietic stem cells. We

have used an MSCV-based oncoretroviral (MGIN) vector and an HIV-1-based lentiviral (HEG1)

vector for delivery and expression of the multimeric hammerhead ribozyme Rz1-7 (Figure 2.2).

Transcription from the MGIN 5’ LTR promoter results in constitutive expression of a long

transcript containing the EGFP ORF, the IRES element, and the NEO ORF. The transcript

produced from MGIN-Rz1-7 also contains the Rz1-7 downstream of the EGFP ORF. In contrast,

the 5’ LTR promoter in the HEG1 and HEG1-Rz1-7 vectors is not expressed in the absence of

HIV-1 Tat protein. Therefore, an internal EF-1α promoter was used to allow strong constitutive

expression of a transcript containing the EGFP ORF or the EGFP ORF and the Rz1-7. CCR5

mRNA cleavage by the Rz1-7 would result in surface CCR5 downregulation, preventing HIV-1

entry.

RT-PCR analyses of total RNA extracted from PM1 cells transduced with MGIN-Rz1-7 or HEG1-

Rz1-7 showed that both vectors were able to express the Rz1-7 (Figure 2.4). The Rz1-7 expressed in

these cells was active, since the CCR5 mRNA (Figure 2.5) and surface CCR5 co-receptor

(Figure 2.6) levels decreased.

As expected, high-levels of progeny virus were produced when the untransduced cells, as well as

MGIN-, MGIN-Rz1-7-, HEG1-, or HEG1-Rz1-7-transduced PM1 cells, were challenged with the

NL4-3 strain, suggesting that the cells were permissive to X4-tropic HIV-1 replication. Control

untransduced PM1 cells and MGIN- or HEG1-transduced cells could be infected by the BaL

strain. However, when the MGIN-Rz1-7-transduced cells were challenged with the BaL strain

(MOIs of 0.225, 0.675, and 2.025), 99-100% inhibition of progeny virus production was

observed for the duration of the experiment (2-3 months post-infection) (Figure 2.7A and B).

Inhibition of replication of the BaL strain was more prominent when the multimeric ribozyme

93

was expressed from the MGIN-Rz1-7 vector than from the HEG1-Rz1-7 vector. When the HEG1-

Rz1-7-transduced cells were challenged with the BaL strain (MOIs of 0.225, 0.675, and 2.025),

90-99% inhibition of virus replication was observed for the duration of the experiment (2 months

post-infection) (Figure 2.7C and D).

In the progeny viruses released from the lentiviral vector-transduced PM1 cells, HEG1 and

HEG1-Rz1-7 vector RNAs could have been packaged with the HIV-1 RNA. The inclusion of Rz1-

7 was not believed to alter progeny virus infectivity since it is not targeted against HIV-1 RNA.

The infectivity of the progeny viruses produced from the BaL virus-infected HEG1 or HEG1-

Rz1-7-transduced PM1 cells was assessed by infecting untransduced PM1 cells. As expected,

similar amounts of progeny viruses were produced in both cases (196-199 pg/ml on day 14 post-

infection), suggesting that the progeny viruses produced from the control or ribozyme-expressing

cells were equally infectious.

PM1 cells have been shown to express CXCR4, CCR1, CCR3, CCR4 and CCR5 receptors277.

Therefore, significant inhibition of HIV-1 BaL virus replication in MGIN-Rz1-7 and HEG1-Rz1-7-

transduced cells (Figure 2.7B and D) indicates that the progeny viruses produced from these cells

do not correspond to escape viruses with altered tropism for CXCR4, CCR1, CCR3 or CCR4 co-

receptors.

PCR analyses at different time intervals confirmed that the inhibition of BaL virus replication in

MGIN-Rz1-7- and HEG1-Rz1-7-transduced cells is at the level of entry, as no or very little

proviral DNA was detected by PCR (Figure 2.8).

94

2.6. Conclusions

This study shows that Rz1-7-mediated downregulation of CCR5 mRNA results in almost

complete inhibition of R5-tropic HIV-1 entry and replication in MGIN-Rz1-7-transduced cells

that are permissive to X4-tropic HIV-1 replication. These results demonstrate the feasibility of a

multimeric hammerhead ribozyme-based strategy in successfully inhibiting HIV-1 entry. Vectors

expressing this multimeric hammerhead ribozyme will be challenged with all major clades of

HIV-1. These vectors will also be tested for inhibition of HIV-1 replication in transduced

peripheral blood mononuclear cells and in the in vitro differentiated progeny of transduced

CD34+ stem cells. Since this multimeric hammerhead ribozyme confers excellent inhibition of

R5-tropic HIV-1 replication up to an MOI of 2.025 (Figure 2.7B and D), we anticipate that it will

be beneficial for HIV-1 gene therapy.

List of Abbreviations

EF-1α, human elongation factor-1α; egfp, enhanced green fluorescence protein; FACS,

fluorescence activated cell sorter; IRES, internal ribosome entry site; LTR, long terminal repeat;

MOI, multiplicity of infection; MSCV, mouse stem cell virus; neo, neomycin

phosphotransferase; ORF, open reading frame; Rz, ribozyme.

Acknowledgements

This work was supported by grants from the Canadian Institutes of Health Research and the

Ontario HIV Treatment Network. R. N. is thankful to the Ontario HIV Treatment Network for a

doctoral fellowship. We thank Dr. R. G. Hawley for providing us with the MGIN and SIN-EF-

EGFP vectors and to Dr. D. Trono for the pHR’CMVLacZ, pCMV∆8.9, and pMD.G vectors. We

are grateful to Dr. A. L. Haenni for excellent scientific discussions and for critical proofreading

95

of this manuscript. We also thank Dr. A. Arora for the HEG1 vector construction and Dr. M.

Ameli for technical assistance. Flow cytometry experiments were performed by Ms. G. Knowles

at the Center for Cytometry and Scanning Microscopy (Sunnybrook Health Sciences Centre),

and FACS analyses were performed by Mr. W. Lee at the CIHR Group in Matrix Dynamics

(University of Toronto). The following reagents were obtained through the AIDS Research and

Reference Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 NL4-3 strain from Dr. A.

Adachi; HIV-1 BaL strain from Dr. S. Gartner; PM1 cell line from Dr. D. Richman, and

pc.CCR5 from Dr. N. Landau.

96

CHAPTER 3

Exploring the Potential of Using Group II Introns to Inactivate HIV-1

Reza Nazari1 and Sadhna Joshi1,2,*

1Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Toronto, Ontario, M5S 3E2, Canada 2Department of Medical Genetics and Microbiology, Faculty of Medicine, University of Toronto, Toronto, Ontario, M5S 3E2, Canada

This chapter has been modified from a manuscript submitted to Journal of General Virology in 2008.

* To whom correspondence and reprint requests should be addressed. Sadhna Joshi, Department of Medical

Genetics and Microbiology, University of Toronto, 150 College St. # 212, Toronto Ontario M5S 3E2, Canada. Tel.

+ 416 978-2499; Fax +416 638-1459; Email: [email protected]

97

3.1. Abstract

Towards the development of a potential gene therapy strategy that could interrupt integration of

HIV-1 proviral DNA during the next round of infection, we have examined whether insertion of

a mobile group II intron into infectious HIV-1 proviral DNA could inhibit virus replication. We

have used introns targeted against two sites within the IN-coding region of the HIV-1 pol gene.

Intron insertion within this region should not interfere with viral RNA and protein synthesis;

however, the progeny virus produced is expected to be non-infectious. Intron insertion into an

infectious HIV-1 proviral DNA clone was allowed to occur in Escherichia coli. The intron-

inserted HIV-1 proviral DNA clones were then isolated and tested for virus replication in

mammalian cells. Similar amounts of HIV-1 RNA, protein, and progeny virus were produced

from HIV-1 proviral DNA as from intron-inserted HIV-1 proviral DNA. However, when the

progeny virus was tested for its infectivity, although the group II intron-inserted HIV-1 RNA

was packaged and reverse-transcribed, the DNA failed to integrate as expected in the absence of

a functional IN, and virus replication was aborted. These results demonstrate that group II introns

can inhibit HIV-1 replication and should be further exploited for HIV-1 gene therapy.

Key Words: HIV-1, gene therapy, interfering RNA, group II intron, Ll.LtrB

98

3.2. Introduction

A number of anti-HIV-1 gene therapy strategies have been developed that inhibit HIV-1

replication post-integration by interfering with the function of HIV-1 RNA and proteins18,278.

However for these strategies to be successful, inhibition must last for the life of the gene-

modified cells, as they all contain the proviral DNA. In contrast, a one-time intervention at the

HIV-1 DNA level might suffice to inhibit virus replication in the gene-modified cells and their

progeny.

Certain group II introns act as mobile genetic elements that not only can be spliced at the RNA

level, but with the help of a protein encoded by the introns, also can insert into specific DNA

target sites239,279,280. Recent advances led to the development of a Lactococcus lactis group II

intron (Ll.LtrB) with novel target DNA specificities248,252,255. Five Ll.LtrB intron insertion sites

have been identified within the HIV-1 DNA237. Intron insertion at two of these sites, located at

nts. 4021 and 4069 within the HIV-1 DNA (nt +1 denotes the first nucleotide of the RNA), was

shown to occur at high frequency in Escherichia coli. Liposomes were then used to deliver the

ribonucleoprotein complex containing the intron targeted against nt. 4069 and the intron-encoded

LtrA protein, as well as a plasmid containing the HIV-1 DNA target site, to a mammalian cell

line. Intron insertion within the target DNA was demonstrated by PCR, followed by DNA

sequencing237. This result demonstrates that the modified group II intron and the LtrA protein are

sufficient to allow intron insertion into mammalian cells.

Mobile group II introns offer the unique advantage of allowing the development of a gene

therapy strategy that could inhibit HIV-1 replication as a result of intron insertion within the

HIV-1 DNA. Intron insertion in the reverse-transcribed HIV-1 dsDNA or in the integrated HIV-1

99

proviral DNA may not only produce non-infectious virus that fails to infect a healthy cell, but

also rescue the HIV-1-infected cells. Furthermore, the reverse-transcribed HIV-1 dsDNA may be

more accessible for intron insertion than the integrated HIV-1 proviral DNA. Therefore, we

believe that HIV-1 represents a perfect model to assess the feasibility of developing group II

intron-based therapies (i.e. gene therapy). An important step towards the development of intron-

based HIV-1 gene therapy is to determine the extent of inhibition of virus replication following

intron insertion within the HIV-1 proviral DNA. Using Ll.LtrB-derived introns targeted against

nts. 4021 and 4069 within the IN-coding region of HIV-1 pol gene, we have tested whether

group II intron insertion within an infectious HIV-1 proviral DNA clone can inhibit virus

replication.

3.3. Materials and Methods

3.3.1. Bacteria and Viruses Strains and Cell Lines

E. coli DH5α was obtained from GIBCO-BRL. E. coli HMS174(DE3) was obtained from

Novagen, USA.

NL4-3 (Cat.# 2479) and BaL (Cat#. 510) were obtained from NIH AIDS Research and

Reference Reagent Program.

PA317, an adherent mouse packaging cell line (expressing MoMuLV gag, pol, and env genes)265

was obtained form ATCC (Cat.# CRL-9078) and used to obtain amphotropic retroviral vector

particles.

100

293T, an adherent human embryonic kidney cell line266, was obtained from Stanford Reference

(Cat.# S97-079) and used to produce lentiviral vector particles.

PM1267, a suspension human CD4+/CCR5+/CXCR4+ T-lymphoid cell line, was obtained from

NIH AIDS Research and Reference Reagent Program and used for expression of Rz1-7,

producing stocks for HIV-1 strains NL4-3 and BaL, and challenge with the same HIV-1 strains.

The adherent 293T and PA317 cells were cultured in complete Dulbecco's modified Eagle's

medium (DMEM), and the PM1 cells in complete Roswell Park Memorial Institute medium

(RPMI-1640). Both media were supplemented with 10% cosmic calf serum (HyClone, USA),

1% antibiotic-antimycotic (Gibco-BRL, USA), and 1% L-Glutamine 200 mM (Gibco-BRL,

USA). Cells were incubated in a 5% CO2 cabinet at 37°C. For freezing, media were removed from

cells by centrifugation at 600 × g for 5 min. Cell pellet was resuspended in cosmic calf serum

containing 10% dimethyl sulfoxide (DMSO), followed by 2 hr at -20°C and overnight at -70°C,

and stored in liquid nitrogen. To revive, frozen cells were thawed at room temperature and mixed

with 3 ml fresh medium, followed by centrifugation as above. Cell pellet was resuspended in the

appropriate amount of fresh medium and transferred to flask or plate.

3.3.2. Oligonucleotides and Primers

All oligonucleotides and primers were synthesized by ACGT Corporation (Toronto, CANADA).

Long oligonucleotides were cartridge-purified. Primers were received in powder form (0.2 µmole).

See table 3.1 for sequences of oligonucleotides and primers.

101

Table 3.1- Oligonucleotides and primers sequences

Name Sequence

β-actin-R 5’-caa-aca-tga-tct-ggg-tca-tct-tct-c-3’

β-actin-F 5’-gct-cgt-cgt-cga-caa-cgg-ctc-3’

DV-5’ 5’-gcc-gta-tac-tcc-gag-agg-3’

IIS-3’ 5’-agg-cgg-cct-taa-ctg-tag-3’

IIS-5’ 5’-atg-ggt-tgg-tca-gtg-ctg-3’

IIS-UP 5’-ttt-gca-gga-ttc-ggg-att-ag -3’

LTR-5’ 5’-gag-agc-tgc-atc-cgg-agt-ac-3’

LTR-3’ 5’-agg-caa-gct-tta-ttg-agg-ctt-aag-c-3’

R5-Ti-F or R5-Ti-5’* 5’-ata-ggt-acc-tgg-ctg-tcg-tcc-atg -3’

R5-Ti-R or R5-Ti-3’* 5’-ggt-cca-acc-tgt-tag-agc-tac-tgc-3’

T7MT Sense:

5’-ggg-ggg-tga-agc-ttc-tcg-agg-agc-tct-aat-acg-act-cac-tat-agg-gag-gtc-gac-gcc-atg-gag-gat-cca-ggg-ccc-tat-tta-aat-ggc-gcg-cca-gcg-gcc-gct-t-3’

T7MT Antisense:

5’-ggg-ggg-ggg-aat-tcc-tcg-agt-taa-tta-aca-cac-aaa-aaa-cca-aca-cac-aga-tgt-aat-gaa-aat-aaa-gat-att-tta-ttt-taa-tta-agc-ggc-cgc-tgg-cgc-gcc-a-3’

T7-Tat-F or Tat-5’* 5’-ata-tca-tat-gta-ata-cga-ctc-act-ata-ggg-cga-ata-ctt-ggg-cag-gag-tgg-aag-c-3’

Tat-R or Tat-3’* 5’-gat-cta-tgc-atg-agc-cag-3’

* Either name has been used in the text.

