Adeno-associated Virus Vectors to Support Clinical StudiesAdeno-associated Virus Vectors to Support Clinical Studies

J. Fraser Wright, Ph.D.

Principal Investigator, AAV Clinical Vector Laboratory

Center for Cellular and Molecular Therapeutics

Children’s Hospital of Philadelphia

American Society for Gene Therapy Annual MeetingNHLBI Gene Therapy Resource Program SessionBoston, May 28, 2008

JF Wright Disclosures

Consultant for:

Tacere Therapeutic (CA)

Genzyme Corporation (MA)

Adeno –associated virus Adeno –associated virus (AAV)(AAV)

• member of Parvoviridae family• ~25 nm diameter (small), non-enveloped (stable)• ssDNA genome of 4.7 kb• dependent upon helper virus for replication

• adenovirus, herpes simplex virus, others

• > 9 distinct serotypes, many capsid variants• no known disease association

• attractive vector for gene delivery because:• lack of pathogenicity• defective self-replication• long-term transgene expression in animal models• various serotypes for different tissues

repcapITRITR ITRITR

Wild-type Wild-type AAVAAV

• Availability of a dedicated facility and staff with extensive experience in the development, manufacturing and certification of AAV vectors for clinical studies;

• Commitment to support NHLBI Investigators in the translation of promising basic / pre-clinical research to clinical trials.

The NHBLI GTRP Clinical AAV Vector Laboratory, part of The NHBLI GTRP Clinical AAV Vector Laboratory, part of The Center for Cellular and Molecular Therapeutics, The Center for Cellular and Molecular Therapeutics, Children’s Hospital of Philadelphia, provides:Children’s Hospital of Philadelphia, provides:

Clinical Vector CoreClinical Vector Core

Center for Cellular and Center for Cellular and Molecular Therapeutics, Molecular Therapeutics, Children’s Hospital of Children’s Hospital of Philadelphia Philadelphia

General layoutGeneral layout

Purification-0.05” WG

Cell culture-0.10” WG

De-gowning-0.10” WG

Gowning-0.125” WG

Staging-0.15” WG

QC Lab0.00” WG

PT1

PT2

PT3

PT1

Overview of AAV2 Vector Overview of AAV2 Vector Biosynthesis MethodBiosynthesis Method

1. . Initiation and propagation of HEK293 Initiation and propagation of HEK293 cells from a Master Cell Bank vial cells from a Master Cell Bank vial

↓↓2.2. Seeding of HEK293 cells in roller bottles Seeding of HEK293 cells in roller bottles

↓↓3. 3. Transfection of HEK293 cellsTransfection of HEK293 cells

↓↓4. 4. Post transfection medium exchange Post transfection medium exchange

in serum free mediumin serum free medium

Overview of AAV VectorOverview of AAV VectorPurification ProcessPurification Process

1. 1. Vector harvest concentration by TFFVector harvest concentration by TFF↓↓

2. 2. Harvest lysis by microfluidization Harvest lysis by microfluidization and clarification by filtrationand clarification by filtration

↓↓3. 3. Vector purification by Vector purification by

ion exchange chromatographyion exchange chromatography↓↓

4. 4. Gradient centrifugationGradient centrifugation↓↓

5.5. Buffer exchange by TFF, Buffer exchange by TFF, formulation, and 0.2formulation, and 0.2µmµm filtration filtration

↓↓6. 6. Final 0.2 µm filtration and vial fillFinal 0.2 µm filtration and vial fill

pAAV

6916 bp

Antiobiotic Resistance

Transgene

5' ITR

3' ITR

pUC origin

polyA

Backbone 2.5 kb

Promoter

f1 origin

pAAV

11277 bp

Antibiotic Resistance

Transgene

5' ITR

3' ITR

pUC origin

polyABackbone 7.1 kb DNA Stuffer

Promoter

f1 origin

- Using various vector generation platform:

- transfection of HEK293 using plasmid pRC, pDG- rAd infection of stably transfected cell lines

- Significant packaging of vector plasmid backbone occurs.

