Manufacturing Viral Gene Therapy Vectors: General Approaches and Challenges John T. Gray

Transcription

1 Manufacturing Viral Gene Therapy s: 1 Dr. Vice President, Research & Development Audentes Therapeutics, Inc. San Francisco, USA Natural virus life cycle Late Early Viral Virally infected cell Naïve cell 2 Production of injectable viral vectors Viral vector producer cell Patient s cell 3 1

2 Production of viral vectors for ex vivo transduction Viral vector producer cell Patient s cell 4 Engineering vector producer cells Introduce necessary genetic material into vector producer cell: 1) Helper components 2) component Viral vector producer cell 5 Stable vector producer cells or Viral vector producer cell 6 2

3 Transient vector producer cells or Viral vector Replicative production producer cell 7 Summary of terminology producer cells Helper components components Stable producer cells Transient production systems Replicative production systems 8 Design strategies for engineering helper cassettes 9 3

4 cis-acting trans-acting Particle assembly Replication complex formation Cellular physiology adjustment 10 cis-acting trans-acting genome genetic cargo 11 Packaging cell cis-acting trans-acting Genetic cargo Helpers 12 4

5 Goals for the design of a replication incompetent viral vector production system genome minimal cis-acting elements Helper only necessary trans-acting factors Optimization of viral pseudoreplication 13 Individual vector production systems First generation adenovirus Helper dependent adenovirus γ-retrovirus Lentivirus Adeno-associated virus 14 Adenoviral vectors Wild type virus ITR E1 ITR Helper ITR E1 HEK293 ITR E1 ITR gene 15 Graham, et al., J Gen Virol, 1977 v36 p59 Haj-Ahmad and Graham, J Virol, 1986 v57 p267 5

6 First generation adenovirus vector production Adenoviral vector Particle producer cell HEK Minimally deleted adenovirus vectors Graham, et al., 1977 Haj-Ahmad and Graham, 1986 x x 17 Helper dependent adenovirus vector production Transient delivery of both a helper virus and the virus vector to enable amplification of both genomes, but selective packaging of only the virus vector genome + Helper Adenoviral vector Particle producer cell 293Cre 18 6

7 Helper dependent adenovirus vector production (2) E1 E1 19 RJ Parks, et al., PNAS, 1996 Figure adapted from Vetrini & Ng Viruses, 2010 Retroviral vectors Wild type virus LTR Ψ gag pol env LTR LTR Ψ gag pol env 20 Mann, Mulligan, and Baltimore, Cell, 1983 v33 p153 Moloney Murine Leukemia Virus Ψ 21 7

8 Stable cell lines gagpol Helper Envelope Helper Therapeutic vector producer cell 22 Packaging cell line Producer clone Cleanroom manufacturing facility The challenge of self-inactivating (SIN) retroviral vector genomes Non-SIN Genomic RNA R U5 U3 R A n Reverse transcription Proviral DNA U3 R U5 U3 R U5 SIN Genomic RNA R U5 ΔU3 R A n 23 ΔU3 R U5 Proviral DNA ΔU3 R U5 SIN retroviral vector production with transient transfection ΔU3 SIN- + + gagpol Helper env Helper γ-retroviral vector producer cell HEK293T 24 JC van der Loo et al., Gene Ther

9 HIV based lentiviral vectors Ψ Late RRE Early RRE 25 HIV based lentiviral vector systems Ψ RRE RRE 26 Lentiviral vector production with transient transfection Transient delivery of plasmids for gagpol helper sequences, rev helper, envelope helper, and vector genome plasmids ΔU3 SIN HIV vector + + gagpol Helper Rev Helper + Env Helper producer cell HEK293T 27 Review: Schweizer & Merten, Curr Gene Ther

10 Lentiviral stable cell lines gagpol Helper Throm, et al., Blood, 2009 env Helper rev Helper Induction R U5 genome ΔU3R producer cell 28 Packaging cell line Producer clone Cleanroom manufacturing facility AAV vector & helper components 29 Adenovirus genome - 35,935 bp Xiao, X, Li, J, Samulski, RJ, J Virol,

11 Plasmid-based AAV vector production Transient delivery of plasmids for AAV helper sequences, Adenovirus helper sequences, and AAV vector sequences. Xiao, X, Li, J, Samulski, RJ, J Virol, AAV Helper AdV Helper AAV vector producer cell HEK Review: Wright, JF, Human Gene Therapy, 2009 Dedicated AAV producer cells Production systems which are dependent upon stable introduction of some vector or helper components into a cell. Production is initiated in the cleanroom by infection with helper virus, which may or may not carry additional components. For example: + Adenovirus helper AAV Helper AAV vector producer cell 32 Reviews: Thorne, et al. HGT 2009 Zhang, et al. HGT 2009 Baculovirus-based AAV vector production Transient delivery of AAV helper and vector genome sequences viabaculovirus vectors into insect cells provides alternative system to mammalian cells for vector production + RepCap Helper AAV vector producer cell Sf9 33 Review: Kotin, RM, Hum Mol Genet

12 cis-acting trans-acting genome genetic cargo 34 Packaging Cell Downstream processing Bulk harvest Cell disruption for non-secreted virions Detergent lysis Physical disruption Supernatant harvest for secreted products Continuous perfusion Batch harvest 35 Downstream processing (2) Purification Early processes developed in academic laboratories: Centrifugation Density gradients Dialysis Modern scalable processes Tangential flow filtration Chromatography (ion exchange, affinity) Single use, disposable units, closed systems 36 12

13 Cleanroom management Raw materials Personnel Air Equipment Quality systems Standard operating procedures Document control Personnel training Production methodology Quality control Release of product 37 Cleanroom manufacturing facility Product testing Physical titer Genome copy numbers Real-time PCR or RT-PCR Blot hybridization Direct dye binding Digital PCR Particles ELISA for capsid UV spectrophotometry Light scattering HPLC Mass spectrometry Product testing (2) Other parameters Infectious titer Potency Gene delivery Gene expression Enzymatic function Identity Homogeneity Contaminants Anything anyone has ever seen, if possibly relevant 13

14 40 Manufacturing Viral Gene Therapy s: Example release testing for AAV vector product Bulk purified material Titer Sterility Residual BSA Residual host cell protein Residual host cell DNA Residual benzonase E1A DNA SV40 DNA Plasmid backbone DNA Capsid DNA Rep-ITR DNA genome sequencing Restriction analysis Capsid Western blot In vitroadventitious virus assay Replication competent virus testing Final vialed product Container integrity Vial seal integrity Sterility ph Titer Endotoxins General safety Potency Thank you