102

3.3.3. Plasmid Constructions

pACD-HIV1-4021s (pACD-I4021s), pACD-HIV1-4069s (pACD-I4069s), pACD-HIV1-54a (pACD-

I54a), pACD-HIV1-2654a (pACD-I2654a), and pBRR-HIV1(3805-4178s)/Tet plasmids237 were

kindly provided by Dr. A. Lambowitz. pACD-I4021sIN, pACD-I4069sIN, pACD-I4021sN, pACD-

I4069sN, pACD-I4021s∆N, and pACD-I4069s∆N were constructed as follows. A pair of single-stranded

oligonucleotides, T7MT sense and T7MT antisense, were designed to contain a T7 promoter, a

multiple cloning site and a transcription terminator (table 3.1). The single-stranded

oligonucleotides were converted to a double-strand fragment (T7MT cassette) by a single round

of PCR (95°C for 60 sec, 56°C for 60 sec, and 72°C for 300 sec). The PCR product was then

purified by agarose gel extraction method and were digested by Hind III and EcoR I restriction

enzymes. T7MT cassette was cloned into the EcoR I – Hind III digested pUC18 to produce pUC-

MCS. The internal ribosome entry site (IRES)268 and the neomycin phosphotransferase (neo) gene

were cloned in this plasmid. An Xho I – Xho I 1336-bp long fragment of pUC-MCS containing

the IRES element and the neo gene were then cloned at the Sal I sites within intron domain IV of

pACD-4021s and pACD-4069s to obtain pACD-I4021sIN and pACD-I4069sIN, respectively. Plasmids

pACD-I4021sN and pACD-I4069sN were obtained by replacing the Sal I – Bsp120 I fragment

containing the IRES element and the neo gene, by the Sal I – Bsp120 I 857-bp long fragment of

pMoTN281 containing the Shine-Dalgarno sequence and the neo gene. Deletion of an Eag I – Eag I

2107-bp long fragment from these plasmids then led to pACD-I4021s∆N and pACD-I4069s∆N.

3.3.4. Intron Insertion Assay

Intron insertion assay was performed as described elsewhere237. Briefly, E. coli HMS174(DE3)

cells were co-transformed with recipient pBRR-HIV1(3805-4178s)/Tet (ApR) and one of the

pACD-based donor plasmids (CmR). An ApRCmR colony, containing both the donor and the

recipient plasmids, was selected. Co-transformed bacteria were induced by culturing in the

103

presence of 0.1 mM isopropyl-β-D-thiogalactopyranosid (IPTG) for 1 hr to allow intron and LtrA

production, and intron insertion. Finally, the number of ApR and TetR colonies was determined and

used to calculate intron insertion frequencies by calculating the percentage of TetR colonies that

allow intron insertion over the total number of ApR colonies that do or do not allow intron

insertion.

3.3.5. Intron Insertion in HIV-1 Proviral DNA Clone and Purification of Intron-inserted HIV-

1 Proviral DNA Clones

E. coli HMS174(DE3) cells were co-transformed with recipient pHIV (an infectious HIV-1

proviral DNA clone, pNL4-3; ApR)275 and one of the two donors, pACD-I4021sN or pACD-I4069sN

(CmRKmR). A single ApRCmR colony was selected and cultured overnight in LB broth containing

Ap and Cm. The cells were then induced with 1 µM IPTG for 1 hr at 37°C, washed with LB broth,

and cultured overnight in LB broth containing Ap and Cm. Plasmid DNA was then extracted

using the QIAprep Spin Miniprep kit (Qiagen, Canada), and digested with Sac II to excise the

donor plasmids; pHIV, pHIV-I4021sN, and pHIV-I4069sN plasmids are insensitive to Sac II

digestion. The DNA was used to transform E. coli DH5α cells. The KmRApRCmS colonies were

identified as they contained pHIV-I4021sN or pHIV-I4069sN. Plasmid DNA from these colonies was

extracted and analyzed by restriction enzyme and PCR analyses.

3.3.6. Transfection of 293T Cells with Group II Intron-inserted HIV-1 Proviral DNA Clones

293T cells (4 × 106 cells/plate) were transfected with pHIV, pHIV-I4021sN, or pHIV-I4069sN, as

described before27. Briefly, the adherent cells were trypsinized and 4 × 106 of them were

transferred to each 10 cm cell culture plate containing 10 ml complete DMEM medium. Cells

were incubated in a 37°C CO2 incubator for 5-6 h till attached to the bottom of the plate. 15 µg

104

of the plasmid DNA were mixed in up to 450 µl H2O by vortexing. 50 µl ice-cold 2.5 M CaCl2

was added and vortexed briefly. 500 µl of ice-cold 2× HeBS (containing 0.283 M NaCl, 1.5 mM

Na2HPO4.7H2O, 0.023 M HEPES, pH 7.05; filter-sterilized) was added drop-wise while mixing

with bubbles made by a transfer pipette and then mixed more by finger tapping. The mixture was

incubated at room temperature for 20 min and then added drop-wise to the cell culture while

rotating the plate in all directions. Cells were incubated in a 37°C CO2 incubator for over night.

The medium was changed next morning. On day 4 post-transfection, the cells and the culture

supernatants were harvested and analyzed as described below.

3.3.7. RT-PCR Analysis to Detect Group II Intron-inserted HIV-1 RNA in Transfected 293T

Cells

Total RNA was extracted from the pHIV-, pHIV-I4021sN-, or pHIV-I4069sN-transfected 293T cells

using the RNeasy kit (Qiagen, Canada). The RNA was incubated with RQI RNase-free DNase

(Promega, USA) for 15 min at 37°C to degrade any residual DNA. To ensure that the DNase

treatment was complete, the RNA samples were analyzed by PCR. Reverse transcriptions were

performed for 1 hr at 37°C using the IIS-3’ primer and the RT-PCR kit (Invitrogen, USA). The

cDNAs were then PCR-amplified using IIS-5’, DV-5’, and IIS-3’ primers for 40 cycles (95°C

for 15 sec, 53°C for 1 min, and 72°C for 45 sec). The β-actin-F/β-actin-R primer pair was used

to amplify the endogenous β-actin gene. The RT-PCR products were analyzed on a 2% agarose

gel along with the λ–Hind III DNA marker (Gibco-BRL, USA).

3.3.8. Intracellular HIV-1 p24 and Progeny Virus Production from Transfected 293T Cells

To determine intracellular p24 concentrations, the transfected 293T cells were scraped from the

plates and washed three times with phosphate buffered saline (PBS). The cell pellets were

105

suspended in 0.5 ml of ice-cold lysing solution (containing Tris-Cl (pH 8.0) 50 mM, NaCl 150

mM, sodium azide 0.02%, phenylmethylsulfonyl fluoride 100 µg/ml, and NP-40 1%) and

incubated at 4°C for 20 min. The cell debris were then pelleted by centrifugation at 13000 × g for

2 min and the amount of intracellular p24 present in the supernatants was determined using the

enzyme linked immunosorbent assay (ELISA) p24 Antigen EIA kit (Beckman Coulter, USA)

Cell culture supernatants containing the progeny viruses were collected from the pHIV-, pHIV-

I4021sN-, or pHIV-I4069sN-transfected 293T cells. The amount of HIV-1 p24 antigen was measured

by the ELISA p24 Antigen EIA kit.

3.3.9. RT-PCR Analysis to Detect Group II Intron-inserted HIV-1 RNA in the Progeny

Viruses Produced from Transfected 293T Cells

Progeny viruses (280 µl) produced from the pHIV-, pHIV-I4021sN-, or pHIV-I4069sN-transfected

293T cells were used for virion RNA extraction by QIAamp Viral RNA minikit (Qiagen, Canada),

followed by DNase treatment as described above. Reverse transcriptions were performed for 1 h at

37°C using the IIS-3’ primer and the RT-PCR kit (Invitrogen, USA). The cDNAs were then

analyzed by PCR using IIS-Up/IIS-3’ primer pair to amplify a 613-bp region within the HIV-1

RNA, and the DV-5’/IIS-3’ primer pair to amplify a 275- or 227-bp region within the I4021sN- or

I4069sN-inserted HIV-1 RNA. PCRs were performed for 40 cycles (95°C for 60 sec, 50°C for 30

sec, and 72°C for 90 sec). The RT-PCR products were analyzed on a 2% agarose gel along with

the λ–Hind III DNA marker.

3.3.10. PM1 Cell Infection with Progeny Viruses Produced from the Transfected 293T Cells

PM1 cells were infected as described before282. Briefly, PM1 cells (6 × 105 cells in 1.5 ml) were

incubated for 1 hr at room temperature with ~10 ng p24 equivalent of progeny viruses from the

106

pHIV-, pHIV-I4021sN-, or pHIV-I4069sN-transfected 293T cells, respectively, followed by three

washes with PBS. The cells were then cultured in 2 ml complete RPMI in a 12-well plate. Every

3-4 days, ~1 ml of cultures was replaced by 1 ml fresh complete RPMI-1640. Samples were

centrifuged at 450 × g for 5 min, and the cells and supernatants were stored at -70°C.

3.3.11. PCR Analyses to Detect Proviral DNA and Reverse-transcribed HIV-1 dsDNA in

Infected PM1 Cells

Genomic DNAs were extracted using the DNeasy Tissue kit (Qiagen, Canada) from PM1 cells on

day 8 post-infection with progeny viruses from transfected 293T cells. PCRs were performed for

40 cycles (95°C for 15 sec, 53°C for 1 min, and 72°C for 45 sec) using the Tat-5’/Tat-3’ primer

pair to detect the HIV-1 sequence, and the R5-Ti-5’/R5-Ti-3’ primer pair to detect the

endogenous ccr5 gene. The PCR products were analyzed on a 2% agarose gel.

To detect the reverse-transcribed HIV-1 dsDNA, the progeny viruses (500 µl) from the pHIV,

pHIV-I4021sN-, and pHIV-I4069sN-transfected 293T cells were first treated with 30 units of DNase

for 30 min at 37°C283 and then used to inoculate PM1 cells. Genomic DNA was extracted 1 hr

post-inoculation using the DNeasy Tissue kit. PCR was performed using the LTR-5’ and LTR-

3’284 primer pair for 40 cycles (95°C for 1 min, 50°C for 30 sec, and 72°C for 30 sec). The PCR

products were analyzed on a 2% agarose gel.

3.3.12. Progeny Virus Production from Infected PM1 Cells

Cell culture supernatants collected from the pHIV-, pHIV-I4021sN-, or pHIV-I4069sN-infected PM1

cells were stored at -70°C. The amount of the HIV-1 p24 antigen was measured by the ELISA

p24 Antigen EIA kit.

107

3.4. Results

3.4.1. Modified Group II Introns

To investigate the potential utility of mobile group II introns in inhibiting HIV-1 replication, two

introns (I4021s and I4069s) were used that are derived from Ll.LtrB. These introns are 956-nt. long.

They are targeted against nts. 4021 or 4069 within the sense DNA strand of the IN-coding region

of the HIV-1 pol gene (Figure 3.1A).

Plasmids pACD-HIV1-4021s and pACD-HIV1-4069s237 (hereafter referred to as pACD-I4021s and

pACD-I4069s respectively) allow T7lac promoter-driven expression of RNA containing the I4021s and

I4069s introns. In these plasmids, the ltrA gene was deleted from its natural location within the

intron domain IV and cloned further downstream of the intron. Both plasmids were shown to

allow intron insertion at the respective HIV-1 DNA target sites in E. coli237.

We modified the pACD-I4021s and pACD-I4069s plasmids to confer a selectable (KmR) phenotype.

To this end, the T7 promoter and the neo gene were cloned in the intron domain IV. The

resulting plasmids were named pACD-I4021sN and pACD-I4069sN.

3.4.2. Modified Group II Intron Insertion Frequencies

To determine the insertion frequencies of the modified introns, E. coli cells were co-transformed

with the donor plasmid (pACD-I4021sN or pACD-I4069sN, allowing intron and LtrA protein

production and providing the T7 promoter) and the recipient plasmid (pBRR- HIV1(3805-

4178s)/Tet, providing the introns’ insertion sites and a promoterless TetR gene).

