Pre-clinical grade Vector Core INSERM, U649

NANTES

Véronique BLOUIN

Laboratoire Thérapie Génique (U649)!

Objectif

Valider les études pré-cliniques

chez le petit et le gros animal

R & D (11 pers)

vecteurs viraux grade

clinique

Outils et procédés de

1- production

2- purification

3- contrôles

Plateforme de production

vecteurs cliniques en 2009

Recherche (18 pers)

Transfert de gènes in vivo

- rétine

(essai clinique)

- cerveau

- muscle

Plateforme de

production (11 pers)

vecteurs viraux

pré-cliniques

AAVr

Adénoviraux

Lentivirus (HIV)

1- Vecteurs Adeno-Associated.

2- Vecteurs Adénoviraux.

3- Vecteurs Lentiviraux.

4- Immortalisation cellules Primaires.

Plateforme de productions de vecteurs pré-cliniques:

5- Détection Ac neutralisants anti-AAV (ou Adv) dans fluides biologiques.

!! Parvovirus member of dependovirus sub-family

!! Replication only when helper virus is present : adenovirus or herpes virus (HSV-1 et –2)

!!11 different serotypes : AAV2 the most documented

!! 70-80% of population seropositive

!!No known pathology associated

!! No enveloped icosahedrical particle

!!20 - 24 nm in diameter, density 1,41g/cm3 (CsCl)

!!1 no enveloped capsid consists of 3 structural proteins VP1, VP2, VP3

!! cellular receptors identified : Heparan Sulfate, !V"5 integrins, R-1 au FGF

AAV Genome

!!Single Stranded linear DNA 4675bp

!!2 ITRs (Inverted Terminal Repeats) 145 bp

!!1 open reading frame (ORFs) rep encoded for 4 functional proteins :

-! Rep 78 and Rep 68 under p5 promoter control

-! Rep 52 and Rep 40 under p19 promoter control

!!1 open reading frame cap encoded for 3 structural proteins VP1, VP2, VP3 under p40 promoter control (in ratio: 90% VP3, 5% VP1, and 5% VP2 )

VP1 (87 kDa)

VP2 (72 kDa)

VP3 (63 kDa)

Poly A

CAP REP

p5 p19 p40

3’ITR 5’ITR

4.7 kb

REP68

REP52

REP40

REP78

AAV-2 genome Organisation :

Fonctional Proteins

Structural Proteins

Recombinant AAV vectors

ITR ITR REP CAP AAV-2 (4.7Kb)

TRANSGENE (4,7 kb) rAAV

ITR sequences are Only retained in recombinant AAV genome.

pAAVr!

transgène!ITR! ITR!

Classic method of rAAV vector production:

Tri-transfection!

pRC"

rep! cap!

pAd!

E4!E2!

VA!

293 (E1)

cells!

AAVr!

Co-transfection!

pDG!

E4!

E2!

VA!

cap!rep!

AAVr!

293 (E1)

cells!

1- transient transfection :

EXTRACTION! 1- cell pellet + supernatant extraction

CLARIFICATION!

CsCl density gradient

(swing rotor) :

Purified rAAV

PURIFICATION!

rAAVr purification process

rAAV crude Lysat

freeze/ thaw centrifugations

CsCl density gradient

(fixed rotor)

Iodixanol gradient

Heparin column!

dialysis dialysis

Polyacrylamid gel/Silver stain revelation

! presence of impurities.

Current improvements:

2- used of DNAse +/- detergent

Empty capsids

Full particles

83 kDa

62 kDa

48 kDa

33 kDa

175 kDa

25 kDa

VP3 VP2 VP1

rAAV characterization

! Vector concentration:

100 ng 0,05 ng

Amount of rAAV plasmid used to construct standard curve.

Different amounts of rAAV vector stock.

Titer : vector genome (vg/ml).

2- Replication or Infectious Center Assay (RCA or ICA).

10-7 10-8 10-9 T-

Infection of 10 fold dilutions of rAAV vector on HeLa rep-cap cells co-infected with wild-type Adenovirus (MOI : 500).

Titer : infectious particles (ip/ml).

1- Dot blot assay for rAAV physical particle titer of rAAV virion that contain vector genomes.

rAAV characterization

! Quality control assays:

ICA: Rep + detection and adenovirus detection Dot blot : transgene detection

! Initial Quality control assays:

- pAAV vector restriction analysis - restriction analysis to control ITRs presence - control of the tolerate insert size.

rAAV features:

! Advantages : - Efficient infection of a large type of cells (include quiescent cells). - Efficient cellular targeting depend on serotype, high capacity to

transduce (AAV5: SNC, AAV1: muscle, AAV4: retina, AAV8: liver, AAV9: heart, brain). - Stable and long transgene expression - Low immunogenicity: the most interesting to correct chronic diseases.

! Disadvantages: - Transgene size limited (4.4 kb)

- To scale up the production and quality control are difficult. - The repeated administration lead to immune response against the rAAV.

- Question of molecular integration in vivo: if AAV is integrative, risk of mutagenesis.

Applications in vivo:

Dog RPE65 -/- deficient

Photorécepteurs!

RPE!

bulle!

72 hours

13 days 30 days

4 days Injection day

8 days

No damage after sub-retinal injection.

