2. Adeno-associated virus

3. 1965

4. Family: Parvoviridae
Genus: Dependoparvovirus
Species: Adeno-associated virus
ss DNA virus
Require proteins of helper virus, adenovirus or herpes virus for replication
Genome:~4.7kb
22nm icosahedral nonenveloped virus

5. AAV genome

6. Rolling hairpin replication of adeno-associated virus
Figure 4 | Rolling hairpin replication of adeno-associated virus. For simplicity, secondary structures at the ends of the single-stranded adeno-associated virus (AAV) genome (called inverted terminal repeats (ITRs)) are shown as T junctions with complementary base pairing. Parental DNA is green and newly synthesized DNA is red. a  | The insert shows a schematic organization of the ITR, and blue circles indicate ITR regions that interact with the protein Rep (replication). Replication is initiated using the host replication machinery and the 3′-OH of the 3′  ITR as a primer. b,c  | Replication continues to the end of the genome, duplicating the 5ʹ-terminal ITR structure. d  | For AAV5, five Rep molecules bind to the 20‑base (GCTC)5 Rep-binding site (RBS) in the 3′  ITR and contact the Rep-binding element (RBE′) hairpin tip. e  | This binding is proposed to provoke a conformational change in the DNA at the palindromic terminal resolution site (trs), and to induce Rep-mediated cleavage at trs to generate a phosphotyrosine bond between Rep and the DNA. f  | Recruitment of the host replication machinery then allows replication of the 3ʹ-terminal structure (a step called terminal resolution). g  | Refolding of the ends generates structures resembling those that are present before replication initiation. h  | The end result is a fully replicated viral genome.
- Conversion of single-stranded to double stranded rAAV DNA is a limiting step
generation of double-stranded rAAV vectors
Second-strand synthesis is a rate limiting
step for efficient transduction by AAV
2013

7. Different tropism among AAV serotypes
AAV 1 – 12 primate serotypes
Different tropism among serotypes
Luciferase protein expression profile of AAV serotypes 1–9
100 days after intravenous injection of 1 × 10^11 particles of AAV1–9
RLU;relative light unit, luciferase assay
Zincarelli et al., 2008

8. Serotype
Tropism
AAV1
Muscle, heart, CNS
AAV2
In vitro, CNS
AAV5
Lung (airway, alveoli), eye, CNS
AAV6
Lung (airway), heart
AAV7
Muscle, liver
AAV8
Liver, muscle, eye, CNS
AAV9
Lung (alveoli), liver, muscle, heart, CNS
AAVrh10
Pleura, CNS

9. Different tropism among AAV serotypes

10. Various AAV receptors AAV2 receptor: heparan sulfate proteoglycan
coreceptor:integrin
AAV5 coreceptor: platelet derived growth factor receptor
AAV1 and 6 receptor:N-linked sialic acid
AAV3, 8, 9:Laminin receptor

11. Cell entry and trafficking
Nuclear-localization signals and phospholipase domain on capsid proteins
allow endosomal escape and nuclear targeting.
Molecular Therapy, 2008

12. Capsid engineering
Mosaic virions by capid DNA shuffling for multiple serotypes
Bi-specific antibody by fusing Fc regions of two different antibodies:
anti-AAV capsid antibody and anti-cell marker antibody
Biotinylating the capsid and streptavidin conjugate carrying growth factor
Attaching ligand to the capsid surface (flexible area) directly

13. Introduction of immunoglobulin G (IgG) binding domain
from Staphylococcus aureus into AAV capsid

14. AAV helper free system

15. AAV vector Replication deficient Insertion up to 3 kb
Long term gene expression
Broad range of infectivity
High titer
Superior biosafety rating
No require cell division
Mostly epichromosomal

16. Current AAV gene therapy
$ 20 million investment to Oxford startup gene therapy company “NightstaRx”
For choroidermia, an interited X-linked form of progressive blindness, caused by mutations to Rab-escort protein 1 (REP1)
AAV2 vector
6 patients had vision improvement and 12 patient is underway
If this is approved, $83,000~$110,000
= cost of treatment of rheumatoid arthritis
Voyager Therapeutics of Cambridge, MA
$45 million from investors Third Rock Venture
AAV 2
Dopa decarboxylase gene for Parkinson’s disease
Regeneron and Avalanche Biotechnologies Announce Collaboration to Develop Next-Generation Gene Therapy Products in Ophthalmology (From Wall Street Journal, 2014)

17. Preclinical trial
100 million engineered adeno-associated viruses
Five that were effective in penetrating the retina
Those able to penetrate the many cell layers of the retina are used to shuttle
a corrective gene into cells, in particular the light-sensitive photoreceptors and
the retinal pigment epithelium
“Building upon 14 years of research, we have now created a virus that you just
inject into the liquid vitreous humor inside the eye and it delivers genes to
a very difficult-to-reach population of delicate cells in a way that is surgically
non-invasive and safe. It’s a 15-minute procedure, and you can likely go home
that day.

18. Targeting the CNS Injection of AAV directly to the brain area
Obstacle of the blood-brain barrier by intravascular vector administration
- Clinical trials - intracranial injections that result primarily in localized
transduction due to limited vector dispersion in the brain parenchyma
an autosomal recessive[2] degenerative disorder that causes progressive damage to nerve cells in the brain. Canavan disease is one of the most common degenerative cerebral diseases of infancy.
Ojala et al., 2014

19. Safety assessment after clinical AAV gene therapy
Source: German Canzer Research Center DKFZ
AAV1-LPLS447X vector system (alipogene tiparvovec)
AAV1-LPLS447X has been developed for the treatment of patients suffering from lipoprotein lipase deficiency (LPLD)
Potential safe random nuclear integration profile and integrations in mitochondria  in five LPLD patients and in an associated mouse study
happen in naturally occurring double strand breaks in the host genome at low frequencies, recent reports in mice in which vector integration events led to oncogenesis, raised some concerns regarding the safety of AAV vectors
Random integration profile in the nuclear genome and hotspots in mitochondria
Nature medicine 2013

20. Clinical results following hepatic artery infusion of AAV2-Factor IX
Blood 2013

21. Strategies to overcome humoral immunity to AAV
Select subjects with low to undetectable anti-AAV Nab
Administer high vector doses
Use empty capsids to adsorb anti-AAV antibodies, thus allowing for vector transduction
Administer immunosupressior to prevent or eradicate humoral immune responses to AAV
Switch AAV serotype or engineer AAV capsids that are less susceptible to Nab
Use repeated plasma exchange cycles to adsorb immunoglubulins and therefore reduce the anti-AAV antibody titer
Use delivery techniques such as balloon catheters followed by saline flushing to isolate the target tissue from the systemic circulation to avoid vector dilution in blood and exposure to NAb

22. Schematic representation of AAV-based strategies for large gene transduction
Intermolecular concatemerization is exploited to transfer large genes in vivo by splitting a large gene expression cassette into halves (<5 kb in size), each contained in one of two
separate (dual) AAV vectors
EMBO 2013