108

Figure 3.1

R

3’ LTRU3 U5R

3’ LTR

---ugg cug cua gau ugu aca cau uua gaa gga gug cgc cca gau agg gug uua agu caa gua guu uaa ------Trp Gln Leu Asp Cys Thr His Leu Glu Gly Val Arg Pro Asp Arg Val Leu Ser Gln Val Val

III VII II V

Cap (A)n

I4021sN intron-inserted 11.1kb HIV-1 RNA

C-terminus of mutant Gag-Pol

Intron domain I

---guu cau cua gcc agu gga uauagu gga uau aua gaa gca gug cgc cca gau agg gug uua agu caa gua guu uaa ------Val His Val Ala Ser Gly Tyr Ile Glu Ala Arg Pro Asp Arg Val Leu Ser Gln Val Val END

-terminus of mutant Gag-Pol (Processing of mutant Gag-Pol will give rise to 107 aa-long integrase)

Intron domain I

SD-neo

T7

U3 U5R

5’ LTRU3 U5R

5’ LTRpro RT

Group II intron-inserted integrase

poltatvpu

rev

rev

tat

env

I4021sN or I4069sN

U3 U5R

3’ LTRU3 U5R

3’ LTR

---ugg cug gau ugu aca cau uua gaa gga gug cgc cca gau agg gug uua agu caa gua guu uaa ---Trp Gln Leu Asp Cys His Leu Glu Gly Arg Pro Asp Val Leu Ser Gln Val Val

III VII II V

(A)n

-

Intron domain I

---guu cau cua gcc agu gga uau aua gaa gca gug cgc cca gau agg gug uua agu caa gua guu uaa ---Gly Ile Glu Ala Val Arg Pro Asp Val Val END

C- -

Intron domain I

neo

a

c

vif

vpr

gag

integrase domain)Group II intron--

Cap (A)n

integrase domain)--

integrase domain)---inserted 11.1 kb HIV-1 RNA Gag and mutant Gag-Pol (with a truncated -

Cap (A)n

Cap (A)n4-5 kb singly spliced HIV-1 RNAs Vif, Vpr, Vpu, and Env 4-

(A)n4-4-4-4-

Cap (A)n2 kb completely spliced HIV-1 RNAs- Tat, Rev, and Nef -

(A)n--------

b

(Processing of mutant Gag-Pol will give rise to 91 aa-long integrase)

END

nef

Group II intron-------inserted 11.1 kb HIV-1 RNA-


R

3’ LTRU3 U5R

3’ LTR

---ugg cug cua gau ugu aca cau uua gaa gga gug cgc cca gau agg gug uua agu caa gua guu uaa ------Trp Gln Leu Asp Cys Thr His Leu Glu Gly Val Arg Pro Asp Arg Val Leu Ser Gln Val Val

III VII II V

Cap (A)n


C-terminus of mutant Gag-Pol

Intron domain I

---guu cau cua gcc agu gga uauagu gga uau aua gaa gca gug cgc cca gau agg gug uua agu caa gua guu uaa ------Val His Val Ala Ser Gly Tyr Ile Glu Ala Arg Pro Asp Arg Val Leu Ser Gln Val Val END

-terminus of mutant Gag-Pol (Processing of mutant Gag-Pol will give rise to 107 aa-long integrase)

Intron domain I

SD-neo

T7

U3 U5R

5’ LTRU3 U5R

5’ LTRpro RT

Group II intron-inserted integrase

poltatvpu

rev

rev

tat

env

I4021sN or I4069sN

U3 U5R

3’ LTRU3 U5R

3’ LTR

---ugg cug gau ugu aca cau uua gaa gga gug cgc cca gau agg gug uua agu caa gua guu uaa ---Trp Gln Leu Asp Cys His Leu Glu Gly Arg Pro Asp Val Leu Ser Gln Val Val

III VII II V

(A)n

-

Intron domain I

---guu cau cua gcc agu gga uau aua gaa gca gug cgc cca gau agg gug uua agu caa gua guu uaa ---Gly Ile Glu Ala Val Arg Pro Asp Val Val END

C- -

Intron domain I

neo

a

c

vif

vpr

gag

integrase domain)Group II intron--

Cap (A)n

integrase domain)--

integrase domain)---inserted 11.1 kb HIV-1 RNA Gag and mutant Gag-Pol (with a truncated -

Cap (A)n

Cap (A)n4-5 kb singly spliced HIV-1 RNAs Vif, Vpr, Vpu, and Env 4-

(A)n4-4-4-4-

Cap (A)n2 kb completely spliced HIV-1 RNAs- Tat, Rev, and Nef -

(A)n--------

b

(Processing of mutant Gag-Pol will give rise to 91 aa-long integrase)

END

nef

Group II intron-------inserted 11.1 kb HIV-1 RNA-


C

B

A

109

Figure 3.1- (A) Structure of the group II intron-inserted HIV-1 proviral DNA. Intron domains I,

II, III, V and VI are shown. Domain IV is modified to contain the T7 promoter and neo gene. (B)

Transcription from the U3 promoter in the HIV-1 5’ LTR generates primary 11.1 kb transcripts

containing the group II intron. Splicing of HIV-1 introns yields 4-5 and 2 kb RNAs, lacking

group II intron. Translation of the group II intron-inserted 11.1 kb HIV-1 RNA yields HIV-1 gag

and mutant gag-pol with a truncated IN domain. Translation of the 4-5 kb RNAs yields HIV-1

virion infectivity factor (Vif), virion protein R (Vpr), virion protein U (Vpu), and envelop protein

(Env). Translation of the 2 kb RNA yields HIV-1 trans-activator of transcription (Tat), regulator

of expression of virion proteins (Rev), and negative factor (Nef). (C) The HIV-1 RNA sequence

adjacent to the I4021sN and I4069sN insertion sites and the amino acid sequence of the C-terminal

region of the mutant gag-pol encoded by pHIV-I4021sN and pHIV-I4069sN, are shown. Processing of

mutant gag-pol produced from pHIV-I4021sN and pHIV-I4069sN gives rise to 91- and 107-aa long

IN, respectively.

110

The intron insertion frequencies were determined by calculating the percentage of colonies that

allow intron insertion over the total number of colonies that do or do not allow intron insertion.

Our results indicate that insertion frequencies of the modified introns that contain the neo gene

(I4021sN and I4069sN) are lower (Figure 3.2, samples 3 and 7) than those of the unmodified introns

(I4021s and I4069s) (Figure 3.2, samples 1 and 5). The decrease in insertion frequencies of the

modified introns, I4021sN and I4069sN, may have resulted from poor TetR gene expression because of

the extra distance created by the presence of neo gene between the T7 promoter and the TetR

gene. This observation is based on the fact that upon neo gene deletion, the insertion frequencies

of I4021s∆N and I4069s∆N introns returned to normal (Figure 3.2, samples 4 and 8), and that inclusion

of IRES element upstream of the neo gene in I4021sIN and I4069sIN reduced the insertion frequencies

even further (Figure 3.2, samples 2 and 6).

3.4.3. Group II Intron Insertion into an Infectious HIV-1 Proviral DNA Clone

Since intron insertion has not yet been demonstrated in mammalian chromosomes, we bypassed

this step by allowing intron insertion in an infectious HIV-1 proviral DNA clone in E. coli,

purifying the intron-inserted HIV-1 proviral DNA clones, and testing them for inhibition of virus

replication in mammalian cells. pACD-I4021sN and pACD-I4069sN were used to allow intron

insertion into an infectious HIV-1 proviral DNA clone in E. coli (Figure 3.3). To this end, the E.

coli cells were co-transformed with pACD-I4021sN or pACD-I4069sN along with pHIV (an infectious

HIV-1 proviral DNA clone, originally referred to as pNL4-3)275. Upon IPTG induction, pACD-

I4021sN and pACD-I4069sN would allow intron insertion at nts. 4021 and 4069 within the IN-coding

region of the HIV-1 pol gene. The intron-inserted pHIV, referred to as pHIV-I4021sN or pHIV-

I4069sN, respectively, would contain nts. 1-4021 or 1-4069 of the HIV-1 genome, the intron

domains I, II, and III, the T7 promoter and the neo gene, the intron domains V and VI, and the

rest of the HIV-1 genome (Figure 3.1A). Neo gene expression from the T7 promoter would

111

0

1020

3040

5060

7080

90

100

1 2 3 4 5 6 7 80

1020

3040

5060

7080

90

100

1 2 3 4 5 6 7 8

% In

tron

inse

rtio

n fr

eque

ncy

0

1020

3040

5060

7080

90

100

1 2 3 4 5 6 7 80

1020

3040

5060

7080

90

100

1 2 3 4 5 6 7 8

% In

tron

inse

rtio

n fr

eque

ncy

Figure 3.2- Insertion frequencies of introns I4021s, I4021sIN, I4021sN, I4021s∆N, I4069s, I4069sIN, I4069sN,

and I4069s∆N are presented (samples 1, 2, 3, 4, 5, 6, 7, and 8, respectively). The insertion

frequencies were determined by calculating the percentage of TetR colonies that allow intron-

insertion over the total number of ApR colonies that do or do not allow intron-insertion. Values

were normalized against those obtained for unmodified introns I4021s (66.4%) and I4069s (45.1%).

112

CmS

CmRKmR

pACD -I4021sN or pACD -I4069sN

VI

IPTG -induction

Isolate plasmid DNADigest donor plasmids with Sac II

(pHIV , pHIV -I4021sN, and pHIV-I4069sN are not digested)Transform DH5α cells, select ApRKmRCmS colonies

pHIV

ApR

pHIV -I4021sN or pHIV -I4069sN isolation and characterization

E. coliDH5α

E. coliHMS174 (DE3)

pHIV

E. coliHMS174 (DE3)

ApR ApRKmR

pHIV -I4021sN or pHIV -I4069sN

ApRKmR


CmRKmR


CmRKmR

or

CmRKmR


CmRKmR


Recipient5’ LTR 3’ LTR5’ LTR 3’ LTR

Sac IISac II Sac IISac II

LtrA

P PT7

I II V VIIIIE1 E2

DonorLtrA

T7

I II V VIIIIE1 E2

DonorLtrALtrA

T7 T7

I II V VIIIIE1 E2

T7Lac T7

I II V VIIIIE1 E2

Donor

SD-neoSD-neoSD-neoneo

LtrA

P PT7

I II V VIIIIE1 E2

DonorLtrA

T7

I II V VIIIIE1 E2

DonorLtrALtrA

T7 T7

I II V VIIIIE1 E2

T7LacT7

I II V VIIIIE1 E2

Donor



PT7

I II V VIIII

T7

I II V VIIII

T7

I II V VIIII

T7

I II V VIIII SD-neoSD-neoSD-neoneo

5’ LTR 3’ LTR5’ LTR 3’ LTRIntron-inserted recipient

5’ LTR 3’ LTR5’ LTR 3’ LTRIntron-inserted recipient

PT7

I II V VIIII

T7

I II V VIIII

T7

I II V VIIII

T7


CmS

CmRKmR


VI

IPTG -induction

Isolate plasmid DNADigest donor plasmids with Sac II

(pHIV , pHIV -I4021sN, and pHIV-I4069sN are not digested)Transform DH5α cells, select ApRKmRCmS colonies

pHIV

ApR

pHIV -I4021sN or pHIV -I4069sN isolation and characterization

E. coliDH5α

E. coliHMS174 (DE3)

pHIV

E. coliHMS174 (DE3)

ApR ApRKmR


ApRKmR


CmRKmR


CmRKmR

or

CmRKmR


CmRKmR



Sac IISac II Sac IISac II

LtrA

P PT7

I II V VIIIIE1 E2

DonorLtrA

T7

I II V VIIIIE1 E2

DonorLtrALtrA

T7 T7

I II V VIIIIE1 E2

T7Lac T7

I II V VIIIIE1 E2

Donor


LtrA

P PT7

I II V VIIIIE1 E2

DonorLtrA

T7

I II V VIIIIE1 E2

DonorLtrALtrA

T7 T7

I II V VIIIIE1 E2

T7LacT7

I II V VIIIIE1 E2

Donor



PT7

I II V VIIII

T7

I II V VIIII

T7

I II V VIIII

T7


5’ LTR 3’ LTR5’ LTR 3’ LTR5’ LTR 3’ LTR5’ LTR 3’ LTRIntron-inserted recipient

5’ LTR 3’ LTR5’ LTR 3’ LTR5’ LTR 3’ LTR5’ LTR 3’ LTRIntron-inserted recipient

PT7

I II V VIIII

T7

I II V VIIII

T7

I II V VIIII

T7


Figure 3.3

113

Figure 3.3- Experimental scheme used to show insertion of I4021sN and I4069sN introns into pHIV

(an infectious HIV-I proviral DNA clone) and isolation of the intron-inserted HIV-1 proviral

DNA clones (pHIV-I4021sN and pHIV-I4069sN). E. coli HMS174(DE3) cells were co-transformed

with the donor plasmid (pACD-I4021sN or pACD-I4069sN) and the recipient plasmid (pHIV). The

donor plasmids are shown to contain the intron domains I, II and III, the T7 promoter, neo gene,

and the intron domains V and VI. In these plasmids, the introns are flanked by the 5’ (E1) and 3’

(E2) HIV-1 exon sequences. These exons are required for the splicing of group II introns. IPTG-

induction allows intron and LtrA protein production and intron insertion into pHIV. Plasmids,

consisting of the donor (pACD-I4021sN or pACD-I4069sN), recipient (pHIV), and the intron-inserted

recipient (pHIV-I4021sN or pHIV-I4069sN), were extracted. They were digested with Sac II, which

excises pACD-I4021sN and pACD-I4069sN, but leaves the pHIV, pHIV-I4021sN, and pHIV-I4069sN

DNA intact. E. coli DH5α cells were then transformed with the resulting DNA and the

ApRKmRCmS colonies were selected. pHIV-I4021sN and pHIV-I4069sN were isolated from these

cells and characterized by restriction enzyme digestion and PCR analyses.

114

confer Km resistance. However, because pACD-I4021sN and pACD-I4069sN present in the co-

transformed cells would also confer Km resistance, Km selection at this stage cannot imply

intron insertion. We, therefore, used the following strategy to isolate pHIV-I4021sN and pHIV-

I4069sN (Figure 3.3). Plasmids were extracted from the IPTG-induced E. coli cells and digested

with Sac II, which excises pACD-I4021sN and pACD-I4069sN, but leaves both the intron-inserted

pHIV-I4021sN and pHIV-I4069sN, and the pHIV DNA intact. The E. coli cells were transformed with

the DNA after digestion with Sac II, and the ApRKmRCmS colonies containing pHIV-I4021sN or

pHIV-I4069sN were selected. Confirmation that the clones contained pHIV-I4021sN or pHIV-I4069sN

was obtained by restriction digestion and PCR analyses.

3.4.4. Group II Intron-inserted HIV-1 Proviral DNA Replication in Mammalian Cells

The intron-inserted HIV-1 proviral DNA clones, pHIV-I4021sN and pHIV-I4069sN, were first tested

for group II intron-inserted HIV-1 RNA production in mammalian cells. To this end, pHIV (as

positive control), pHIV-I4021sN, and pHIV-I4069sN were used to transfect human 293T cells. Total

RNA from these cells was extracted on day 4 post-transfection and analyzed by RT-PCR. As

expected, a 423-bp RT-PCR product was detected in the control pHIV-transfected 293T sample

(Figure 3.4A, lane 1). A 275-bp and a 227-bp RT-PCR product arising from group II intron-

inserted HIV-1 RNA was detected in pHIV-I4021sN- and pHIV-I4069sN-transfected 293T samples

(Figure 3.4A, lanes 2 and 3). Similar amounts of RNA were present in all samples analyzed by

RT-PCR (Figure 3.4B, lanes 1-3).

