Prof. Józef Dulak Department of Medical Biotechnology Faculty of Biochemistry, Biophysics and Biotechnology Room 3.025/3.07 Phone 664-63-75 Email: [email protected] 9th November 2015 Viral vectors Part II Adenoviral vectors
Prof. Józef Dulak Department of Medical Biotechnology
Faculty of Biochemistry, Biophysics and BiotechnologyRoom 3.025/3.07 Phone 664-63-75
Email: [email protected]
9th November 2015
Viral vectorsPart II
Adenoviral vectors
2
Vectors
Non-viral/plasmids Viral
RNA DNA Retroviruses(including lentiviruses)
AdenoviralAAV Herpes
„naked” DNA
Lipoplexes
Viroplexes(lipoplexes enriched In specific viral proteins)
complexes withother chemicals
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Viral vectors
Integrating Non-integratingLentiviral -retroviral -AAV (limited)
AdenoviralHSV Baculoviral
Integration depends on:-LTR sequences and integrase (retroviruses) - ITR seqeuences and rep proteins (AAV)
Adenoviruses
• First isolated from adenoidal cells in 1957 (searching for viruses causing common cold)
• Medium-sized, non enveloped DNA viruses (70-90 nm in diameter)
• Classified in five genera with all human AdV serotypes belonging to the genusMastadenovirus
• HAdV are further grouped within species Human mastadenovirus A to G, basedon the phylogeney, genome organisation, G+C content, hemagglutinationpattern and other biological properties
• At present, 56 distinct serotypes belonging to HAdV A to G have been described
• Infection and diseases of different organs
• HAdV infection poses a risk for immune compromised individuals; infections aremostly subclinical in immunocompromised subjects
Based on Alonso-Padila et al., Hum Gene Ther Oct 2015
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Adenoviral serotypes and disorders caused by them
• Incubation period – 2-14 days
• Majority in the first 5 years of life; peak around 2 years
• Common – 5-15% of upperrespiratory tract and approx. 15% of lower respiratory tractinfections during childhood
• During winter time usually
• Generally mild and self-limiting
• Primary adenovirus infectionresults in production of neutralisingantibodies, which are thoughtto confer life long immunity
Based on M. Tebruegge & N. Curtis, Pediatr Infect Dis Journal 31: 626-627, 2012
G
X
56 (57) subtypes
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Adenoviruses
Adenovirions are icosahedral in shape, 70–90 nm in diameter and not enveloped. The viral genome is large, consisting of a double-stranded DNA molecule of 36–38 kb in size.
Hexon protein • The most abundant• Contains hypervariable
regions which areserotype specific
• Are considered as major immune determinants
Fiber protein • The main determinant of
serotype tropism
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Adenoviruses
Genom: 36 kbp, more than 50 proteinsE1 region– contains genes regulating the expression of genes
necessary for viral replication
E2 i E4 regions – together with E1 are required for viralreplication
E3 region– is not required for replication, modulates response of cell to infections
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Binding and internalization of adenovirus
Type 2 & 5 are mostly used in gene therapy
Cellular uptake mechanisms of Ad5 adenoviruses
AC Bradshaw, A Baker, Vascular Pharmacology 2013
10Waddington & Baker, 2008
In vivo infection with Ad5 adenoviruses
importance of coagulation factor X (FX) in adenovirus(Ad) serotype 5-mediated livertransduction in vivo. FX binds to the adenovirushexonhypervariableregions (HVRs).