!! 50 different serotypes

!! Icosahedrical particle : 70-100nm in diameter

!! 1 proteic capsid (87%): 7 different polypeptides

!! 1 DNA core (13%): DNA DB linear + 4 different proteins include the Terminal protein involved in the DNA viral replication

!! some carbohydrates (fibers)

!! Capsid: 252 capsomers

!! Proteic fiber : variable size depend on serotype

Adenoviral genome

- Double Stranded linear 36 kb

- 2 ITRs (100-140bp)

- 5 early genes (E1A, E1B, E2, E3, E4)

- 2 immediate-early genes pIX and IVA2

- 1 # packaging sequence : interaction with capsid and packaging direction

- 1 late gene encoded for 5 transcripts L1 to L5 (capsomer, fiber, core)

- 1 VA gene

"E1! "E3!

insert!

First generation!

$E1! $E4!E2 ts! "E3!

insert!

Second generation!

35 kb insert!

Gutless: 3rd generation!

Late Genes

E3

Adenoviral genome (36kb)

recombinant Ad

Ad5 wt

non replicative ADENOVIRUS

!! E1 region is deleted and replaced by the therapeutic gene !! E1 region induces the viral replication cycle and cellular apoptosis.

Adenovirus purification on CsCl density gradient

Purification:

2 CsCl gradients

Gradient 1

Gradient 2

Crude lysate

defective AdV

recombinant AdV (1,35)

Exclusion Column

Purified recombinant

Adenoviral vectors.

Adenoviral vector characterization

! Vector concentration

1- Calculate viral titer : viral titer (vp/ml) = OD260 x viral dilution x 1.1 x 1012

2- Infectious Center Assay (ICA). titration on HEK 293 cells and hybridization by a DBP probe (ip/ml).

! Final Quality control assays:

- host cell DNA contamination: PCR albumin - PCR E4 gene - E1 gene contamination (E1 and TG PCR)

- Replication-Competent Viruses (RCV)

! Initial Quality control assays:

- pshuttle restriction analysis - pAd restriction analysis - plaque purification (selection of E1-/TG+ viral clone)

Adenoviral vector features:

! Advantages: - Efficient infection of a large type of cells (include quiescent cells) in

vivo and ex vivo. - No cellular genome integration - post-mitotic cells are also infected - no disturb host cell genome

- Transient transgene expression - Capacity to transfer large DNA sequence (until 30 kb, commonly 7-8 kb)

! Disadvantages: - Large tropism : risk of no control of dissemination. - Transient expression (2 weeks), disappear when cells die - need several administrations - genetic exchanges with endogen adenovirus : - loss of transgene or generation of replicative vectors

- viral protein expression by infected cell: immune response.

In vivo applications :

% Transfer in hepatocarcinoma of woodchuck.

-! Lentivirus member of retrovirus family.

-! Enveloped particle 120 nm in diameter.

-! 1 lipidic and glycoproteic envelope (2 different polypeptides): surface

glycoprotein (SU or gp 120) and transmembrane glycoprotein (TM or gp41).

-! 1 viral core or nucleocapsid supporting 2 SS RNA molecules and the core

viral protein.

-! 1 proteic matrix mainly made of non glycosylated protein (P17).

3 structural genes de!: Gag, Pol et Env

2 transactivation genes!: Tat et Rev viral cycle essential

4 «!accessory!» genes !: Nef, Vpu, Vpr, Vif non essential to virus replication.

-! Lentiviral vectors are produced by transient transfection (tri- or quadri-) or with packaging cell lines

- Lentiviral vectors are purified and concentrated by 2 ultra- centrifugation steps

Pseudo-typed particles, envelope VSV-G, the most stable and resistant at different stage of ultracentrifugation

Transfection

! Vector concentration

Calculate viral titer : - viral capsid p24 protein titration (Elisa Kit) Titer : µ& p24/ml. theoretical ratio : 1 µ& p24 = 1010 vp

- Transducing unit (FACS) Titer : Transducing Unit (TU/ml).

- Q-PCR on HeLa cells infected

Lentiviral vector characterization

! Quality control assays

- Replication-Competent Viruses (RCV)

Lentiviral vector features:

! Advantages:

- Efficient infection ex-vivo.

- Efficient integration with transgene integrity

- Transgene persistence

- Low immunogenicity.

! Disadvantages: - Transgene size limited (8-9 kb max) - Very difficult to produce, the quality control and storage. - Low transduction efficiency in vivo.

- Question of molecular integration in vivo: randomly integration - risk of transgene inactivation if integrated in silence cellular genome. - activation of undesirable gene (oncogene). - risk of changing gene function.

Application in vivo

! Transgenese: rat transgenese plateform Ignacio Anegon

! Transduction in hepatic diseases situation In Crigler-Najjar model : Gunn rat

-! Using Lenti-UGT1A1 vector to correct

rat congenital hyperbilirubinemy .

Transduced Fibroblasts:

! Transduction of rat primary fibroblasts

- Using Lenti-GFP reporter vector.

Protocol: - Series of infections - Antibiotic selection (neomycin) - Constitution of a cell bank from the selected population or a specific cell clone.

Vector used in routine: - MLV vector expressing the thermo-sensitive

SV40 Large T antigen (TsA58)

Quality control: - SV40 Tts immuno-fluorescence - Mycoplasma

Custom-made vectors: - HIV vector expressing immortalizing genes (HPV E6/E7, Tert, AdE1…)

Application in vitro

! Immortalization of Canin MPSVII primary hepatocyte cells - Using Lenti-vector SV40T and/or Lenti-vector hTert.

24H post-isolation 24H post–infection, MOI 30

Hepatocytes:

Clusters 30 days post–infection

Immortalized Hepatocytes

Different cell clones isolated : some hepatocyte markers are lost

Bilan 2009 de l’activité:

http://www.vectors.nantes.inserm.fr"