Issues in production of viral gene transfer vectors. Stefan Kochanek Department of Gene Therapy Ulm University

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1 Issues in production of viral gene transfer vectors Stefan Kochanek Department of Gene Therapy Ulm University

2 Only few positive results in gene therapy so far - many early phase, few late phase clinical trials - only approved gene therapy products - based on adenovirus Gendicine Shenzen Sibiono GeneTech Co. (2003) Oncorine H101 Shanghai Sunway Biotech Co. (2005)... and availabe only in China

3 Most frequently used viral vectors Adenovirus (double stranded DNA) Adeno-Associated Virus (single stranded DNA) Retrovirus (single stranded RNA)

4 Some comparisons... Route of application Adeno - episomal in vivo AAV - episomal in vivo Retro/Lenti - integrating ex vivo (mainly)

5 Some comparisons... Route of application Needed amounts per dose (VP) in humans Adeno - episomal in vivo >10 14 AAV - episomal in vivo >10 14 Retro/Lenti - integrating ex vivo (mainly) >10 9 (i.u.)

6 Some comparisons... Route of application Needed amounts per dose (VP) in humans Production yield/cell (VP) Adeno - episomal in vivo > AAV - episomal in vivo > Retro/Lenti - integrating ex vivo (mainly) >10 9 (i.u.) (i.u.) High cell numbers needed for production of the product

7 Adenovirus vectors Adenovirus (double stranded DNA) Genome Vectors - double stranded DNA - mostly had5 based - also other human serotypes (vaccine) - also from other species (vaccine) Use - tumor therapy, genetic vaccine (in vivo)

8 Production of adenovirus vectors 293 cells - academic institutions PER.C6 - industry

9 293 cells: generation of replication competent adenovirus (RCA) In 293 cells: through homologous recombination between vector and cellular DNA 293 cellular DNA E1 E1-deleted vector!e1 RCA ITR " E1 ITR

10 PER.C6 cells: no generation of RCA In PER.C6 cells: no homologous recombination between vector and cellular DNA PER.C6 cellular DNA E1 E1-deleted vector!e1

11 Very rare events in PER.C6 at large scale production Heterologous recombination events may rarely lead to helperdependent E1-positive region containing viral particle (HDEP) Murakami et al. 2004

12 Overall Production/cell lines cave RCA PER.C6 - industry standard Purification CsCl Chromatography - academic units - industry Storage Different buffers/freezing Quality control Assays well established Reference material (ARMWG) available

13 Overall Production/cell lines cave RCA PER.C6 - industry standard Purification CsCl Chromotagraphy - academic units - industry Storage Different buffers/freezing Quality control Assays well established Reference material (ARMWG) available field is quite mature, but...

14 Production is not the real problem Route of application Required amounts per dose (VP) in humans for liver transduction Production yield/cell (VP) Adeno in vivo > consider - a human liver has 1.5 x cells - human body about cells

15 Production is not the problem Route of application Required amounts per dose (VP) in humans for liver transduction Production yield/cell (VP) Adeno in vivo > consider - a human liver has 1.5 x cells - human body about cells Efficacy is very low! Why?

16 Efficacy is low because of many barriers - Interaction with cellular and non-cellular compartments CAR on Erys, Complement activation, Receptor issues... anti-ad antibodies, serum proteins anti-ad cellular immune response blood vessel issues/basement membrane extracellular matrix - Physical issues (diffusion, pressure) -... To get adenovirus to work we need to work on the virus

17 AAV-based vectors Adeno-Associated Virus Genome Size Use Promising - Single-stranded or double stranded DNA genome - 20 nm - genetic diseases (in vivo) - many different serotypes with different tropism

18 Genetic map of AAV ITR p5 p p ITR RNAs polya 4.2 kb Rep kb Rep kb Rep kb Rep kb VP1 2.3 kb VP2 2.3 kb VP3 3 functions needed for production ITR origin of replication, packaging signal Rep proteins replication Cap (VP 1-3) capsid proteins

19 AAV production systems 1. Stable rep/cap cell line (HeLa) - infection with 2 viruses a) Adeno WT b) Adeno-AAV vector 2. Helper-free transfection method (293, 293-T) - transfection 3 plasmids a) AAV packaging plasmid (rep/cap) b) AAV vectors c) Adenovirus plasmid (E2A, E4, VA) 3. Stable rep/cap + vector cell line (HeLa) - infection with 1 virus a) Adeno WT 4. HSV-based system (Vero, BHK) - infection 2 viruses a) HSV-rep/cap b) HSV-AAV vector 5. Baculovirus-based system (SF9) - infection with 2 to 3 viruses a) Baculo rep b) Baculo cap c) Baculo AAV vector

20 Production based on 3 different Baculoviruses

21 Advantages/disadvantages of the baculovirus system Advantages - cells derived from insects - safety - scalable system Disadvantages - stability of Baculovirus during amplification

22 Helper-virus free production method Needed a) vector plasmid b) rep/cap plasmid c) Ad helper plasmid (E2A, VA, E4)

23 Advantages/disadvantages of the transfection system Advantages - no virus involved Disadvantages - limitation in scale up

24 Overall Production/cell lines different Purification CsCl/iodixanol Chromatography - depending on system - academic units - industry Quality control Assays established Reference material (ARMWG) available (AAV2, AAV8) field is developing well, but...

25 field is developing well, but... still very large amounts of vector are needed one reason the relatively high particle/i.u. ratio (>100) while AAV wildtype has a low particle/i.u. ratio needed is a better understanding of the reasons for the differences in biology/infectivity of AAV wildtype versus AAV vector

26 Conclusions on viral vectors Vector systems at different levels of development Very high cell numbers for production required due to - low in vivo activity: Adenovirus, AAV - low production yield: Retro/lentivirus Adenovirus AAV - production overall quite mature cells only at small scale (RCA) - PER.C6 well developed (industry standard) minor issues with HDEPs - strategies to overcome barriers a/o achieve specific gene transfer are essential - developing fast, many serotypes - plasmid transfection at smaller scale for some areas (e.g. eye) - scale-up is still an issue with room for improvements - improving infectivity (particle/i.u.ratio) of vectors would be a major advance Retro/lentivirus - developing fast/many clinical trials - room for improvements for up- and downstream

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