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Dr Agnieszka JaźwaProf. Józef Dulak
Department of Medical BiotechnologyFaculty of Biochemistry, Biophysics and Biotechnology
Room 3.025/3.07 Phone 664-63-75
Email: jozef.dulak@uj.edu.pl
16th November 2015
Viral vectorsPart III
Adeno-associated viral 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
3
Viral vectors
Integrating Non-integratingLentiviral -retroviral -AAV (limited)
Adenoviral HSV Baculoviral
Integration depends on:-LTR sequences and integrase (retroviruses) - ITR seqeuences and rep proteins (AAV)
4
AAV
adenovirus
Adeno-associated viruses – AAV
5
Infectious cycle of AAV
6
Adeno-associated viruses – AAV
Small, non-pathogenic single stranded DNA viruses.
There are at least 13 different serotypes of AAVs. The most common are AAV1 and AAV2 – 70-80% of human population is seropositive to this serotypes
For replication require additional genes delivered by other viruses (adenoviruses or herpes simplex viruses; also CMV and HPV)
Genome AAV – 4681 nucleotides, at both ends there are 145 nt-long ITR (inverted terminal repeats)
AAV infect both dividing and non-dividing cells
Entering into the cells is dependent on binding to the receptor – different serotypes use various receptors
AAV integrate into cellular geneome – in human this integrations is strictly specific, at chromosome 19 (AAVS1 – AAV integration site-1).
7
removal of rap and cap genes transgene insertion
AAV vectors
ITRITR
Verma & Weitzman, Ann Rev Biochem 2005
Essential and non-essential elementsin different viral vectors
ITR – necessary in cis – initiation of replication
- packaging signal - integration into genome
Scheme of the AAV genome
Ploquin et al. in : Advanced Textbook on Gene Transfer, Imperial College London, 2014
Rep78 & Rep 68 – DNA binding, helicase and endonuclease activity; essential for replication Rep52 & rep 40 - involved in packaging viral DNA
VP1-VP3 – structural viral proteinsAAP – assembly activating protein
10
Ways of production of AAV vectors
- dependent on helper vector
- helper-vectors independent
11
Construction of AAV vectors – system with helper adenoviral
packaging cells
co-infection with adenovirus
recombinant AAVadenovirus
capsid proteins
Difficulty in purification of AAVs from adenoviruses
12
Vectors in AAV helper-free system
13
Helper-free production of AAV vectors (2)
Strategies of production of AAV vectors
Rutkowski et al., Biotechnologia 2007
Packaging cells
Three vs two plasmid systems
Purification on density gradients
Helper-vector independent AAV production
Tomczyk M., Praca magisterska, WBBiB UJ, 2013
Media change(24 h)
Transfection
Collection of cells 72 h after
transfection
Preparation of lysates from collected cells
AAV vectors production
Purification of AAV vectors
Tomczyk M., Praca magisterska, WBBiB UJ, 2013
Ultracentrifugation in iodixanol gradient
Concentration of AAV preps
Tomczyk M., Praca magisterska, WBBiB UJ, 2013
10-times dilution of AAV preps
Centrifugation in MILLIPORE columns
Washing with PBS, collection
Titration of AAV vectors
Tomczyk M., Praca magisterska, WBBiB UJ, 2013
AAV vectors
Digestion with DNase I
Inactivation of DNase I
Digestion with proteinase K
Phenol:chloroform extraction
qPCR
Titer
cmr.asm.org
SYBR Green-based quantitative PCR (qPCR)
CMV Standard Curve
rAAV-CMV-Luc
Melting Curves
Titration of AAV vectors with qPCR
Tomczyk M., Praca magisterska, WBBiB UJ, 2013
22
Site-specific integration
• AAV integrates usually stably into a specific site on chromosome 19q13.3 (AAVS1)
• Integration region- AAVS1 (RBS,TRS)
• Rep78 and Rep68 bind to a 109 bp DNA fragment near AAVS1 and can mediate complex formation (DNA of chromosome 19 and AAV harpin DNA)
• Viral DNA replication within AAVS1 are likely involvedin site-specific integration;
AAV2 virus has been associated with oncogenic insertional mutagenesis in human HCC
Hepatocellular carcinomas (HCCs) are liver tumors related to various etiologies, including alcohol intake and infection with hepatitis B (HBV) or C (HCV) virus.