The pHIV-, pHIV-I4021sN-, and pHIV-I4069sN-transfected 293T cells were then tested for virus

replication. Similar amounts of gag protein were produced in these cells (Figure 3.5A, samples

1-3). The amount of progeny produced was also measured. Similar amounts of progeny viruses

were produced from cells transfected with pHIV (positive control), pHIV-I4021sN, or pHIV-I4069sN

115

1 2 3 4 5 6

-

-

-

-HIV 1 RNA 423 bp

275 bp227 bpinserted HIV 1

RNA

a

β actin mRNA 353 bpc

423 bp

275 bp227 bp

a 423 bp

275 bp227 bp

a

353 bpc

353 bpb

cHIV 1

virion RNA 613 bp

Group II intron 275 bp227 bpinserted HIV-1

virion RNA

275 bp227 bp275 bpd

-

-

-

-Group II intron

1 2 3 4 5 6

-

-

-

-HIV 1 RNA 423 bp

275 bp227 bpinserted HIV 1

RNA

a

β actin mRNA 353 bpc

423 bp

275 bp227 bp

a 423 bp

275 bp227 bp

a

353 bpc

353 bpb

cHIV 1

virion RNA 613 bp

Group II intron 275 bp227 bpinserted HIV-1

virion RNA

275 bp227 bp275 bpd

-

-

-

-Group II intron

D

C

B

A

Figure 3.4- RT-PCR analyses for determining intron insertions: (A) HIV-1 RNA or group II

intron-inserted HIV-1 RNA production in 293T cells transfected with pHIV (lane 1), pHIV-

I4021sN (lane 2), or pHIV-I4069sN (lane 3). Total RNA extracted from these cells on day 4 post-

transfection was analyzed by RT-PCR. RT-PCR amplification of the HIV-1 RNA by the IIS-

5’/IIS-3’ primer pair gave rise to a 423-bp product. This primer pair was designed to hybridize

with the HIV-1 RNA sequences that flank the intron insertion sites. RT-PCR amplification of the

I4021sN and I4069sN intron-inserted HIV-1 RNAs by the DV-5’/IIS-3’ primer pair gave rise to 275-

bp and 227-bp products, respectively. This primer pair was designed to hybridize within the

intron domain V and HIV-1 RNA further downstream of the intron insertion sites. PCR

amplification of pHIV DNA (lane 4) by the IIS-5’/IIS-3’ primer pair and of pHIV-I4021sN and

pHIV-I4069sN DNA (lanes 5 and 6) by the DV-5’/IIS-3’ primer pair was done in parallel. (B) The

endogenous β-actin mRNA from pHIV- (lane 1), pHIV-I4021sN- (lane 2), or pHIV-I4069sN- (lane 3)

transfected 293T cells was RT-PCR amplified as control. (C) HIV-1 RNA packaging in the

progeny viruses released from 293T cells transfected with pHIV (lane 1), pHIV-I4021sN (lane 2),

or pHIV-I4069sN (lane 3). Virion RNA extracted from these progeny viruses was analyzed by RT-

PCR using IIS-Up-5’ and IIS-3’ primers. PCR amplification of pHIV DNA (lane 4) by the IIS-

Up-5’/IIS-3’ primer pair was done in parallel. (D) Group II intron-inserted HIV-1 RNA

116

packaging in the progeny viruses released from 293T cells transfected with pHIV (lane 1),

pHIV-I4021sN (lane 2), or pHIV-I4069sN (lane 3). Virion RNA extracted from these progeny viruses

was analyzed by RT-PCR using DV-5’ and IIS-3’ primers. The RT-PCR products were analyzed

on 2% agarose gels. RT-PCR product sizes are indicated on the left of the gels. PCR

amplification of pHIV-I4021sN and pHIV-I4069sN DNA (lanes 5 and 6) by the DV-5’/IIS-3’ primer

pair was done in parallel.

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2 32 3

bB

aA

Figure 3.5- HIV-1 p24 antigen detection in cell lysates (A) and progeny viruses released into the

cell culture supernatants (B) from pHIV- (sample 1), pHIV-I4021sN- (sample 2), and pHIV- I4069sN-

(sample 3) transfected, and untransfected (sample 4) 293T cells on day 4 post-transfection.

118

(Figure 3.5B, samples 1-3). No gag protein or progeny virus could be detected in the

untransduced 293T samples (Figures 3.5A and B, sample 4).

Progeny viruses from pHIV-, pHIV-I4021sN-, and pHIV-I4069sN-transfected 293T cells were tested

for the HIV-1 RNA and group II intron-inserted HIV-1 RNA packaging. Virion RNA was

extracted from progeny viruses and analyzed by RT-PCR. When an HIV-1 RNA-specific IIS-

Up-5’/IIS-3’ primer pair was used, a 613-bp product was detected upon RT-PCR analysis of

virion RNA from the progeny of pHIV-transfected 293T cells (Figure 3.4C, lane 1). No RT-PCR

product could be detected when virion RNA from the progeny of pHIV-I4021sN- or pHIV-I4069sN-

transfected 293T cells was analyzed (Figure 3.4C, lanes 2 and 3). When a group II intron-

inserted HIV-1 RNA-specific DV-5’/IIS-3’ primer pair was used, 275- and 227-bp products were

detected upon RT-PCR analysis of virion RNA from the progeny of pHIV-I4021sN- and pHIV-

I4069sN-transfected 293T cells, respectively (Figure 3.4D, lanes 2 and 3). No RT-PCR product

could be detected when virion RNA from the progeny of pHIV-transfected 293T cells was

analyzed (Figure 3.4D, lane 1). These results demonstrate that the group II intron-inserted HIV-1

RNA was packaged.

3.4.5. Group II Intron-inserted Progeny Virus Replication during the Second Round of

Infection

The infectivity of the progeny viruses was assessed by infecting the human CD4+ T lymphoid

(PM1) cells. Since the group II intron-inserted HIV-1 RNA was packaged, reverse transcription

should have occurred. The presence of reverse-transcribed HIV-1 DNA was detected by PCR

analysis of the genomic DNA isolated 1 hr post-infection. A 220-bp product resulting from the

HIV-1 dsDNA was amplified from the genomic DNA of PM1 cells that were infected with the

progeny from pHIV- (positive control), pHIV-I4021sN-, or pHIV-I4069sN-transfected 293T cells

119

(Figure 3.6A, lanes 1-3). As expected, no PCR product was amplified from the genomic DNA

obtained from uninfected PM1 cells (Figure 3.6A, lane 4). We then tested for the presence of

integrated proviral DNA by PCR analysis of genomic DNA isolated on day 8 post-infection from

uninfected and infected PM1 cells. No proviral DNA could be detected in PM1 cells infected

with the progeny from pHIV-I4021sN- or pHIV-I4069sN-transfected 293T cells (Figure 3.6B, lanes 2

and 3). As expected, a 424-bp product resulting from HIV-1 proviral DNA amplification was

detected in control PM1 cells that were infected with the progeny from the pHIV-transfected

293T cells (Figure 3.6B, lane 1). No PCR product was detected in the uninfected PM1 sample

either (Figure 3.6B, lane 4). The amount of DNA analyzed by PCR was similar in all cases

(Figure 3.6C, lanes 1-4).

Infected PM1 cells were then tested for progeny virus production (Figure 3.7). As expected,

virus production, extensive cell death, and syncytia were observed in control PM1 cells that were

infected with the progeny virus from the pHIV-transfected 293T cells.

No virus was produced from PM1 cells infected with progeny virus from pHIV-I4021sN- or pHIV-

I4069sN-transfected 293T cells. These PM1 cells were healthy with no cell death, no syncytia, and

no progeny production for the length of the experiment (up to 62 days in two independent

experiments).

120

Reverse-transcribedHIV-1 ds DNA

HIV-1 provirus DNA

CCR5 DNA

220 bp

465 bpc

a

b


HIV-1 provirus DNA

CCR5 DNA

220 bp

1 2 3 4 5

465 bp

424bp

c

a

b

c

a

b


HIV-1 provirus DNA

CCR5 DNA

220 bp

465 bpc

a

b


HIV-1 provirus DNA

CCR5 DNA

220 bp

1 2 3 4 5

465 bp

424bp

c

a

b

c

a

b

C

B

A

Figure 3.6- PCR analyses for determining intron inserted pHIV-1: (A) Detection of reverse-

transcribed HIV-1 dsDNA in PM1 cells infected with DNase-treated progeny from pHIV-

(positive control, lane 1), pHIV-I4021sN- (lane 2), or pHIV-I4069sN- (lane 3) transfected 293T cells.

One hr post-infection, the genomic DNA was extracted from the infected (lanes 1-3) or

uninfected (negative control, lane 4) PM1 cells. The DNA was analyzed by PCR using the HIV-

1 DNA specific LTR-5’/LTR-3’ primer pair to detect the HIV-1 and group II intron-inserted

HIV-1 dsDNA. PCR amplification of pHIV DNA (lane 5) by the LTR-5’/LTR-3’ primer pair

was done in parallel. (B) Detection of integrated proviral DNA in PM1 cells infected with

progeny from pHIV- (positive control; lane 1), pHIV-I4021sN- (lane 2), or pHIV-I4069sN- (lane 3)

transfected 293T cells. Genomic DNA was isolated from infected (day 8 post-infection; lanes 1-

3) and uninfected (lane 4) PM1 cells. They were analyzed by PCR using the HIV-1 Tat-5’/Tat-3’

primer pair to detect the presence of both HIV-1 and group II intron-inserted HIV-1 proviral

DNA. PCR amplification of pHIV DNA (lane 4) by the Tat-5’/Tat-3’ primer pair was done in

parallel. (C) The endogenous CCR5 DNA was amplified from PM1 cells infected with the

progeny from pHIV- (lane 1), pHIV-I4021sN- (lane 2), or pHIV-I4069sN- (lane 3) transfected 293T

cells. CCR5 DNA was also analyzed from uninfected (lane 4) PM1 cells. The PCR products

were analyzed on 2% agarose gels. PCR product sizes are indicated on the left of the gels.

121

0100200300400500600700

0 10 20 30 40 50 60 70

Days post-infection

HIV

-1 p

24 (n

g/m

l)

Figure 3.7- Progeny virus production by PM1 cells infected with the progeny virus from pHIV-

(— —), pHIV-I4021sN- (—▲—), or pHIV-I4069sN- (— —) transfected 293T cells. Virus

production from infected PM1 cells is reported until day 62 post-infection.

122

3.5. Discussion

In the present study, we have used HIV-1 as model to address a number of key questions

regarding the therapeutic application of a mobile group II intron. Two Ll.LtrB-derived introns

were used that are targeted against nts. 4021 or 4069 within the sense DNA strand of the IN-

coding region of the HIV-1 pol gene. pACD-I4021sN and pACD-I4069sN were engineered to express

introns that confer a selectable phenotype to the intron-inserted HIV-1 proviral DNA clones.

Insertion frequencies of the modified introns (I4021sN and I4069sN) were lower than those of the

unmodified introns or introns from which the neo gene had been deleted. These results indicate

that intron design (modifications within the intron domain IV) may directly (due to inadequate

RNA configuration) or indirectly (due to poor TetR gene expression) affect intron insertion

frequency.

pACD-I4021sN and pACD-I4069sN were used to allow intron insertion into an infectious HIV-1

proviral DNA clone in E. coli. The infectious HIV-1 proviral DNA clone (pHIV) and intron-

inserted HIV-1 proviral DNA clones (pHIV-I4021sN and pHIV-I4069sN) were then tested for virus

replication in 293T cells. Similar amounts of WT or group II intron-inserted HIV-1 RNA (Figure

3.4A), gag protein (Figure 3.5A), and progeny viruses (Figure 3.5B) were produced in all cases.

These results are in line with what we had anticipated. All viral RNAs and proteins produced in

pHIV-I4021sN- and pHIV-I4069sN-transfected 293T cells are expected to be WT, except for the

increased length of the group II intron-inserted HIV-1 RNA, and the gag-pol precursor that

would be truncated beyond the intron insertion site within the IN domain (Figure 3.1B).

Therefore, progeny viruses should have been produced in the pHIV-I4021sN- and pHIV-I4069sN-

transfected cells. However, these viruses were expected to be non-infectious because of the

increased length of the intron-inserted HIV-1 RNA (11.1 kb instead of 9.2 kb) that may prevent

123

encapsidation and/or the absence of a functional IN that may prevent integration even if the RNA

was packaged and reverse-transcribed. Since the insertion sites for the two introns are located in

the IN-coding region, a 91-aa long (aa 1-80 of the IN + 11 aa from the I4021sN intron) or a 107-aa

long (aa 1-96 of the IN + 11 aa from the I4069sN intron) truncated IN should be produced in the

pHIV-I4021sN- and pHIV-I4069sN-transfected 293T cells, respectively (Figure 3.1C); the full-length

IN is 298-aa long.

The intron-inserted HIV-1 RNAs were packaged in the progeny viruses released from the pHIV-

I4021sN- or pHIV-I4069sN-transfected 293T cells (Figure 3.4D). The infectivity of the progeny

viruses was then assessed by infecting the human CD4+ T lymphoid (PM1) cells. Although the

group II intron-inserted HIV-1 RNAs were reverse-transcribed (Figure 3.6A), no proviral DNA

could be detected (Figure 3.6B). The presence of reverse-transcribed HIV-I4021sN and HIV-I4069sN

dsDNA accompanied by the absence of integrated proviral DNA indicates that the reverse-

transcribed dsDNA could not integrate. These results suggest that the 91-aa and 107-aa long INs

(in the progeny from pHIV-I4021sN- and pHIV-I4069sN-transfected 293T cells) were non-functional.

