Mehdi Gasmi, PhD

Improvements towards the industrialization ofrecombinant AAV vector manufacturing forclinical useASGCT/ESGCT EARLY PHASE CLINICAL TRIALS TRAINING COURSEESGCT Annual Meeting, Versailles, France Oct. 25-29, 2012Mehdi Gasmi, PhD.

Adeno-associated VirusAdeno-associated virus, ~20 nmAdenovirus, ~90nm‣Non pathogenic parvovirus (ssDNA), non inflammatory, non integrative (vector)‣Various natural human and animal serotypes (AAV2, AAV1, AAV8..etc)‣Hybrid vectors, self complementary(ds), chimeric serotypes, directed evolution….

AAV vectors in the clinic‣~30% protocols approved to date worldwideOrph. Dis, cardiovasc., neurodegenerative…etc‣First approved product (EU): AAV1-LPL (uniQure)‣Late stage: Ocular disease AAV2-RPE65‣PhI/II: Hemophilia B (AAV8)

3 PlasmidsrAAV-most common production processAAVVectorRep-CapAdenovirus Helper2 or 3 plasmidsHuman cell lineHEK293Calcium PhosphateTransfectionAnchorage dependentSerumPurificationX.Xiao, RJ Samulski and othersConfidential

Transfection for rAAV production at GNTHQ PlasmidsPurified from E.coli WCBsHEK293WCBE.coli WCBE.coli WCBCell thaw/amplification to 24 CF10DMEM-2% FCSpAd helperpRep-Cap3 PlasmidTransfectionE.coli WCBCa.Phosph.pTransgeneHarvest at 72 hpost transfectionTotal ~ 25L bulk harvestConfidential

vg titer (vg/mL)rAAV by transfection: YieldsExample : Results for AAV2 (technology transfer batches)24 CF10 (n = 3)CriteriaValuesTiterVolume1 to 3x10 12 vg/mL~10 mLTotal VG 1 to 3x10 131E+133.5E+121E+121.3E+121.2E+121E+11Ocular disease dose ~10 11 vg/eye=>okSystemic indication (e.g. DMD) ~10 14 vg/patient=>X1E+10T.11044.Dev T.11045.Dev T.11057.Dev

Transfection in suspension: lack of scalabilityLa tourMontparnasseX 24 CF10X 75 patientsOcular Disease(LCA)X 1 patientDuchenne’sMuscularDystrophy

AAV by transfection-Conclusions‣Process OK for GMP- early stage (FBS)‣Multiple manipulations of CF10 : risk of microorg.contamination‣Process approved for clinical trials‣OK for applications that do not require large amounts ofvectors (retina, CNS…etc)‣Not compatible for applications like DMDlarge muscle tissue=> high titer, large volumes‣Need for a scalable system, compliant with cGMPConfidential

Baculovirus Technology‣rAAV production system developed in collaboration with R. Kotin(NIH)‣Non pathogenic insect virus that does not infect human cells‣Cell substrate (Sf9)•Non tumorigenic•Allows rAAV replication/production without helper virus•Robust growth in defined media•No fetal bovine serum requiredConfidenti

The Baculovirus SystemAutographa californica multiple nuclear polyhedrosisvirus (AcMNPV)rBac-Rep/CappA Cap Pp10 PpHReppArBac-rAAVITRAAV-vectorITRProduced inE.coliProduced inSf9 cellsSf9 cellsCulture in disposable bioreactors in serum-free conditions

AAV –Bac systemSf9 MCB/WCBAmplificationBaculovirus MVBsProductionBac Rep/CapBac transgeneInfection200 L disposablebioreactorsCellAmplification

Total purified vgScalability of the rAAV-BAC processTotal vg / bioreactor size1.0E+161.0E+151.0E+141.0E+132L (n=3) 10L 50L (n=10) 200L (n=4)

vg titer (vg/mL)rAAV-Bac: YieldsResults for AAV2/8 (3 technology transfer batches, n=3)CriteriaTiter (VG/mL)Valuesx10 13 vg/mLVolume~200 mL1.00E+145.10E+132.90E+134.30E+13Total VG x10 151.00E+131.00E+121.00E+111.00E+10Multiple batches of 200L runs needed for ~10 14 vg/patient=>Pool of 3 batchesConfidential

AAV-Bac: Conclusions‣Easy scale up, up to Nx200L‣No serum=> better safety risk profile‣Vector quality similar to transfection production‣Process already approved for registered product‣One extra raw material to make (MVB, >3 mo.) and control•Compared with plasmid transfection

Comparison rAAV transfection vs. rAAV-BacCriteriaTransfectionBaculovirussystemScale-up No YesPurity Profile of DP Good GoodValidation Potential Poor HighMfg costs(Low demand)Mfg costs(High demand)$ $$$*$$$ $*Regulatory (Quality) OK for PhI/II (serum) Approved (EU)ConclusionVery cumbersomeprocess but useful toget to the clinic fast.Difficult to go pastearly phases ofdevelopmentBetter option forhigh-doseindications. Longerlead-time fromtransgene to product(MVB)

Further improvementsbreakthroughtechnologyTour Montparnasse1000 L Bulk~1,25x10 15166 Wks (>3 y)Bioreactor 200L200 L Bulk~ 2 E x10 156 WksBioreactor 20L20 L Bulk~ 2x10 156 Wks

Detailed presentation of Genethon’s rAAV-BAC systemDr. Otto W. MertenINV069Sat. 27 Oct. 16h30-Session 4B“GMP and bioprocess development for GT vectors”

ACKNOWLEDGEMENTSGénéthon:Otto MertenStéphanie BucherEmmanuel GalèneLionel GalibertCécile Geny-FiammaAlexandra GrolhierAurélien JacobNicolas LaroudieThanh Hoa LeChristophe LecomteNicolas MarceauLoïc MillotAnthony RamirezMohamed RifkiChristel RivièreAntonietta Zanta…Fulvio MavilioAlain Schwenck- Atlantic GT/Inserm:Philippe Moullier- NIH (USA):Rob Kotin-NRC (Canada):Amine Kamen- University of Wageningen (NL):Monique van OersBaculogenes