Some human Ad bind to CD46 or CD80/86
CD46 – inhibitory complement receptorCD80/CD86 – on B cells and antigen
presenting cells
11Verma & Weitzman, Ann Rev Biochem 2005
Essential and non-essential elements in different viral vectors
11 structural proteins in the adenowirus virion: - 7 form the capsid- 4 are packed with the DNA in the core
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Adenoviruses and adenoviral vectors• Genome consists a 36 kb double-stranded linear DNA with ITR sequences at
each end, with:
Early genes (responsible for viral gene transcription, DNA replication, host immune suppression and host cell apoptosis
Late genes (coding proteins required for virus assembly)
• E1 early gene is essential for the subsequent adenoviral gene expression
transgene
region E1Adenoviral DNA
DNA of 1st generation vector
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Production of DE1 adenoviral vectors
transgeneITR ITR
293 cells (stably transfected with DE1 region)
E1 E1 E1 E1 E1
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Homologic recombination inHEK 293 packaging cells
Packaging cell
HEK 293
Shuttle vector
transgene
Risk of generation of replication active viruses
Construction of adenoviralvectors of 1st generation by homologous recombination
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Production of adenoviral vectors withouthomologous recombination (1)
E1 genes are deleted from adenoviral genomeHEK 293 cells provide in trans the required E1 genes
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Production of adenoviral vectors withouthomologous recombination (2)
Swa I digestion – to reduce the frequency of non-recombinant clones
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Production of adenoviral vectors withouthomologous recombination (3)
ΨITR ITR
ΨITR ITR
E2E1 E3
E4
L1 L2 L3 L4 L5
Adenoviral genome
ΨITR ITR
ΨITR ITR
± ∆E3(3.1 kb)
∆E1(3.2 kb)
± ∆E2b(1.6 kb)
± ∆E2a(1.8 kb)
± ∆E4(2.8 kb)
∆E1(3.2 kb)
± ∆E3(3.1 kb)
AdV
1st generation
2nd generation
Capacity≈ 8.2 kb
≈ 14 kb
≈ 37 kb
Generations of adenoviral vectors
Stopa et al., Biotechnologia 2007
3rd Generation
„gutless vectors”Helper-dependant AdV
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1. Many types of cells can be transduced depending on the route of administration. Infection by adenovirus is not cell-cycle dependent
2. Direct injection into the peritoneum, kidney, pancreas, cerebral spinal fluid, skeletal muscle, brain, cardiac muscle, coronary artery and many other tissues, results in transgene expression.
3. However, intravenous injection into rodents results primarily in transgeneexpression in the liver and spleen.
4. Following infection, target gene is transiently expressed at high levels since many cells receive multiple copies of the recombinant genome.
5. Typically, transgene expression that results from the first generation of adenoviral vectors is transient regardless of the route of administration and the type of cells. However persistent expression in non-dividing cells has been observed in vivo
5. Expression usually peaks in 1–7 days and declines rapidly to an undetectable level by 2–4 weeks
Transduction with adenoviral vectors
0
20
40
60
80
100
B16 HaCaT NIH 3T3 HMEC-1 HUVEC COS-7liczb
a tr
ansd
ukow
anyc
hko
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B16 HaCaT NIH 3T3
HMEC-1 HUVEC COS-7
Various cells are transduced with different efficacy with Ad
Stopa et al., Biotechnologia 2007
100 MOI
1 MOI
HaCaTCOS-7
10 MOI
0,1 MOI
100 MOI
1 MOI
10 MOI
0,1 MOI
Increasing the titer of a vector improves the transduction efficacy
Stopa et al., Biotechnologia 2007
MOI – multiplicity of infection(infection units/ml)
# of positive cellsDdilution x V
= titer (IU/ml)
V- volume of virus dilutionadded to cells
0
500
1000
1500
2000
2500
3000
3500
4000
HaCaT HMEC-1 COS-7 HUVEC
IL-6
[pg/
ml]
kontrola
10 MOI
100 MOI
##
#
#
#
Proinflammatory effect of adenoviral vectors
Stopa et al., Biotechnologia 2007
Mechanisms of cytokine induction by Ad vectors
In splenic macrophages
GR Nemerow,Mol Ther. 2009 Sep; 17(9): 1490–1491.