Clonal integration of AAV2 was found in 11 of 193 HCCs. These AAV2 integrations occurred in known cancer driver genes, namely CCNA2(cyclin A2; four cases), TERT (telomerase reverse transcriptase; one case), CCNE1 (cyclin E1; three cases), TNFSF10 (tumor necrosis factor superfamily member 10; two cases) and KMT2B (lysine-specific methyltransferase 2B; one case), leading to overexpression of the target genes.
Nault et al., Nat Genet, 2015
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AAV vectors features- due to the lack of Rep68 and Rep78 the specific integrationinto chromosome 19 is lost
- unspecific integration (low efficacy, about 5-10%) - this unspecific integration preferentially occurs at the
transcriptionally active genes. However, there is doubt whether the integration can be oncogenic
- episomal expression
- however, because of non-immunogenic nature the episomal expression in non-dividing cells can be long-term
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AAV vectors, in contrast to adenoviral, can provide long-term expression
Champion et al., Circulation 2003
Mouse heart
Vasculogenesis
EC
P
EndothelialProgenitor
Cell
Capillaryblood vessel
Angiogenesis
EC
P
EC
EC
P
EC
P
Capillaryblood vessel Network of capillaries
VEGF
Arteriogenesis
EC
P
EC
SMCSMC
SMCSMC
F
F
Primaryblood vessel
Mature artery
increased blood flowVEGF + PDGFVEGF + Ang-1
EC – endothelial cellP – pericyteF – fibroblastVEGF – vascular endothelial
growth factorPDGF – platelet-derived
growth factorAng-1 – angiopoetin-1
Mechanisms of blood vessels formation –role of VEGF
Long-term VEGF-A expression from AAV2 vector promotes aberrant angiogenesis and fibrosis in skeletal muscle
Karvinen et al., Gene Ther, 2011
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How to deal with a small capacity of AAV vectors?
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AAV- concatamerisation
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AAV serotypes
13 serotypes are known
AAV-2 serotype is the most commonly used
Different serotypes can employ various receptors to enter the cells
- AAV-2: heparan sulphate αVβ5 integrin FGFR-1; HGFR
AAV3 - HSPG- AAV-1,4,5 & 6– sialic acid (5-acetylneuraminic acid)- AAV-5 co-receptor: PDGF-B receptor- AAV8 – LamR - AAV9 – N-linked galactose residues
Glycan structures bound by AAV serotypes
Mietzsch et al., J Virol, 2014
A CB
FED
IHG
A CB
FED
IHG
Transduction efficacy of AAV2 vectorsdepends on cell type
HEK 293
HeLa
HaCaT
Not transduced 1000 MOI 10 000 MOI
Jaźwa A., Praca doktorska, WBBiB UJ, 2008
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Endothelial
Bronchial epithelial
Vascular smooth muscle
Skeletal muscle
Different transduction efficiency of AAV-2 viral vectors
Cells physiologically refractory to AAVs:1. endothelial Cells2. fibroblasts3. various types of stem cells
C) mosaic capsid
19/1 3/1 1/1 1/3 1/19
Different ratios of plasmids
Rep1 Cap1 Rep2 Cap2
B) bi-specific antibody
cell-surfacereceptor (for e.g.endothelial cell)
A) transcapsidation
AAV95’
transgene
3’AAV2
ITR
ITR Rep Cap
AAV2/9
D) chimeric capsid
5’
Rep Cap
3’
ITR
ITR
SIGYPLP
AAVsig
Methods enhancing the effectiveness of AAV vectors
Jazwa et al.,, Curr Gene Therapy, 2007
35Su et al., Gene Therapy 2006
AAV1 provides earlier and higher expression in the heart than AAV2
1 day
14 days
14 days
Normal heart
Ischemic heart-expression in myocytes around the scar
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AAV8 provides higher expression in the rat heart
Palomeque et al.. Gene Therapy 2007
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AAV9 is even more effective than AAV8 even at lower doses
systemic delivery
Ianagaki et al., Mol Therapy 2006
CA Pacak & BJ Byrne , Mol Ther 2011
AAVs provide long term expression in cardiac cells
AAV serotype 9 heart transduction
AAV serotype 9 (AAV2/9) vectors coding forluciferase gene (AAV2/9-CMV-Luc) were delivered into the jugular vein (sytemic injection)
AAV serotype 9 efficiently and specifically transduces the hart following systemic injection to the circulatory system via the jugular vein
Pacak et al, Cardiovasc Res, 2006
Bioluminescence as a tool to evaluate the transduction efficiency and establish the safe and efficient dose of the vector
AAV9-CMV-Luc(5 x 10^10 viral genomes/mouse)
AAV9-CMV-Luc(5 x 10^11 viral genomes/mouse)
IVIS Lumina (7 days post-transduction)
PBheart
liver
lungsfatskeletal muscle
kidney
bones(hind limb)
spleenovaries
kidney
2 min exposure time
2 min exposure time
2 min exposure time
AAV9-CMV-Luc(1 x 10^11 viral genomes/mouse)
Jaźwa & Tomczyk, unpublished data
Liver enzymes are increased following AAV9 transduction
Therefore, the cardiotropic properties of AAV9 appear to be undermined.