Point mutations, insertions and deletions during HIV-1 reverse transcription285 could have

removed the group II introns and yielded a WT HIV-1 dsDNA. The absence of integrated

proviral DNA despite the formation of reverse-transcribed dsDNA further indicates that even if

these group II introns were deleted during reverse transcription, the resulting dsDNA could not

integrate due to the absence of a functional IN.

No virus was detected in PM1 cells infected with the progeny from pHIV-I4021sN- or pHIV-

I4069sN-transfected 293T cells (Figure 3.7). These PM1 cells were healthy with no cell death, no

syncytia, and no progeny production for the length of the experiment. PM1 cells are highly

permissive to HIV-1 replication. Therefore, if any WT HIV-1 had been produced as a result of

124

group II intron self-splicing in the pHIV-I4021sN- or pHIV-I4069sN-transfected 293T cells, or if

escape mutants had been generated during reverse transcription, progeny should have been

detected after two months. A concern with sense DNA targeting introns is that they may splice out

of the HIV-1 transcripts, enabling the group II intron-inserted HIV-1 proviral DNA to complete a

normal virus life cycle. Our results indicate that self-splicing of the particular introns used here is

not a concern for inhibition of HIV-1 replication.

In conclusion, we have shown here that the Ll.LtrB-derived I4021sN and I4069sN introns are capable

of conferring “complete” inhibition of HIV-1 replication at the intended step. These results

demonstrate that group II introns provide a powerful tool that should be further exploited for

HIV-1 gene therapy.

Acknowledgements

This work is supported by grants from the Canadian Institutes of Health Research and the

Ontario HIV Treatment Network. R.N. is thankful to the Ontario HIV Treatments Network for a

doctoral fellowship. We are grateful to Anne Lise Haenni (Institute Jacques Monod, Paris,

France) for critical proofreading of this manuscript. pACD-HIV1-4021s, pACD-HIV1-4069s,

and pBRR-HIV1(3805-4178s)/Tet were obtained from Alan Lambowitz (Institute for Cellular

and Molecular Biology, Austin, Texas). PM1 cell line and pNL4-3 were obtained through the

AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH.

125

CHAPTER 4

GENERAL DISCUSSION AND FUTURE DIRECTIONS

126

4.1. Discussion and Thesis Summary

To date, none of the anti-HIV drugs can eradicate the HIV-1 infection, no matter how early

treatment is initiated, or how long treatment is continued286. The reason is that the proviral DNA

persists in some cells. These cells, acting as viral reservoirs, can start virus production as soon as

the suppressive therapy stops. It has been shown that these cells, which are estimated to be about

0.05% of the total resting CD4+ T-cell population (~106 cells)287, are established early in HIV-1

infection288. Besides, genetic variations help the virus escape from the host immune system.

A mutation in CCR5, which results in a deletion of 32 nt. in the coding region, produces a non-

functional allele (CCR5∆32). About 10% of Caucasians carry this ∆32 allele and about 1% of

them are homozygotes. The ∆32/∆32 homozygotes are not usually susceptible to infection. R5-

tropic HIV-1 strains, which use CCR5 for entry, are favored for infection. Despite several

hypotheses that try to explain this phenomenon, the reason is not yet fully understood. Although

other CCR co-receptors, such as CCR1 and CCR3, can be used instead of CCR5 by some HIV-1

strains in vitro, there is no evidence of such infection and pathogenesis in vivo. The ∆32

heterozygotes are not completely protected, but progression to AIDS is slowed and delayed in

them108.

Once the infection is established, in 50% of patients, the viruses evolve from the R5-tropic strain

to an X4-tropic strain. The reason for such evolution is not understood, and its mechanism seems

to be mutations in viral glycoprotein gp120. The R5-tropic strains are less cytopathic in vitro and

have been shown to possess less replication capacity, killing, and syncytia formation, compared

to the X4-tropic strains. Therefore, shift from R5-tropic to X4-tropic results in a faster

progression to AIDS.

127

In infected individuals, one of the important keys that are involved in disease progression is

thought to be the activation of the immune system leading to production of inflammatory

cytokines and T-cell death/apoptosis289. HIV-1-infected macrophages may show abnormality in

function and production of cytokines, which may cause pathological and patho-physiological

problems. HIV-1 proviral DNA remains in the infected macrophage genome and the progeny

virus will be collected in and released by intracellular vesicles, without killing the cells or

producing high number of progeny virus, which makes the macrophages appropriate reservoirs

for virus. High levels of inflammatory cytokine production push both the infected and uninfected

cells to a pro-apoptotic state290.

During disease progression, some infected CD4+ T-cells produce high levels of viral proteins

and die after infection. A smaller number of infected T-cells, mainly the naïve and memory

CD4+ cells, will still carry the proviral DNA, but may remain as an HIV-1 reservoir and produce

little or no virus. The number of other uninfected CD4+ and CD8+ T-cells will also decline due

to apoptosis, which occurs either by cell-cell contact291 or by binding the HIV-1 Tat protein to

CD4 receptor of uninfected cell292. Also, the rate of CD4+ and CD8+ T-cell production will be

lowered by effects on bone marrow and/or thymus caused by the infection291. Hematopoiesis is

shown to be inhibited by secretion of certain hematopoietic factors, such as tumor growth factor-

α, from infected stromal fibroblasts and macrophages in bone marrow291.

As mentioned in the introduction, HIV-1 PIC, containing the reverse-transcribed dsDNA bound

to viral matrix protein p17, IN, RT, and Vpr, has the ability to integrate the HIV-1 DNA into the

chromosomes of non-dividing cells, which enables the HIV-1 to infect a wider number and type

of target cells. This ability is provided by Vpr functions, which, induces breaks in the nuclear

membrane293. The increase in numbers of such breaks may also induce cell death.

128

Increasing the amount of unintegrated DNA in re-infected cells294, as well as cytotoxic T

lymphocytes (CTL) attacks to the infected cells that express viral proteins295, are other reported

causes of infected cell death.

Nef is also an important HIV-1 protein involved in immunodeficiency and cell death, as well as

promoting more viral production. Although, by downregulating the surface CD4 expression, Nef

helps the infected cell escape being killed by CTL response, it enhances the expression of

apoptosis-inducing proteins, such as FasL and TNF-α, that induce apoptosis in uninfected cells

through a cell-cell contact296.

Knowing the above, if any therapeutic strategy is to be designed against HIV-1, in addition to

inhibition of viral replication, it must be able to stop cell death resulting from any of described

mechanisms. HIV-1 therapy strategies that focus on inhibiting proviral integration or disrupting

integrated viral DNA seem to be preferred because they would prevent viral gene activities.

Once no viral protein is produced, the infected cell may have a chance to remain and function in

the immune system. Our results demonstrated that group II introns, which are modified to target

integrated viral DNA, can be a good candidate for such strategy.

When HAART was developed in mid 1990s, its activity against HIV-1 was so promising that

people estimated the virus would be eliminated soon. Now, not only does the virus still infect

new patients, but also emergence of multi-drug resistant strains, HAART toxicity* and metabolic

* HIV/AIDS drug side effects may include appetite loss, nausea and vomiting, diarrhea, fatigue, gas and

bloating, headaches, nightmares and sleeping difficulties, hair loss, skin problems, muscles aches and

pains, sexual difficulties, liver toxicity, insulin resistance and diabetes, pancreatitis, kidney stones,

peripheral neuropathy, lose of bone density and osteonecrosis, body distortions, and cardiac

complications304.

129

complications are common problems in HIV/AIDS patients receiving HAART regimens. The

HAART therapy fails in about 50% of patients in less than two years286, and made the fatal

infection to become a chronic illness, which has increased the rate of transmission! According to

WHO estimates, 5 million people were diagnosed with new HIV-1 infection globally in 2005

and ~40,000 new infections occur yearly in the USA. Thus, other therapeutic approaches are

required to stop new infections along with treating the patients.

Since high levels of resistance to infection by HIV-1 was observed in CCR5∆32 homozygous

individuals, inhibiting viral entry through blockage/deletion of CCR5 may be the best possible

approach to eliminate and cure HIV-1 infection. This approach has many supporting reasons: 1)

CCR5 appearse to be dispensable. 2) viral shift to a more pathogenic X4-tropic strain is not a

concern anymore because it is known that R5-tropic strains alone can also produce AIDS.

Furthermore, resistance of a R5-tropic virus to a CCR5 inhibitor via changes in gp120 was

shown to result in a more efficient binding to CCR5 rather than a switching towards CXCR4

usage297.

Different classes of HIV-1 entry inhibitors, such as attachment inhibitors, CCR5 antagonists, and

fusion inhibitors have been developed. However, some anti-CCR5 drugs, such as aplaviroc and

vicriviroc, were ceased at different stages of phase IIb and III trials due to hepatotoxicity and

inferior efficiency. For these reasons, we and other researchers have focused on gene therapy

strategies to downregulate CCR5 expression. In table 4.1, I have summarized gene therapy

strategies that were developed and tested for downregulation of CCR5 expression. In addition to

their inefficiency in conferring complete CCR5 downregulation and durable inhibition of HIV-1

replication at high virus concentrations, each of them faced obstacles that slowed their

progression to clinical trials. For example, intrakines have off-target cellular effects that cause

130

immune system complications; intrabodies failed to protect the target cells when exposed to

higher virus concentrations; siRNAs were not durable and had off-target gene regulation effects;

and the antisense RNA was not efficient enough. Since ribozymes have not shown any off-target

gene regulation, cellular toxicity and immune system induction, they might be better candidate

tools for HIV-1 gene therapy. CCR5 downregulation by ribozymes is extensively studied.

Monomeric and multimeric ribozymes are designed to target CCR5 mRNA, however, none of

the studied monomeric or tirmeric ribozymes were completely efficient in CCR5 downregulation

and inhibition of HIV-1 replication. Therefore, we proposed that a multimeric ribozyme,

containing 7 ribozyme may function more efficiently.

131

Ref

.

Yan

g et

al.

(199

7)16

6

Schm

idtm

aye

rova

et

al.

(199

6)16

7

Tedl

a et

al.

(199

6)17

1

Kel

ly e

t al.

(199

8)14

4

Yan

g et

al.

(199

7)16

6

Dis

adva

ntag

e(s)

Into

lera

nce

to

ligan

ds o

ver-

ex

pres

sion

Tim

e of

add

ition

of

β-c

hem

okin

e is

im

porta

nt.

Upr

egul

atio

n of

tra

nscr

iptio

n fa

ctor

s via

G-

prot

ein

sign

alin

g is

a

fact

or15

3 .

Leak

age

of

intra

cellu

lar

chem

okin

es

indu

ces

infla

mm

ator

y re

spon

ses.

Res

ults

Cel

l gro

wth

failu

re

Stim

ulat

atio

n of

H

IV-1

repl

icat

ion

No

inhi

bitio

n or

vira

l at

tach

men

t

Bot

h en

hanc

emen

t an

d in

hibi

tion

of

infe

ctio

n

Dec

reas

e in

HIV

-1

repl

icat

ion

and

sync

ytia

form

atio

n

Vir

us

R5-

tropi

c H

IV-1

Cel

l(s)

PM1

Mac

roph

ages

Chr

onic

ally

in

fect

ed P

M1

Mon

ocyt

e,

MD

M a

nd

CD

4+ T

-cel

l

PM1

Vec

tor/

Pr

omot

er

pCM

V a

nd

LNC

X

(RV

)*

The

rape

utic

m

olec

ule

RA

NTE

S an

d M

IP1-α

RA

NTE

S

RA

NTE

S

RA

NTE

S M

IP1-α,

and

M

IP1-β

RA

NTE

S-K

DEL

and

M

IP1-α-

KD

EL

Tab

le 4

.1. S

umm

ary

of g

ene

ther

apy

appr

oach

es to

dow

nreg

ulat

e C

CR

5 su

rfac

e ex

pres

sion

.

Cla

ss o

f th

erap

eutic

m

olec

ule

Liga

nd

Mon

oclo

nal

ant

ibod

y * (R

V) =

retro

vira

l vec

tor;

(LV

) = le

ntiv

iral v

ecto

r; PI

= p

ost-i

nfec

tion;

(AV

) = a

deno

vira

l vec

tor;

Rz

= rib

ozym

e

132

Ref

.

Ros

chke

et

al.

(200

4)14

6

Cas

tagn

a et

al.

(200

5)16

,147

Stei

nber

ger e

t al.

(200

0)15

1

Swan

et a

l. (2

006b

)175

Cag

non

et

al.

(200

0)17

7

Dis

adva

ntag

e(s)

Pote

ncy

of a

gent

for

dura

tion

and

mag

nitu

de

of re

cept

or

inte

rnal

izat

ion.

Nee

ding

hig

h co

ncen

tratio

n, p

oten

cy

of a

gent

for d

urat

ion

and

mag

nitu

de o

f re

cept

or in

tern

aliz

atio

n.

Shor

t per

iod

of

inhi

bitio

n (1

0 da

ys P

I)

40%

inhi

bitio

n of

HIV

-1

repl

icat

ion

(day

20

PI)

20-5

0% in

hibi

tion

of

vira

l rep

licat

ion

(at 0

.3,

1 an

d 1.

5 ng

p24

of

BaL

)

Res

ults

Phas

e I c

linic

al tr

ial

Phas

e II

clin

ical

tria

l

Inhi

bitio

n of

CC

R5

expr

essi

on, s

yncy

tia

form

atio

n, a

nd H

IV-

1 in

fect

ion

Dis

rupt

ion

of C

CR

5 ex

pres

sion

50-6

0% re

duct

ion

of

CC

R5

expr

essi

on,

com

plet

e in

hibi

tion

of H

IV-1

repl

icat

ion

Vir

us

SF16

2 an

d JR

-CSF

(R

5) M

OI

of 0

.01

BaL

(1 n

g p2

4 eq

uiva

lent

)

BaL

(0.0

5-0.

1 ng

p24

eq

uiva

lent

)

Cel

l(s)

PM1

CD

4+ T

-cel

l an

d C

D34

+-de

rived

m

acro

phag

e

SupT

1,

PM1,

M

DM

s and

m

icro

glia

l

Vec

tor/

Pr

omot

er

Bab

e-Pu

ro

(RV

)

CA

D-R

5 (L

V)

SV40

-ba

sed

(RV

)/ C

MV

The

rape

utic

m

olec

ule

HG

S004

Pro-

140

ST6

(aga

inst

C

CR

5 N

-te

rmin

al)

ST6

(aga

inst

C

CR

5 N

-te

rmin

al)

2C7-

KD

EL

T

able

4.1

. con

tinue

d.

