L4. Viral Vectors_student Copy

8/8/2019 L4. Viral Vectors_student Copy

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L4. Viral Vectors

Junghae Suh, Ph.D.

BIOE 422/522Fall 2010


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Advantages of Viral Vectors

High efficiency- nature engineered

Many can transduce non-dividing cells as well Transduction- viral, transfection- nonviral

Can manipulate precisely w/ genetic engineering


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Viral Vectors

(Davidson & Breakefield, Nat Rev Neuro, 2003)


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Retrovirus Structure

100nm diameter

Lipid envelope + protiencapsid

Moloney murine

leukemia virus (MoMLV)

Reverse transcriptase Where virus gets name

Carries out RNA-

dependent DNA synthesis

Error-prone high

mutagenesis rate

Cool fact: Howard Temin &

David Baltimore received

Nobel Prize in Physiology

& Medicine in 1975 for

discovering RT

(Principles of Virology, Flint et al.)


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Retrovirus Genome

ssRNA genome- 10kb

3 genes: Gag-group specific antigens (core

protiens)

Pol: reverse transcriptase

Env: viral envelope protiens

LTR: long terminal repeats Needed for integration of genome intochromosome

Promotoer and enhancer function

Polyadenylation site for viral mRNA

Integrates randomly into

chromosomes Insertional mutagenesis

LTRs may activateprotooncogene contribute tocancer



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Retrovirus Production

Producer cells based on293 (aka phoenix cells)

Cells express gag-pol & env Proteins provided in trans

Transfect producer cellswith plasmid encoding geneof interest Gene placed between 2 ltrs

Contains packaging signal

LTR and packaging signal arecis-acting

Use nonviral transfection

Retroviruses bud off ofproducer cells

(www.stanford.edu/group/nolan/retroviral_systems/phx.html)


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Retrovirus

3,151 publications

Peaked in 2000

Decreasing

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Adenovirus Structure

80-120 nm in diameter

No lipid envelope, only protien capsid Many serotypes exist

Infects dividing and non-dividing cells

(retroviruses only infect dividing cells)

Does not integrate into chromosomes

High immunogenicity (causes disease) Causes diarrhea

Causes 10% of upper respiratory

infections

Cool fact: virus isolated from adenoid

tissue in 1953; found to cause upper

respiratory infections in WWII soldiers



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Adenovirus Vectors

(Danthinne & Imperiale, Gene Ther, 2000)

E1 protiens are strong

activators, can be provided intrans 293 cells (already have E1 in them)

E3 proteins to fight host celldefenses, not needed for virus

production in vitro

1st generation- highimmunogenicity in vivo b/c viralprotein production

2nd generation- packaging cellsprovide additional viral protiensin trans.

Gutted vectors (helper-dependent vectors) only containcis-acting elements ITR

Packaging signal

Needs replication-competent Ad Co-infection


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Insert Gene of Interest (GOI) into Adenovirus?

Clone GOI into Ad genome Problematic b/c genome is very large, few restriction sites

Clone GOI into shuttle plasmid, then move into AD

genome using homologous recombination

Use homologous recombination in packaging cells to

move GOI into Ad genome- time consuming

Use double-recombination in E.coli- much faster Stratagene AdEasy system- insert 7.5 kb into Ad genome


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Adenovirus Production

(Stratagene AdEasy system for producing generation 1 Ad vectors)


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(Stratagene AdEasy system)


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(Stratagene AdEasy system)


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Adenovirus

11,286 publications

Peaked in ~2003

Decreasing

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(Xie,Qin

ge

tal.,

PNAS

2002

)

Adeno-Associated Virus (AAV)

25 nm in diameter

No lipid envelope, only protein capsid Many serotypes exist (species for viruses) Can transduce dividing and non-dividing

cells Cannot replicate by itself

Dependovirus Requires adenovirus of herpes simplex

Mainly exists episomally in circular forms,low chance of random insertion, wildtypeintegrates site specifically into chromosome19

No known pathology Immunogenicity Cool fact: discovered as contaminant of

adenovirus stock


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AAV Genome

ssDNA genome- 4.7 kb 2 genes- rep & cap

Limited cargo capacity- 4.5 kb

Replace both viral protiens w/ GOI GOI between ITFs

Irts- packaging siganl

Provide rep and cap in trans

(Principles of Virology, Flint et al.) (Robbins et al., TIBTech, 1998)


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AAV Production

Ad HelperProteins

AAV.lacZ

Rep - Cap

CaPO4Transfection

293T Cell

LysisSeparationPurification

AAV particles

Triple transfection method

1 plasmid- GOI between

ITRs

1 plasmid- rep and cap

1 plasmid- adenovirus

proteins

230 cells- have E1 already


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AAV Production

(Xiao et al., J Virol, 1998)


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AAV Production

(Stratagene AAV Helper-Free system)


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AAV Production

Stratagene AAV

Helper-Free system


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AAV Production


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Adeno-associated Virus

2,946 publications

Reached plateau

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Oncolytic Adenovirus

Virotherapy: use replication

and lytic properties of AD for

therapy, not delivering

therapeutic genes

Changes in tumor allows forselective virus replication

Oncolysis of tumor cells

allows spread of virus.


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ONYX-015 virus

2 mutations in E1B-55 kDa protein E1B-55k normal binds and inactivates p54

Ricus defective in E1B-55k requires cells w/ low p53 function to

replicate Other factors involved

Replicates selectively in tumor cells

Approved for head and neck cancer in China

Shanghai Sunway Biotech Co. Ltd.


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380 publications

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