Cours AAV 2005.PPT [Lecture seule]

Thomas M. (Thomas Matthew) St. John

Description

Adeno-associated virus1Initial discovery196521. AAV biology3Adeno-Associated Virus- Classification : parvovirus – Dependovirus- Defective virus = Productive infection requires host cell factors and the presence of a helper virus (adenovirus, herpes virus, papillomavirus)-Non pathogenic human (highly in dogs)- Sites and sources : tonsillar, respiratory tract, normal flora - Structurally simple, non-enveloped virus, physico-chemically stable- The capsid which is 22 nm in diameter, contains a linear single-stranded DNA genome of 4.7kb- At least eleven (and may be more) AAV serotypes have been identified among human and non-human primates- AAV has a broad tropism, transduce non dividing cells4AAV Life CycleAdAAV AAV HSVRescue+ Ador HSVLatency Replicative phase- persistance of viral DNA in an episomal - synthesis of Rep and Cap proteinsor integrated form (chr.19 AAVS1) - AAV assembly in the nucleus- no viral gene expression5Second-strand synthesis is a rate limiting step for efficient transduction by rAAV6Self-complementary or double-stranded AAV-2 vectors- Conversion of single-stranded to double stranded rAAV DNA is a limiting stepgeneration of double-stranded rAAV vectors- rAAV DNA of less than half the wtAAV genome lenght can be packaged asa dimer or a diploid monomer.4.7 kb single-stranded DNA 2.3 kb dimer or diploid monomer DNA7In vivo behaviour of scAAV versus rAAVscAAVrAAVITR ITR ITR ITR2.35 kb4.7 kbscAAVrAAVMcCarty et al. ...

Informations

• Publié par: Vowyar
• Nombre de lectures: 74
• Langue: English

Extrait

Adeno-associated virus

Initial discovery

1965

1. AAV biology

Adeno-Associated Virus

- Classification : parvovirus  Dependovirus

- Defective virus = Productive infection requires host cell factors and the presence of a helper virus (adenovirus, herpes virus, papillomavirus)

-Non pathogenic human (highly in dogs)

-Sites and sources: tract, normal floratonsillar, respiratory

- Structurally simple, non-enveloped virus, physico-chemically stable

- The capsid which is 22 nm in diameter, contains a linear single-stranded DNA genome of 4.7kb

- At least eleven (and may be more) AAV serotypes have been identified among human and non-human primates

- AAV has a broad tropism, transduce non dividing cells

AAV

AAV Life Cycle

Rescue

+ Ad or HSV

Latency - persistance of viral DNA in an episomal or integrated form (chr.19 AAVS1) - no viral gene expression

AAV

Ad HSV

Replicative phase - synthesis of Rep and Cap proteins - AAV assembly in the nucleus

Second-strand synthesis is a rate limiting step for efficient transduction by rAAV

Self-complementary or double-stranded AAV-2 vectors

- Conversion of single-stranded to double stranded rAAV DNA is a limiting step

generation of double-stranded rAAV vectors

- rAAV DNA of less than half the wtAAV genome lenght can be packaged as a dimer or a diploid monomer.

4.7 kb single-stranded DNA

2.3 kb dimer or diploid monomer DNA

ITR

In vivo behaviour of scAAV versus rAAV

rAAV

4.7 kb

ITR

McCartyetal.GeneTher.2001

scAAV

rAAV

scAAV ITR ITR

2.35 kb

Fu etal.Mol. Ther. 2003

ITR

p5

REP78 REP68 REP52 REP40

p19

REP

AAV-2 genome

p40

CAP

PolyA

ITR

4.7 kb

AAVS1

VP1 VP2 VP3

1 1 10

2. rAAV

production

wt AAV  ITR  (4.7Kb)

rAAV

Recombinant AAV vectors

REP

TRANSGENE

CAP

ITR

The ITRs are the sole viral elements retained in rAAV vectors = « gutless » vector