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

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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)

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AAV

adenovirus

Adeno-associated viruses – AAV

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Infectious cycle of AAV

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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).

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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

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Ways of production of AAV vectors

- dependent on helper vector

- helper-vectors independent

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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

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Vectors in AAV helper-free system

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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

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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

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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

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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

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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

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Texts to read !