Cla

ss o

f th

erap

eutic

m

olec

ule

Mon

oclo

nal

antib

ody

Int

rabo

dy

* (R

V) =

retro

vira

l vec

tor;

(LV

) = le

ntiv

iral v

ecto

r; PI

= p

ost-i

nfec

tion;

(AV

) = a

deno

vira

l vec

tor;

Rz

= rib

ozym

e

133

Ref

.

Mar

tinez

et

al. (

2002

)36

Qin

et a

l. (2

003)

37

And

erso

n et

al.

(200

3)15

9

And

erso

n et

al.

(200

5)16

3

Li e

t al.

(200

6)15

2

Dis

adva

ntag

e(s)

79%

inhi

bitio

n of

HIV

-1

repl

icat

ion

on d

ay 2

PI

A d

efic

ient

HIV

-1 w

as

used

.

Onl

y 32

% in

hibi

tion

of

HIV

-1 re

plic

atio

n on

da

y 5

PI

Low

inhi

bitio

n of

C

CR

5 ex

pres

sion

and

on

ly 3

3% v

iral

repl

icat

ion

on d

ays 3

-7

PI

~55%

inhi

bitio

n of

vi

ral r

eplic

atio

n on

day

12

PI,

~87%

hom

olog

y to

CC

R2a

and

CC

R2b

m

RN

A

Res

ults

48%

redu

ctio

n of

C

CR

5 ex

pres

sion

90%

redu

ctio

n of

C

CR

5 ex

pres

sion

72%

redu

ctio

n of

C

CR

5 ex

pres

sion

98.1

% re

duct

ion

of

CC

R5

expr

essi

on

Vir

us

BaL

(MO

I 0.

03-0

.24)

Mod

ified

H

IV-1

, ex

pres

sing

H

SA

inst

ead

of

VPR

BaL

(MO

I of

0.0

01)

BaL

(MO

I of

0.0

1)

CN

9700

1 (R

5, M

OI

of 0

.01)

Cel

l(s)

U87

(e

xpre

ssin

g C

D4,

CC

R5

and

CX

CR

4)

CD

4+ P

BL

PBM

Cs

PBM

Cs

U93

7

Vec

tor/

Pr

omot

er

FG12

(LV

)

HIV

-7-

GFP

-XH

R

(LV

)/Pol

-II

I, U

6 an

d H

1

pAD

(AV

)

The

rape

utic

m

olec

ule

RN

AR

53i

(aga

inst

nt.

+554

to +

572

of C

CR

5)

siR

NA

(186

) (a

gain

st n

t. +1

86 to

+20

4 of

CC

R5)

Bis

peci

fic

siR

NA

(aga

inst

C

CR

5 an

d C

XC

R4)

siR

NA

(aga

inst

C

CR

5)15

9

653-

nt. l

ong

antis

ense

RN

A

(aga

inst

nts

. 18

7-83

9 of

C

CR

5-co

ding

re

gion

)

T

able

4.1

. con

tinue

d.

Cla

ss o

f th

erap

eutic

m

olec

ule

RN

Ai

(siR

NA

)

ant

isen

se

RN

A

* (R

V) =

retro

vira

l vec

tor;

(LV

) = le

ntiv

iral v

ecto

r; PI

= p

ost-i

nfec

tion;

(AV

) = a

deno

vira

l vec

tor;

Rz

= rib

ozym

e

134

Ref

.

Qur

eshi

et

al.

(200

6)15

3

Cag

non

et

al.

(200

0)17

7

Bai

et a

l. (2

000)

155

Li e

t al.

(200

5)19

0

Li e

t al.

(200

5)19

0

Bai

et a

l. (2

001)

157

Dis

adva

ntag

e(s)

68%

inhi

bitio

n of

vira

l re

plic

atio

n on

day

7 P

I

Mut

ant R

z sh

owed

the

sam

e ef

ficie

ncy

50%

inhi

bitio

n of

HIV

-1

repl

icat

ion

by m

utan

t R

z

Inhi

bitio

n w

as sl

ight

ly

bette

r tha

n ob

tain

ed b

y m

utan

t Rz

Hig

h m

ount

of p

roge

ny

viru

s pro

duct

ion

(1-1

0 ng

/ml o

n da

y 7

and

500

ng/m

l on

day

28 P

I)

30%

inhi

bitio

n of

HIV

-1

repl

icat

ion

on d

ay 4

PI

Res

ults

50-9

0% re

duct

ion

of

CC

R5

expr

essi

on

70%

redu

ctio

n of

C

CR

5 ex

pres

sion

, 1-

3 da

ys d

elay

in H

IV-

1 re

plic

atio

n

70%

inhi

bitio

n of

H

IV-1

repl

icat

ion

on

day

7 PI

Inhi

bitio

n of

HIV

-1

repl

icat

ion

on d

ay 1

7 PI

99%

inhi

bitio

n of

H

IV-1

repl

icat

ion

10-1

5% re

duct

ion

of

CC

R5

expr

essi

on

Vir

us

SF16

2 (3

ng

p4

equi

vale

nt)

BaL

(MO

I 0.

001)

BaL

(MO

I of

0.0

2)

BaL

(MO

I of

0.0

2)

JR-F

L (M

OIs

of

0.01

and

0.

05)

BaL

(MO

I of

0.0

01)

Cel

l(s)

PBM

Cs

HO

S-C

D4.

CC

R5

PM1

CD

34+-

deriv

ed

mac

roph

ages

T ce

lls a

nd

CD

34+-

deriv

ed

mac

roph

ages

CD

34+-

deriv

ed

mac

roph

ages

Vec

tor/

Pr

omot

er

Bab

e-Pu

ro

(RV

)

pG1N

a (R

V)

HIV

-7-

GFP

(LV

)

LN (R

V)

and

MN

D

(RV

)

The

rape

utic

m

olec

ule

Mon

omer

ic

CC

R5R

z*

(aga

inst

nts

. 26

2-26

4 of

C

CR

5 m

RN

A)

Mon

omer

ic R

z (a

gain

st n

ts. 2

3 of

CC

R5

mR

NA

)

Trim

eric

Rz

(aga

inst

nts

. 17

, 153

and

24

9 in

CC

R5

OR

F)

T

able

4.1

. con

tinue

d.

Cla

ss o

f th

erap

eutic

m

olec

ule

Rib

ozym

e

* (R

V) =

retro

vira

l vec

tor;

(LV

) = le

ntiv

iral v

ecto

r; PI

= p

ost-i

nfec

tion;

(AV

) = a

deno

vira

l vec

tor;

Rz

= rib

ozym

e

135

In our study, we investigated downregulation of the CCR5 expression by a heptameric

hammerhead ribozyme, containing 7 ribozymes that target nucleotides 17, 380, 390, 520, 556,

811, and 824 within the CCR5 ORF. These ribozymes were constructed in two sets: Set A,

containing Rz1, Rz2, and Rz3, and Set B containing Rz4, Rz5, Rz6, and Rz7. Each set of

multimeric ribozymes were tested for in vitro cleavage activity by providing with the CCR5

target RNA. The results revealed that the multimeric ribozymes were active (Figure 2.1). Then

the ribozymes of both sets were combined to make the heptameric hammerhead ribozyme, Rz1-7.

Rz1-7 was cloned into a MSCV-based oncoretroviral vector (MGIN), which contained egfp,

IRES, and neo, to produce MGIN-Rz1-7 vector. Constitutive activity of MGIN 5’ LTR results in

production of a long transcript containing the EGFP ORF, the Rz1-7, the IRES element, and the

Neo ORF. IRES facilitates protein expression from the Neo ORF as the second ORF on the same

transcript. Retroviral particles containing either MGIN or MGIN-Rz1-7 were produced by

transfection of an amphotropic packaging cell line, PA317, followed by G418 selection. The

viral particles were used to transduce CCR5+/CXCR4+/CD4+ human lymphoid cell line, PM1,

and the stably transduced cells were purified via FACS. The purification was performed two

times with 2-3 months interval. PCR analysis of stably transduced PM1 cells’ genomic DNA

showed the presence of the MGIN or MGIN-Rz1-7 vector DNAs (Figure 2.3A). RT-PCR analysis

on stably transduced PM1-MGIN and PM1-MGIN-Rz1-7 cells’ total RNA determined the

presence of vectors’ transcripts and expression of Rz1-7 (Figure 2.4A).

Lentiviral vectors are capable of transducing the resting cells by passing through the nuclear

envelope. Therefore, an HIV-1-based vector, HEG1, was constructed in our laboratory. It

contains the egfp gene under the control of the EF-1α promoter. Since HIV-1 5’ LTR promoter

depends on the HIV-1 Tat protein for activity, and this protein does not exist in uninfected cells,

136

EF-1α promoter serves as a strong internal promoter. I cloned the Rz1-7 gene downstream of the

egfp gene to construct HEG1-Rz1-7. Therefore, the transcript produced from EF-1α promoter will

contain both EGFP ORF and Rz1-7. Tat-dependent activity of HIV-1 5’ LTR promoter of the

HEG1-Rz1-7 vector in infected cells will also results in a long transcript containing the HIV-1 5’

sequence of gag gene and RRE element, the EGFP ORF and Rz1-7. HEG1 or HEG1-Rz1-7 vector

particles were used to transduce PM1 cells and the stable transductants were sorted by FACS.

The sorting was performed two times with 2-3 months interval. PCR analysis of stable

transductants’ genomic DNAs showed the presence of HEG1 and HEG1-Rz1-7 vectors DNAs

(Figure 2.3B). RT-PCR analysis of total RNA of PM1-HEG1-Rz1-7 cells also revealed the

expression of Rz1-7 in the cells (Figure 2.4B).

Total RNAs from the PM1-MGIN-Rz1-7 and PM1-HEG1-Rz1-7 cells were used to obtain Rz1-7

cDNAs via in vitro reverse-transcription reactions. These Rz1-7 cDNAs were used as templates to

produce Rz1-7 RNAs during in vitro transcription reactions. Cleavage activity of Rz1-7 originated

from PM1-MGIN-Rz1-7 and PM1-HEG1-Rz1-7 cells were demonstrated in vitro by its ability to

cleave in vitro-transcribed CCR5 mRNA.

RT-PCR analysis on both PM1-MGIN-Rz1-7 and PM1-HEG1-Rz1-7 cells’ total RNA also showed

that the CCR5 mRNA has been downregulated in the Rz-expressing cells (Figure 2.5A and B).

Untransduced PM1 cells, as well as MGIN-, MGIN-Rz1-7-, HEG1-, and HEG1-Rz1-7-transduced

PM1 cells became infected when challenged with NL4-3, an X4-tropic HIV-1 strain (Figure

2.7A and B). This confirms that the infection with an X4-tropic strain can still occur because this

strain uses CXCR4 co-receptor, which was not our target. Transducing the PM1 cells with the

vectors and expression of Rz1-7 did not alter the CXCR4 expression.

137

Untransduced PM1 cells, as well as MGIN- and HEG1-transduced PM1 cells were also infected

by BaL, an R5-tropic HIV-1 strain, indicating that these cells are susceptible to the R5-tropic

strains due to expression of CCR5 co-receptor on the cells’ surface (Figure 2.7A and B). This

experiment also showed that transduction of PM1 cells with empty MGIN and HEG1 vectors did

not affect the surface CCR5 expression. However, a high level of resistance was observed when

MGIN-Rz1-7- and HEG1-Rz1-7-transduced PM1 cells were challenged with BaL strain. The

challenging experiments lasted two to three months. Three different MOIs (0.225, 0.675, and

2.025) of BaL strain, which are quite high in comparison with other published

studies36,152,163,190,298, were used in our experiments. 99-100% inhibition of progeny virus

production was observed when PM1-MGIN-Rz1-7 cells were challenged with BaL strain at those

MOIs (Figure 2.6). In the case of PM1-HEG1-Rz1-7 cells, the inhibition was 80-99% (Figure

2.6). These levels of inhibitions are also among the highest reported inhibitions in all studies that

focused on inhibition at the level of virus entry36,37,152,153,157,163,176,177,190,298 (see table 4.1).

The incomplete inhibition in our study could have occurred for two reasons: first, the FACS

sorting did not result in a 100% purification of the EGFP-expressing cells, therefore, some

untransduced cells were still present in the sorted cells population. Second, the vectors

expression may have been silenced due to integration into the heterochromatin regions of some

host cells’ chromosomes, therefore, despite the presence of vector DNA in the host genome, the

Rz1-7 expression could have been downregulated due to silencing effects.

To confirm that the inhibitions were through blockade of R5 virus entry, PCR analysis were

performed on genomic DNAs extracted from infected PM1-MGIN and PM1-MGIN-Rz1-7 cells

on 4 and 43 days post-infection and from infected PM1-HEG1 and PM1-HEG1-Rz1-7 cells on 4

and 29 days post-infection. Although a tat gene fragment of HIV-1 proviral DNA was amplified

138

from infected PM1-MGIN and PM1-HEG1 cells’ DNAs, no HIV-1 proviral DNA could be

detected in infected PM1-MGIN-Rz1-7 and PM1-HEG1-Rz1-7 cells’ DNAs (Figure 2.5A and B).

To show that the resistance was not due to any effects on the virus infectivity, similar amount of

progeny virus produced on day 17 post-infection from the BaL virus-infected untransduced cells,

as well as HEG1- and HEG1-Rz1-7-transduced PM1 cells (at an MOI of 2.025) were used to

infect plain PM1 cells. On 14 days post-infection, similar amount of viral p24 was detected in

three cultures, which confirms the infectivity of the progeny viruses produced from control and

Rz1-7-expressing cells.