• Activation of TLR9 (in dendritic cells)- production of IFN-1α
• Activation of NLPR3 inflammasomein macrophages & triggering secretionof IL-1β
• after systemic injection – Kupffer cells• Activation of splenic macrophages
(figure & description)
Ad penton base interaction with integrin αvβ3 promotes virus internalization into early endosomes. (2) Partialdisassembly of the virion in the low-pH environment of the endosome allows release of the viral membrane lytic protein that disrupts the lipid bilayer. (3) Membrane disruption acts asa signal for increased transcription of pro-interleukin-1αmessenger RNA (pro-IL-1α mRNA) as well as increased cytokine protein production. (4) Proteolytic cleavage of pro-IL-1α by neutralproteases results in nuclear localization of the N-terminal (NTP) fragment and secretion of the mature IL-1α protein. (5) binding of IL-1α to its receptor (IL-1R1) induces further signaling thatproduces a defined set of cytokines and chemokines
24Elkon et al. 1997. Proc Natl Acad Sci USA 94: 9814-9819, Jay et al. 1999. Blood 94: 3968-3975.
Adenoviral vectors of first generationcan induce inflammatory reaction in liver
Ad-injection control
control
low dose Ad
high dose Ad
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Short expression of transgene after adenoviral gene transfer
Serotype change – does not help much
Consequences of proinflammatory response induced by the Ad vectors
• Limited usefullness to the clinic, therefore the majority of clinical studies uselocal rather than systemic delivery (eg. directly into the tumor)
Disadvantage of local injection:
• suboptimal vector spread• vector does not reach distant, migrating tumor cells
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ITRNecessary for production of a transgene ITRTherapeutic gene
Ψ sequence
Stuffer (intron)Or the whole transgene
Helper-dependent adenoviral vectors
Gutless vectors
DNA recognition site for recombinase enzymes. The DNA recombinases have a similar basic recognition site, as shown here for the Cre enzyme. Two palindromic sequences (loxP sites for the enzyme Cre) are separated by a DNA core. The core sequence can vary, whereas the palindromic sequences must contain a subset of the nucleotides shown to support integration.
Gorman, Curr Opinion Biotech 2000
Use of Cre recombinase for conditional knockoutsRecombinase from P1 bacteriophage
Cre recombinase mediated deletion
ADS Ryding et al., J Endocrinol 2001
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Cre recombinase – to manipulate genes
Recombinase from P1 bacterophage
Therapeutic gene
Gutless vector
Packaging
293 Cre cells
Helper vectorψ+
ψ-
ψ+
ψ+
Packaging of gutless vectors
loxP
Weeks after Treatment0 1 2 3 4 5 6 7
0
200
400
600
800
1000
2010
First generationGutlessControl
*
*
Gutless vectors are less inflammatory than 1st generationadenoviral vectors
Kim (Jozkowicz) et al. PNAS 2001
Gutless vectortransgene ITRstufferITR
First generation vectortransgene ITRITR
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Role of apolipoprotein E (ApoE) protein
Produced mainly by hepatocytes (>70%)
Major structural component of mammalian lipoproteins: chylomicronremnants, VLDL, IDL and HDL
Ligand of mediates their cellular uptake of chylomicrons, VLDL, IDL andHDL by interaction with VLDL-R, LDL-R, and LRP
Decreased apoE activity is associated with type III hypercholesterolemia and accelerated atherosclerosis
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En face preparations of oil red O stained aortas from C57BL6/J and ApoE KO mice at 26 weeks of age (from Behr-Roussel et. Al, 2006).
The Apolipoprotein E knockout mouse model is one of the most widely used experimentalmodel of atherosclerosis. These mice rapidly develop atherosclerotic lesions that resemblehuman lesions evolving over time from initial fatty streaks to complex lesions.