Spotchem(Automated
Dry ChemistrySystem)
Jaźwa & Tomczyk, unpublished data
In humans, only 1.5% to 2% of the total genome codes for proteins.
BUT a large amount of it (about 98%) is transcribed.
The missing link in the human genome
How can the disparity between the number of sequences transcribed andtranslated be explained?
the RNA which is an end in itself
e.g.: long intergenic ncRNAs long intronic ncRNAs telomeric ncRNAs transcribed from subtelomeric promoters (telomeric repeat-containing RNA) Pseudogene transcripts circular RNAs enhancer RNAs transcribed ultraconserved regions
Non-coding RNAs (ncRNAs)
small ncRNAs(sncRNAs)
long ncRNAs(lncRNAs)
e.g.:miRNAs (18-25 nt) siRNAs Piwi-interacting RNAs (piRNAs) small nuclear RNAs (snRNAs) small nucleolar RNAs (snoRNAs) promoter-associated small RNAs
after Kumarswamy & Thum, Circ Res, 2013
Non-Coding RNAs
(18-200 nt) (200 nt-100 kb)
Role of miRNAs in regulation of gene expression
K. Goljanek –Whysall et al., Clinical Science 2012
Endogenous miRNAs can be exploited to regulate transgene expression according to tissue
Brown & Naldini, Nature Reviews Genetics, 2009
Skeletal muscle- and liver-specific silencing of m206TS-3G– and miR-122TS–regulated AAV9 vectors
Geisler et al., Molecular Ther, 2013
Important rate-limiting step for transduction
Improving the efficacy of AAV vectors
McCarty et al., Gene Ther. 2003
RBE – Rep-binding element trs – terminal resolution site
!
Self complementary AAV
In scAAV one of the ITR sequences has been devoid of TRS (terminal resolution site), what eliminated the site of cutting for Rep protein (which has an activity of endonuclease). In this way to the capsid is packed ds DNA
McCarty et al., Gene Ther. 2003
Transduction efficiency of ssAAV and scAAV vectors in human fibroblast cells (10000 vector particles/cell)
Han Z. et al. Mol Genet Metab. 2008 April 93(4): 381-387
Self complementary AAV
scAAV vs ssAAV
Transgene expression evenly distributed among hepatocytes throughout the liver Transduce muscle cells 10 – 15 timesmore efficiently Greater saturation of transduced neuronal cells within limited area Transduce 2500-fold more retinal cells per particle at 5 weeks after infection Threefold increase in transduction after single infection in bone marrow-derived dendritic cells
There are, however, cell types that do not show improved transduction: polarized airway epithelial cell, primary B-cell chronic lypmhocytic leukemia cells Smaller capacity: only ~2,2 kb
Advantages Limitations
51
Features of AAV vectorsAdvantages
1. Long term expression 2. High efficiency of transduction of many cell types3. Non-pathogenic viruses. Low risk of cellular immune response, which is
additionaly limited by removal of viral sequences
Limitations
1. Unspecific integration 2. Small capacity – max. 5 kb in ssAAV (and only ~2,2 kb in scAAV)3. Low efficiency of transduction of certain cell types – targeting might be
required4. Difficulty of production in sufficient titer for in vivo work 5. Risk of humoral immunity: antibodies detect capsid proteins
Anti-AAV neutralising antibodies in human population against various AAV serotypes
MA Kotterman & DV Schaffer, Nature Reviews Genetics, 2014
53
Application of AAV in clinical gene therapy
1. Nervous system diseases – Canvan disease2. Cystic fibrosis 3. Haemophilia – transfer of factor IX 4. Muscular dystrophy5. Leber’s congenital amaurosis (blindness)6. Cardiovascular diseases
54Nature Genetics,
Key features of viral vectors
retroviruses
adenoviruses
AAV
naked DNA
liposomes
8 years ago...
Types of vectors used in clinical trials of gene therapy
2014 2015
Increasing use of AAV vectors
58
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