Since PM1 cells express variety of other chemokines receptors, such as CXCR4, CCR1, and

CCR3277, in addition to CCR5, the fact that the infection of an R5-tropic HIV-1 strain was

inhibited in the anti-CCR5 Rz1-7-expressing cells suggests that the inhibition was through

blockade of the entry and other chemokine receptors could not participate in infection with the

R5 strain. It can also be concluded that during two to three-month long experiments, the R5

strain did not evolve to gain the ability of using other chemokine receptors (i.e. CXCR4) for

infection.

Using the anti-CCR5 ribozyme, we were able to show that virus replication was inhibited at the

level of entry. However, one may ask what can be done if the virus still manages to enter the cell

or an X4-tropic virus infects the CXCR4-expressing cell.

The efficacy of drug and gene therapy strategies, which use interfering molecules that degrade

HIV-1 RNA or inactivate the downstream proteins, will depend on the levels of HIV-1

RNA/proteins expression. We believe that if, by any means, the virus manages to enter the cell

139

despite CCR5 downregulation, the best way to overcome the virus replication may be

inactivation of proviral DNA or blocking its integration to cells’ chromosomes. By doing this, no

viral RNA and protein would be produced and competition of therapeutic gene expression with

viral gene expression would not be necessary any more. Note that single or double LTR circles,

as well as noncircular forms, cannot express virus in vitro, unless integrated into the host

chromosome288. Therefore, we investigated whether an HIV-1-targeting group II intron can

insert into HIV-1 proviral DNA and inactivate the HIV-1 IN gene. Before analyzing our results, I

bring your attention to table 4.2 that summarizes some other gene therapy studies, which targeted

pre-integration and integration stages of HIV-1 life cycle. The gene therapy strategies that focus

on pre-integration and integration stages can be classified based on the molecules they target.

The targets include released viral RNA, RT, IN, PIC, HIV-1 dsDNA, and integrated proviral

DNA.

140

R

ef.

Sarv

er e

t al

. (1

990)

207

Yam

ada

et

al.

(199

4)21

0

Leav

itt e

t al

. (1

994)

211

Li e

t al.

(199

8)21

4

Yu

et a

l. (1

995a

)212

Yu

et a

l. (1

995b

)213

D

isad

vant

age(

s)

Shor

t-ter

m

inhi

bitio

n

Shor

t-ter

m

inhi

bitio

n

Shor

t-ter

m

inhi

bitio

n

Shor

t-ter

m

inhi

bitio

n

Shor

t-ter

m

inhi

bitio

n

Shor

t-ter

m

inhi

bitio

n

R

esul

ts

97%

resi

stan

ce to

in-

com

ing

HIV

-1 fo

r 7

days

PI*

95%

inhi

bitio

n of

pr

ogen

y vi

rus

prod

uctio

n til

l day

35

PI

Inhi

bitio

n of

pro

geny

vi

rus p

rodu

ctio

n fo

r 10

day

s PI

Inhi

bitio

n of

pro

geny

vi

rus p

rodu

ctio

n fo

r 20

-35

days

PI

95%

inhi

bitio

n of

pr

ogen

y vi

rus

prod

uctio

n fo

r 10

days

PI

Inhi

bitio

n of

pro

geny

vi

rus p

rodu

ctio

n til

l da

y 25

PI

V

irus

HX

B2

HX

B2

BaL

or

isol

ated

fr

om

patie

nts

BaL

C

ell(s

)

HeL

a C

D4+

Jurk

at

PBLs

CD

34+-

deriv

ed

mac

roph

ages

CD

34+-

deriv

ed

mac

roph

ages

CD

34+-

deriv

ed

mac

roph

ages

V

ecto

r/

Prom

oter

LNL6

(RV

)*/

tRN

AV

al

LNL6

(RV

) /tR

NA

Val

LNL6

(RV

) /tR

NA

Val

LNL6

(RV

) /V

A1

LNL6

(RV

) /V

A1

The

rape

uti

c m

olec

ule

HH

Rz*

ag

ains

t HIV

-1

gag-

codi

ng

regi

on

HPR

z*

agai

nst n

t. +1

11 a

t HIV

-1

LTR

HPR

z ag

ains

t H

IV-1

pol

-co

ding

regi

on

Tab

le 4

.2. S

umm

ary

of g

ene

ther

apy

appr

oach

es to

inhi

bit H

IV-1

repl

icat

ion

at p

re-in

tegr

atio

n or

inte

grat

ion

stag

es.

Tar

get

mol

ecul

e

Rel

ease

d vi

ral

RN

A

*HH

Rz

= ha

mm

erhe

ad ri

bozy

me;

HPR

z =

hairp

in ri

bozy

me;

PI =

pos

t-inf

ectio

n; R

V =

retro

vira

l vec

tor;

+sss

DN

A =

plu

s-st

rand

stro

ng

stop

DN

A

141

Ref

.

Wes

taw

ay

et a

l. (1

995)

216

Din

g et

al

. (1

998)

217

Din

g et

al

. (1

998)

217

Peng

et

al.

(199

7)21

8

Jacq

ue e

t al

. (2

002)

34

Cap

odic

i et

al.

(200

2)21

9

Dis

adva

ntag

e(s)

Shor

t-ter

m

inhi

bitio

n

Shor

t-ter

m

inhi

bitio

n

Shor

t-ter

m

inhi

bitio

n

Shor

t-ter

m

inhi

bitio

n

Shor

t-ter

m

inhi

bitio

n

Res

ults

83%

redu

ctio

n of

in

fect

ivity

of p

roge

ny

viru

s

Inhi

bitio

n of

pro

geny

vi

rus p

rodu

ctio

n fo

r 21

day

s PI

Inhi

bitio

n of

pro

geny

vi

rus p

rodu

ctio

n fo

r 21

day

s PI

Inhi

bitio

n of

vira

l re

plic

atio

n fo

r 140

da

ys P

I

30 to

50-

fold

re

duct

ion

of p

roge

ny

viru

s pro

duct

ion

on

day

2 PI

75-9

6% d

ecre

ase

in

prog

eny

viru

s pr

oduc

tion

for 3

day

s PI

Vir

us

NL4

-3 (3

0 ng

p24

eq

uiva

lent

)

NL4

-3 (3

0 ng

p24

eq

uiva

lent

)

NL4

-3

(MO

I of

0.01

)

pHIV

NL-

GFP

IIIB

and

B

aL

Cel

l(s)

PBLs

PBLs

Jurk

at

CD

4+ M

AG

I

U87

-CD

4+-

CX

CR

4+,

U87

-CD

4+-

CC

R5+

and

C

D4+

T c

ells

Vec

tor/

Pr

omot

er

Fuse

d to

tR

NA

Lys3

MO

TN a

nd

MO

TiN

(RV

)

MO

TN a

nd

MO

TiN

(RV

)

LSN

P (R

V)

/tRN

AM

et

The

rape

utic

m

olec

ule

HH

Rz*

ag

ains

t HIV

-1

PBS

regi

on

Ant

isen

se

RN

A a

gain

st

HIV

-1 ψ

-gag

Ant

isen

se

RN

A a

gain

st

HIV

-1 U

3-ga

g-en

v

Sens

e R

NA

ag

ains

t LTR

, se

nse

RN

A

agai

nst

+sss

DN

A*

siR

NA

s ag

ains

t LTR

, vi

f and

nef

siR

NA

s ag

ains

t LTR

an

d ga

g

Tab

le 4

.2. c

ontin

ued.

Tar

get

mol

ecul

e

Rel

ease

d vi

ral R

NA

*HH

Rz

= ha

mm

erhe

ad ri

bozy

me;

HPR

z =

hairp

in ri

bozy

me;

PI =

pos

t-inf

ectio

n; R

V =

retro

vira

l vec

tor;

+sss

DN

A =

plu

s-st

rand

stro

ng

stop

DN

A

142

Ref

.

Mac

ieje

ws

k et

al.

(199

5)22

0

Shah

een

et

al.

(199

6)22

1

Stra

yer e

t al

. (2

002)

223

Josh

i et a

l. (2

002)

225

Sura

bhi e

t al

. (2

002)

226

Dis

adva

ntag

e(s)

Shor

t-ter

m in

hibi

tion

Cha

lleng

ing

with

vi

ruse

s at M

OI o

f 1.0

sh

owed

no

inhi

bitio

n.

Cha

lleng

ing

with

vi

ruse

s at h

ighe

r TC

ID50

show

ed n

o in

hibi

tion

Shor

t-ter

m

inhi

bitio

n.

Shor

t-ter

m

inhi

bitio

n, Ju

rkat

ce

lls n

eede

d 20

-fol

d co

ncen

trate

d si

RN

As

for s

ame

inhi

bitio

n.

Res

ults

Com

plet

e in

hibi

tion

of v

iral r

eplic

atio

n fo

r 35

days

PI

80-9

7% in

hibi

tion

of

prog

eny

viru

s pr

oduc

tion

for 1

5-22

da

ys P

I

Hig

h in

hibi

tion

of

prog

eny

viru

s pr

oduc

tion

23-9

6% re

duct

ion

of

vira

l rep

licat

ion

for

22 d

ays P

I

90%

inhi

bitio

n of

pr

ogen

y vi

rus

prod

uctio

n fo

r 13

days

PI

Vir

us

IIIB

, RF,

57

1, a

nd

MN

(MO

Is

of 2

)

NL4

-3

(MO

Is o

f 0.

012

and

0.00

6) a

nd

R7-

HX

B2

(MO

Is o

f 0.

012

and

0.00

6)

NL4

-3 (4

0-10

0 TC

ID50

)

R3B

(MO

Is

of 0

.1 a

nd

50)

Cel

l(s)

MO

LT-3

ly

mph

oid

cell

line

SupT

1

SupT

1

Jurk

at a

nd

293T

MA

GI

Vec

tor/

Pr

omot

er

pMEP

4 (E

BV

-ba

sed

vect

or)

SLX

CM

V

(RV

)/CM

V

SV40

-bas

ed

(RV

)

pcD

NA

3.1/

CM

V

The

rape

utic

m

olec

ule

Ant

i-RT

Fab

Ant

i-RT

intra

body

Ant

i-RT

mA

b (1

E8)

Ant

i-RT

apta

mer

s

siR

NA

s ag

ains

t RT

Tab

le 4

.2. c

ontin

ued.

Tar

get

mol

ecul

e

Rev

erse

tra

nscr

ipta

se

*HH

Rz

= ha

mm

erhe

ad ri

bozy

me;

HPR

z =

hairp

in ri

bozy

me;

PI =

pos

t-inf

ectio

n; R

V =

retro

vira

l vec

tor;

+sss

DN

A =

plu

s-st

rand

stro

ng st

op

DN

A

143

Ref

.

Levy

-Min

tz

et a

l. (1

996)

227

Bou

Ham

dan

et a

l. (1

999)

224

Gol

dste

in e

t al

. (20

02)22

8

Fass

ati e

t al

. (20

03)19

8

Fass

ati e

t al

. (20

03)19

8

Dis

adva

ntag

e(s)

Shor

t-ter

m in

hibi

tion

Onl

y 18

day

s del

ay in

sy

ncyt

ia fo

rmat

ion

whe

n ch

alle

nged

with

th

e hi

gher

co

ncen

tratio

n of

viru

s

Shor

t-ter

m in

hibi

tion

No

inhi

bitio

n at

MO

Is

>1

Low

inhi

bitio

n

Res

ults

92%

inhi

bitio

n of

vi

ral r

eplic

atio

n fo

r 25

day

s PI

Dec

reas

e in

viru

s pr

oduc

tion

whe

n ch

alle

nged

with

the

low

er c

once

ntra

tion

of v

irus

85%

inhi

bitio

n of

vi

ral r

eplic

atio

n fo

r 14

day

s PI

80-9

0% in

hibi

tion

of

vira

l rep

licat

ion

(with

vi

ral M

OI o

f 0.0

1)

2-38

% in

hibi

tion

of

vira

l rep

licat

ion

Vir

us

NL4

-3

(MO

Is o

f 0.

08)

NL4

-3

(0.0

5 an

d 0.

5 pg

p24

eq

uiva

lent

)

NL4

-3 (8

00

TCID

50)

MO

Is o

f 0.

01 a

nd >

1

Cel

l(s)

PBM

Cs

SupT

1

Hum

an

thym

ic g

rafts

of

thy/

liv-

SCID

-hu

mic

e

NP2

, HeL

a,

HeL

a-C

D4

HeL

a

Vec

tor/

Pr

omot

er

SLX

CM

V

(RV

)/CM

V

SV40

-bas

ed

(RV

)

SV40

-bas

ed

(RV

)

The

rape

utic

m

olec

ule

Ant

i-IN

an

tibod

ie

(scF

v33

and

scFv

33/n

u)

Ant

i-IN

an

tibod

y (s

cFv3

3)

Ant

i-IN

an

tibod

y (s

cFv3

3)

siR

NA

aga

inst

hu

man

imp7

(n

ts. 1

392-

1414

)

siR

NA

aga

inst

hu

man

imp9

(n

ts. 5

27-5

47)

Tab

le 4

.2. c

ontin

ued.

Tar

get

mol

ecul

e

Inte

gras

e

Pre-

inte

grat

ion

com

plex

*HH

Rz

= ha

mm

erhe

ad ri

bozy

me;

HPR

z =

hairp

in ri

bozy

me;

PI =

pos

t-inf

ectio

n; R

V =

retro

vira

l vec

tor;

+sss

DN

A =

plu

s-st

rand

stro

ng st

op

DN

A

144

Ref

.

Ciu

ffi e

t al.

(200

5)19

9

Levi

n et

al.

(199

7)23

0

Dis

adva

ntag

e(s)

Incr

ease

of p

rovi

ral

inte

grat

ion

into

GC

-ric

h D

NA

regi

ons o

f hu

man

chr

omos

omes

Res

ults

6-10

% re

duct

ion

of

prov

iral i

nteg

ratio

n in

to L

EDG

F/p7

5-re

gula

ted

gene

s and

ot

her t

rans

crip

tion

units

Inhi

bitio

n of

vira

l re

plic

atio

n an

d re

duct

ion

of p

roge

ny

viru

s inf

ectiv

ity

Vir

us

Cel

l(s)

Jurk

at

Div

idin

g C

D4+

cel

ls

Vec

tor/

Pr

omot

er

The

rape

utic

m

olec

ule

shR

NA

ag

ains

t hum

an

LED

GF/

p75

Ani

t-MA

in

trabo

dy

Tab

le 4

.2. c

ontin

ued.