PATHOPHYSIOLOGICAL FEATURESCardiovascular features:•Apolipoprotein E deficiency directly results in the increase of plasma levels of LDL andVLDL.• Very high cholesterol level (500-600 mg/dl instead of 50-100 mg/dl)•Spontaneous development of atherosclerotic lesions throughout the arterial tree appearingfirst in the aortic arch in young mice and progressing in the thoracic and abdominal aorta inolder mice
Apolipoprotein E knockout mouse
Maeda et al., Atherosclerosis 2007
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HdAd-gen-ApoE (33.4 kb)
ITR ψ+Genomic ApoE
LSE
HdAd-PEPCK-apoE (34.3 kb)ITR ψ+ ITR
PEPCKpromoter
ApoE cDNA
hGH polyAAI Intron
ITR
Structure of Adenoviral Vectors
Kim (Jozkowicz) et al. PNAS 2001
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Gutless adenovirus
Cell Culture
ApoE-/- mouseFed a regular diet
Expression of mRNA and proteinPlasma apoEPlasma CholesterolLipoprotein ProfileALT and AST levelsAtherosclerosis development
Scheme of experiment
Kim (Jozkowicz) et al. PNAS 2001
Effect of single injection with apoE gutless vector
0 112 224 336 448 560 627 784Plas
ma
Apo
E[m
g/dl
]
2
4
6
8
10
0
AdE1-apoEHdAd-PEPCK-apoEHdAd-gen-apoE
Days after injection
HdAd-gen-apoEapoE-control
Kim (Jozkowicz) et al. PNAS 2001
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Very high transduction efficiency
Broad host species and cell type range
Can transduce mitotic and post-mitotic cells
Can harbor ~ 35 kb (!) of transgene
Do not integrate with genome
Do not produce any viral proteins
Show significantly reduced immunogenicity in vivo (combinationwith tissue specific expression may be helpful)
Drawback: Difficult for producing in high titers Capsid proteins still induce inflammatory response (can be
partially circumvented by pharmacologicial immunosuppression
Helper-dependent adenoviral vectors
Ad5The majority of clinical trials employing adenoviruses utilise vectors based on adenovirus serotype 5 (Ad5) of class C, that has been well-characterised.
PROBLEMS with Ad5:
• The CAR-mediated interaction of the Ad5 fiber knob domain with circulating erythrocytes -> attenuates virus bioavailability
• capsids can be bound by platelets -> platelet agglutination and sequestration by the liver
• high frequency in human population -> neutralising antibodies inhibtAdV-5 transductionn efficacy, linked to the capsids protein composition
• activation of the innate immune response by Ad5 -> strong inflammatory response and the clearance of the virus from the circulation, linked to the capsids protein composition
• hepatotoxcity
Based on: AC Bradshaw, AH Baker , Vasc Pharmacol 58: 174-181, 2013
Rare Ad
At present 56 distinct serotypes belonging to human-AdV (classes A to G) have been described.
Rare and nonhuman serotype vectors exhibit low seroprevalence, frequently possessing the added advantage of increased transduction efficacy of cells that do not express CAR due to their alternate receptor usage
• the first non-AdV-C serotype to be constructed was E1a-deleted HAdV-7 (HAdV-B)• HAdV-3 and HAdV-35 have been tested in human subjects as oncolytic vector• AdV-35 and AdV-26 have been tested in several phase 1 clinical trials as candidate
vaccine component against Mycobacterium tuberculosis, Plasmodium falciparum, HIV, and Ebola
• An alternate strategy for improving vascular cell gene transfer is the use of rare or nonhuman serotype adenoviruses.