- This vector-related impurity is present at ~ 5% of vg in gradient-purified rAAV

- Predicted to be >10% in rAAV co-purified with empty capsids

(Smith et al, 2003; Chadeuf et al, 2005)

Oversized plasmid backbone reduces unintended encapsidation of plasmid DNA ~7.5 fold (to < 1%)

Oversized cis plasmid backbone reduces Oversized cis plasmid backbone reduces encapsidated DNA impuritiesencapsidated DNA impurities

Vector Lot Serotype Transgene size (bp

Backbone size (bp)

HEK DNApg /109vg

Plasmid DNApg /109vg

06002 AAV2 4297 6980 27.0 11.0

003A AAV2 4297 6980 20.9 13.0

NHP AAV2 4297 6980 38.5 13.3

0802 AAV6 4297 6980 7.5 15.8

0803 AAV6 4297 6980 6.3 17.7

avg (± sd) 14.2 (± 2.6)

0801 AAV2 4406 2620 16.9 88.3

N0701 AAV6 4679 2638 16.4 122

0701 AAV6 4679 2638 10.1 77.6

0702 AAV6 4811 2620 12.8 103

0703 AAV6 4811 2620 8.7 147

avg (± sd) 107.6 (± 27.6)

Use of oversized vector plasmid backbone reduced residual plasmid DNA 7.5-fold (p<0.001)

Orthogonal purification steps required to remove empty capsidsOrthogonal purification steps required to remove empty capsids

kDa

94

7667

53

43

30

VP1 (87)

VP2 (73)VP3 (61)

MW

M

Co

l L

1

Co

l L

2

Co

l L

3

Co

l L

4

Cs

Cl

MW

M

(5e10 vg)

Osmolarity (mOsm)

100 200 300 400

Ave

rag

e p

art

icle

ra

diu

s - R

h (

nm

)

0

20

40

60

80

100

120

140

Ionic strength (mM)

50 100 150 200 250

Na chloride Na phosphate Na sulfateMg sulfate Na citrate glycerol

A B

Ionic strength (µ) = ½ ∑cizi2Osmolarity = ∑ci

Formulation ionic strength and vector aggregationFormulation ionic strength and vector aggregation

PARAMETERPARAMETERIdentity: Identity:

AAV capsid proteinvector genomegenome sequence

Purity:Purity:Protein impurities

Residual plasmid DNAResidual mammalian DNAResidual cesium chlorideResidual Benzonase

Potency:Potency:vector genomesin vitro transductioninfectivity titer

Safety:Safety:Adventitious virusesMycoplasmaWT AAVUSP sterilityEndotoxinpHOsmolalityAggregationAppearance

METHODMETHOD

SDS-PAGE SS / WBrestriction digest / SBDNA sequencing / 4-fold redundancy

SDS-PAGE SS / CB optical Density 260 / 280nmQ-PCRQ-PCRICP-MSELISA

Q-PCRtransduction / transgene ELISAlimiting dilution / Q-PCR

in vitro assay for viral contaminantagar cultivable and non-cultivable ICA with Adfour media, direct inoculationLALpotentiometryosmometrydynamic light scatteringvisual inspection

AAV vector characterization: AAV vector characterization: Certification for clinical useCertification for clinical use

- Characterize AAV vectors - Characterize AAV vectors manufactured for clinical studiesmanufactured for clinical studies

- Assess clinical lot consistency- Assess clinical lot consistency

- Ensure successful process - Ensure successful process transfer from research to clinical transfer from research to clinical manufacturing / comparabilitymanufacturing / comparability

2x1010 vg

VP1VP2 VP3

MWM

Template for Template for Clinical AAV Vector Clinical AAV Vector Quality Control Quality Control TestingTesting

AcknowledgementsAcknowledgements

GTRP Co-Investigators: Katherine HighGuang Qu

Clinical Vector Core: Bernd HauckOlga ZelenaiaJitin BajajXingge Liu

Process Development Guang QuJinmin Zhou

Regulatory Affairs Jennifer McDonnellGreg Podsakoff

Research Core Shangzhen ZhouSonali JoyceAlex Tai