Tar

get

mol

ecul

e

Pre-

inte

grat

ion

com

plex

*H

HR

z =

ham

mer

head

ribo

zym

e; H

PRz

= ha

irpin

ribo

zym

e; P

I = p

ost-i

nfec

tion;

RV

= re

trovi

ral v

ecto

r; +s

ssD

NA

= p

lus-

stra

nd st

rong

stop

D

NA

145

As mentioned, despite the fact that the proviral DNA is thought to be the best gene therapy

candidate, it was not successfully targeted so far. This will highlight the importance and novelty

of our study of inactivating the HIV-1 proviral DNA by group II introns. We used the

Lactococcus lactis group II intron (Ll.LtrB) as a therapeutic tool that has an outstanding

advantage: since the Ll.LtrB group II intron RNP recognizes a 12-16 base pair region on the

target DNA site, its RNA sequence can be easily altered to base-pair with the flanking sequences

of the intron’s LtrA protein binding site. Previously, Dr. A. Lambowitz and his team had shown

that group II introns could be designed and modified to be able to insert into the HIV-1 proviral

DNA237. They cloned a ~370-bp cDNA fragment from nt. 3805 to 4178 (respect to nt. 1 of HIV-

1 RNA) of HIV-1 IN domain of pol region into the pBRR vector upstream of a promoter-less tetR

gene to make the pBRR-HIV1(3805-4178s)/Tet, a selectable ‘recipient’ plasmid. They also

cloned the introns targeting nts. 4021 and 4069 of HIV-1 provirus sense-strand DNA into the

pACD expression vector (donor) under the control of a T7 promoter to produce pACD-HIV1-

4021s and pACD-HIV1-4069s, respectively. The introns were modified to contain an internal T7

promoter in the intron’s domain IV. When E. coli cells were co-transformed with both donor and

recipient vectors, the introns were expressed (by induction with IPTG) and inserted into the

target sequence at a frequency of 66% and 63%, respectively. Since the promoter-less tetR gene

could be expressed by the intron’s internal T7 promoter, the frequency of insertion could be

measured by the ratio of TetR colonies to the total of TetR and TetS ones. However, the authors

did not assess the insertion and its frequency into a full-length HIV-1 clone. Because both intron

and target were cloned into bacterial vectors, to be able to test whether the intron-LtrA protein

(RNP) complex could function in human cellular environment, they co-transfected 293T cells

with liposomes carrying the pre-expressed RNP and the recipient vector, separately. The

insertion was detected by PCR analysis237. However, there is a doubt whether the fusion between

146

donor- and recipient-carrying liposomes did not occur outside the cell, which might result in a

better target accessibility for the intron.

In our study, we investigated if the intron can target the full-length HIV-1 clone, if further

modifications to the intron to have a selectable marker will alter the intron’s function and

insertion rate, and if the intron insertion will inhibit HIV-1 replication.

Both pACD-HIV1-4021s and pACD-HIV1-4069s plasmids were modified by cloning a

kanR/neoR gene in intron’s domain IV, downstream of the internal T7 promoter to produce

pACD-I4021sN and pACD-I4069sN (Figure 3.3). When E. coli cells were co-transformed with the

pBRR-HIV1(3805-4178s)/Tet recipient and each of the modified introns, we have shown that

upon IPTG induction, the insertions occurred, but at very lower frequencies compared to those of

unmodified introns (Figure 3.2). This might have happened because of either changes in the

tertiary structure of intron RNA due to cloning of the neoR gene in the intron or lower expression

of the tetR gene due to the additional distance made by cloning the neoR gene downstream of the

T7 promoter.

To investigate the insertion of modified introns into the full-length HIV-1 proviral DNA clone,

E. coli cells were co-transformed with pNL4-3 as the recipient and either of pACD-I4021sN or

pACD-I4069sN donor plasmids, followed by IPTG induction. The frequency of insertions could

not be measured because the recipient plasmid would not demonstrate any selective advantage

upon intron insertion. However, the insertions were detected by plasmid purification,

endonuclease digestions, and PCR analyses.

147

The intron-inserted HIV clones, pHIV-I4021sN and pHIV-I4069sN, were then tested for their ability

to produce progeny virus by transfecting the 293T cells. Since the sizes of the intron-inserted

HIV-1 RNA were about 2 kb larger than the wild-type RNA, we doubted whether or not they

could be packaged in the virions. It appeared that both pHIV-I4021sN and pHIV-I4069sN could

produce the same amount of progeny virus as the control pHIV-1-transfected 293T cells (Figure

3.5). However, the intron-containing progeny viruses were not infectious when PM1 cells were

challenged with them (Figure 3.7). This was anticipated because the intron insertion into the IN

domain would cause formation of an early stop codon and production of 88-aa or 96-aa long

truncated INs, which could not function normally in the integration process. This was proved by

PCR analysis on the infected PM1 cells’ DNAs. Although we could detect the HIV-1 cDNA

molecules in PM1 cells at 1 hr post-infection (meaning accomplishment of reverse-transcription

process), no HIV-1 proviral DNA could be detected 8 days post-infection. No sign of infection

was observed in a more than 2-month long experiment. The results were supported by high cell

viability and lack of syncytia formation in the cultures. The length of experiment was important

to show that the introns, which were inserted into the sense strand of target DNA, could not

splice out to produce wild-type HIV-1 RNA. This study showed that inactivation of IN by

specific introns could result in inhibition of HIV-1 infection in the second host cell, or as we call,

“the second round of infection”.

We also tried to test the anti-sense targeting introns, I54a and I2654a, which targeted the nucleotides

54 and 2654 on antisense strand of HIV-1 proviral DNA. We modified the introns to contain the

kanR/neoR gene, as well as a transcription terminator in the reverse orientation. The advantage of

presence of the terminator would be lack of viral RNA production in the first infected cell, which

would result in inhibition at “the first round of infection”. However, due to very low frequency

of insertion, this part of study could not be continued.

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4.2. Future Directions

Gene therapy holds promise for prevention of HIV infection and control of established

infection. However, there were obstacles that made it progress slowly. The hematopoietic stem

cells that produce CD4+ cells needed to be identified and purified. Culture, transduction, and

induction of modified stem cells to the progeny cells must be established. Appropriate vectors

were needed to be developed with the following specifications: efficient and easy production,

safety, long-term and regulated gene expression, and specific cell targeting. It has been shown

that lentiviral vectors may cause gene silencing when the transduced cells are being cultured for

longer times. Also, integration of lentiviral vectors into various DNA sequences of the

mammalian cells’ genome, where they may be close to a growth-promoting gene, can cause

pathological disorders. An example is induction of leukemia in children treated for severe

combined immunodeficiency using retroviral vector-mediated gene transfer299. And, at last, but

not least, a therapeutic gene that confers complete resistance to HIV-1 needed to be developed.

In this project, I targeted the cellular CCR5 receptor, whose downregulation is strongly believed

to produce the most effective barrier against HIV-1 entry. I used multimeric hammerhead

ribozyme, which is proved to be one of the most efficient therapeutic molecule used in anti-

HIV-1 gene therapies. To deliver the anti-CCR5 Rz1-7 to the cells, I also used and modified a

self-inactivating HIV-1-based vector. Another self-inactivating lentiviral vector, SIN-EF-G-I300,

which contains an insulator to inhibit gene silencing and oncogene activation, has also been

used in our laboratory for cloning the anti-CCR5 Rz1-7 to produce SIN-EF-G-I-Rz1-7 vector.

Anti-HIV-1 ribozymes, Rz1-9 and Rz1-14, which were previously developed in our laboratory26,28,

were also cloned in this vector to obtain SIN-EF-G-I-Rz1-9 and SIN-EF-G-I-Rz1-14 vectors.

These clonings were done by Masoud Ameli and Xue Zhong Ma in our laboratory. I have

149

obtained vector particles for these vectors, however the titers were low and, therefore, high

concentration vector particles will be produced again to be able to obtain an appropriate

transduction frequency. Further experiments with three new vectors on hematopoietic stem cells

will be done in collaboration with Dr. J.J. Rossi (Division of Molecular Biology, City of Hope

National Medical Center, Duarte, CA, USA) and Dr. R. Akkina (Department of Pathology,

Colorado State University, CO, USA).

Considering the possibility that mutant HIV-1 strains may acquire the ability to infect the cells

with downregulated CCR5 via other co-receptors, a combination strategy has been thought as a

back-up. This would combine anti-CCR5 and anti-HIV-1 multimeric hammerhead ribozymes in

the SIN-EF-G-I vector. By this means, if the virus passes the first barrier at the level of entry and

enters the cell, the genomic or transcribed RNAs will be cleaved by the anti-HIV-1 ribozymes.

Cells will be transduced with the SIN-EF-G-I-Rz1-7-Rz1-9 vector particles and will be examined

for ribozymes expression and inhibition of viral entry and progeny virus production. The vector

particles will also be sent to our collaborators for further tests on hematopoietic stem cells.

In my second project, where inactivation of HIV-1 proviral DNA by modified group II introns

was tested, I showed that the intron insertion into the sense strand of the integrase domain of the

pol region could inhibit the HIV-1 infection. The inhibition of progeny virus replication from

the intron-inserted HIV-1 clones has been shown to occur in the second round of infection. It is

necessary, for gene therapy purposes, to inhibit the HIV-1 replication in the first round of

infection. Therefore, I also used another strategy, in which I modified the I54a and I2654a introns

that target the HIV-1 proviral DNA antisense strands at nts. 54 and 2654, respectively. The

modification included cloning the neoR gene and a mammalian transcription terminator in the

reverse orientation. When the intron inserts into the anti-sense strand of HIV-1 proviral DNA,

150

the terminator will be positioned in the forward orientation and will stop transcription.

Therefore, the transcription of HIV-1 RNA will be truncated, especially when intron integrates

after nt. 54, at the R region of the HIV LTR. However, I54a and I2654a intron-inserted HIV clones

could not be obtained due to the original low frequency of insertion at these sites. Further

modification to the intron’s EBS1, EBS2, and δ sequences, in order to obtain more sequence

match with the insertion site, may increase the insertion frequency.

Further modifications to the I4021s and I2654a introns were also done in our laboratory (by Masoud

Ameli and Zue Zhong Ma) to clone an anti-HIV-1 Rz1-926,28 into the introns. A ds-Red gene,

which expresses a red fluorescent protein, was cloned into the intron to facilitate sorting of

mammalian cells that allow intron insertion. Cloning the anti-CCR5 Rz1-7 into the introns is also

planned.

In the experiments reported in this thesis, intron insertions were allowed to take place in E. coli.

For future application of these introns in anti-HIV gene therapy, it is crucial to determine

whether intron expression and insertion can take place in mammalian cells. To this end, I have

recently shown that when 293T cells were co-transfected with I4021s and pBRR-HIV1(3805-

4178s)/Tet plasmids along with pCMV-T7Pol (a T7 RNA Polymerase-expressing plasmid)301, the

intron was expressed and inserted into the recipient plasmid in the mammalian cells. Experiments

are underway to determine the intron insertion frequency in mammalian cells. An ectopic I4021

intron insertion site was identified in the mammalian genome. Therefore, it will be ideal to

investigate whether intron insertion can take place in the chromosomal DNA in mammalian cells

that are co-transfected. If intron insertion at this site is detected, we will also determine whether

this takes place in all of the cells.

151

Intron insertion into the pHIV-1 clone in the co-transfected 293T cells with I4021s, pHIV-1 and

pCMV-T7Pol is planned. If successful, intron insertion into HIV-1 proviral DNA, which is

integrated into the cell line’s genome, will be investigated. However, for this purpose, the LtrA

protein will be modified to be codon-optimized and contain a nuclear localization signal, so that

it can be expressed in mammalian cells and be transported to the nucleus in its RNP form.

Following the successful demonstration of inhibition of HIV-1 replication through an intron

insertion approach, studies will be continued to determine the usefulness of this gene therapy

strategy in the inhibition of HIV-1 replication in transduced peripheral blood T lymphocytes

and in the progeny of transduced stem cells.

Inactivation of HIV-1 proviral DNA by introns has an advantage upon using retroviral or

lentiviral vectors for expressing other anti-HIV-1 genes: the Ll.LtrB group II intron RNP

recognizes a 12-16 base pairs on the target DNA site. Therefore, the insertion will be more

accurate compared to retroviral and lentiviral vectors, which usually integrate into the host

genome randomly and may cause unwanted gene disruption and malignancy.

The retrotransposition properties of group II introns are now being explored as potential tools in

genetic engineering and genomic analysis. The possibility of designing L. lactis group II intron

by modifying EBS–IBS and δ-δ’ regions to disrupt an unwanted or malfunction gene or to

introduce foreign sequences under the control of selected promoters raises a spectrum of

interesting applications in genetic engineering and gene therapy. In the future, efforts will have

to be focused on developing derivatives of the L. lactis mobile group II intron that can transfer a

wider variety of insert sequences without loss of repair efficiency. Moreover, development of

transfer and expression methods that allow for group II mobilization into mammalian

chromosomal DNA will be essential. The observation that L. lactis group II introns can mobilize

152

into plasmid DNA in mammalian cells is encouraging in this regard237. Moreover, the potential

utility of other group II introns and other mobile genetic elements for genetic repair should be

explored. Many mobile genetic elements exist naturally in the human genome302,303, suggesting

that if they can be controlled appropriately, they may become useful therapeutic agents for repair

of the mutant genetic instructions associated with many human maladies.

Although the development of HIV-1 gene therapy strategies from the laboratory design through

to the clinical trials, is an expensive process, the end result will be more beneficial. By the time

HIV-1 gene therapy strategies become a permanent solution, they can serve as a back-up to cope

with the current drug resistance or toxicity. In the future, when these therapies have been

developed, they can serve as the ultimate therapy in developed and developing countries.

153

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RNA AND DNA INACTIVATION STRATEGIES TO · PDF fileRNA AND DNA INACTIVATION STRATEGIES TO ON VIA tly in use for HIV/AIDS therapy, a number of gene therapy strategies have been designed

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