Introduction of the HAdV-5 derived ORF6 protein into the viral backbone of the selected serotype allowed efficient production of non-HAdV-5 replication-deficient vector using existing HAdV-5 E1-complementing cells such as HEK2
Based on: AC Bradshaw, AH Baker , Vasc Pharmacol 58: 174-181, 2013
Pseudotyped Ad
Ad5 vectors can be pseudotyped with fibers from rare serotype adenoviruses, which confers high-affinity binding to receptors other than CAR (CD46 and desmoglein 2). In conjunction with the substitution of Ad5 hexon hyper viarableregions with the HVR regions of rare serotype adenoviruses => can lead to almost complete evasion of anti-Ad5 neutralising antibodies
Ad35 fiber show affinity to CD46 – Ad5 pseudotyped with this fibre posses high affinity to endothelial cells and smooth muscle cells
Neutralization bypass strategy was not achieved when the HAdV-5 fiber knob domain was swapped using a knob domain from a different AdV serotype
For instance, ex vivo transduction of human airwayepithelium was significantly improved with HAdV-5 vectors pseudotyped with the fiber protein derived fromHAdV-35 (HAdV-5F35
Based on: AC Bradshaw, AH Baker , Vasc Pharmacol 58: 174-181, 2013
The fiber protein is known to be the main determinant of serotype tropism
• Ad-5 fiber binds to CAR• Ad-B binds to CD46 or desmoglein• Ad-D binds to sialic acid
Engineering Ad5
Incorporation of new targeting peptides such as RGD4C, smooth muscle cell targeting peptides into the fiber HI loop enhanced adenovirus uptake into vascular smooth muscle cells and intact vein graft tissue
Combining the SIGYPLP vascular-targeted peptide insertion with fiber mutations that block CAR binding resulted in further enhancement of vascularendothelial cell gene transfer.
Based on: AC Bradshaw, AH Baker , Vasc Pharmacol 58: 174-181, 2013
Adenoviral vector retargeting
Based on: AC Bradshaw, AH Baker , Vasc Pharmacol 58: 174-181, 2013
Adenovirus to target cancer cells– oncolytic adenoviruses
S. Kochanek. in : Advanced Textbook on Gene Transfer, Imperial College London, 2014
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General features of adenoviral vectorsCause benign infection
Well characterized and easily manipulated
Stability and high titers of recombinant vectors
Ability to infect a broad range of cell types, including dividing and nondividing cells
High efficiency of cellular uptake of insert capacity (up to 37 kb; ~ 7kb for first generation vector)
Little risk of random chromosomal integration
Safety—lack of association with oncogenicity
Very imunogenic
systemic inflammatory response…
Adenoviral vectorsVery efficient, transduce many cell types, provide high level of expression
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Adenoviral vectors of the first generationGreat:
• Very high transduction efficiency• Broad host and cell type ranges• Can be prepared in high titers• Can transduce mitotic and post-mitotic cells• Do not integrate with genome• Can harbor ~ 7 kb of transgene
But:• Strong immune response to viral proteins eliminate virally transduced cells
within 30 days• Neutralizing antibody response prevents readministration of adenovirus
vector of the same serotype
Thus:Adenoviral vectors provide the high but transient (<4 weeks) transgene expression
Ornithine transcarbamylase deficiency – gene therapy
Jesse Gelsinger1999
It has to be stressed that this tragic event was an isolated case of seriousunwanted side effects of adenoviral vectors.
Adenoviral vectors are now the most commonly used vectors in gene therapyclinical trials
retroviruses
adenoviruses
AAV
naked DNA
liposomes
10 years ago...
Types of vectors used in clinical trials of gene therapy
2014 2015
Texts to read !
Gene. 2013 Aug 10;525(2):162-9. doi: 10.1016/j.gene.2013.03.137.
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Application of adenoviral vectors in gene therapy
• Gene therapy of inborn errors in metabolism – lack of OCT
• Gene therapy of monogenic diseases – cystic fibrosis
• Gene therapy of cardiovascular diseases – transfer of angiogenic genes
• Gene therapy of cancer – it is possible that toxicity and immongenicitywill enhance the therapeutic effectiveness
- oncolytic adenoviral vectors- suicide gene therapy
Exam – planned on 1st February (Monday), room D107, 1 pm