issues in production of viral gene transfer vectors · issues in production of viral gene transfer...
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Issues in production of viral gene transfer vectors
Stefan KochanekDepartment of Gene Therapy
Ulm University
- 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
Only few positive results in gene therapy so far
Most frequently used viral vectors
Adenovirus
(double stranded DNA)
Adeno-Associated Virus
(single stranded DNA)
Retrovirus
(single stranded RNA)
Some comparisons ...
Adeno
- episomal
AAV
- episomal
Retro/Lenti
- integrating
in vivo
in vivo
ex vivo
(mainly)
Route of
application
Some comparisons ...
Adeno
- episomal
AAV
- episomal
Retro/Lenti
- integrating
in vivo
in vivo
ex vivo
(mainly)
Route of
application
1010 - >1014
1010 - >1014
108 - >109 (i.u.)
Needed amounts
per dose (VP)
in humans
Some comparisons ...
Adeno
- episomal
AAV
- episomal
Retro/Lenti
- integrating
in vivo
in vivo
ex vivo
(mainly)
1010 - >1014
1010 - >1014
108 - >109 (i.u.)
Route of
application
Needed amounts
per dose (VP)
in humans
Production
yield/cell (VP)
104 - 105
104 - 105
100 - 101 (i.u.)
High cell numbers needed for production of the product
Adenovirus vectors
Adenovirus
(double stranded DNA)
Genome - double stranded DNA
Vectors - mostly hAd5 based
- also other human serotypes (vaccine)
- also from other species (vaccine)
Use - tumor therapy, genetic vaccine (in vivo)
Production of adenovirus vectors
293 cells - academic institutions
PER.C6 - industry
!E1
E1
293 cellular DNA
ITR " ITRE1
RCA
E1-deleted vector
293 cells: generation of replication competent adenovirus (RCA)
In 293 cells: through homologous recombination
between vector and cellular DNA
!E1
E1
PER.C6 cellular DNA
E1-deleted vector
PER.C6 cells: no generation of RCA
In PER.C6 cells: no homologous recombination
between vector and cellular DNA
Very rare events in PER.C6 at large scale production
Heterologous recombination events may rarely lead to „helper-
dependent E1-positive region containing viral particle (HDEP)“
Murakami et al. 2004
Overall
Production/cell lines
293 - cave RCA
PER.C6 - industry standard
Purification
CsCl - academic units
Chromatography - industry
Storage
Different buffers/freezing
Quality control
Assays well established
Reference material (ARMWG) available
Overall
Production/cell lines
293 - cave RCA
PER.C6 - industry standard
Purification
CsCl - academic units
Chromotagraphy - industry
Storage
Different buffers/freezing
Quality control
Assays well established
Reference material (ARMWG) available
field is quite mature, but...
Production is not the real problem
Adeno in vivo > 1014 104 - 105
Route of
application
Required amounts
per dose (VP)
in humans
for liver transduction
Production
yield/cell (VP)
consider - a human liver has 1.5 x 1011 cells
- human body about 1013 cells
Production is not the problem
Efficacy is very low ! Why ?
consider - a human liver has 1.5 x 1011 cells
- human body about 1013 cells
Adeno in vivo > 1014 104 - 105
Route of
application
Required amounts
per dose (VP)
in humans
for liver transduction
Production
yield/cell (VP)
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
AAV-based vectors
Adeno-Associated Virus
Genome - Single-stranded or double stranded DNA genome
Size - 20 nm
Use - genetic diseases (in vivo)
Promising - many different serotypes with different tropism
3 functions needed for production
ITR origin of replication, packaging signal
Rep proteins replication
Cap (VP 1-3) capsid proteins
ITR10 20
polyA
40 50 60 70 80 90ITR
p19 p40p5
30
Rep78
Rep68
Rep52
Rep40
VP1
VP2
VP3
Genetic map of AAV
RNAs
4.2 kb
3.9 kb
3.6 kb
3.3 kb
2.6 kb
2.3 kb
2.3 kb
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
Production based on 3 different Baculoviruses
Advantages/disadvantages of the baculovirus system
Advantages
- cells derived from insects - safety
- scalable system
Disadvantages
- stability of Baculovirus during amplification
Helper-virus free production method
Needed
a) vector plasmid
b) rep/cap plasmid
c) Ad helper plasmid
(E2A, VA, E4)
Advantages/disadvantages of the transfection system
Advantages
- no virus involved
Disadvantages
- limitation in scale up
Overall
Production/cell lines
different - depending on system
Purification
CsCl/iodixanol - academic units
Chromatography - industry
Quality control
Assays established
Reference material (ARMWG) available (AAV2, AAV8)
field is developing well, but...
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
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 - production overall quite mature
- 293 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
• AAV - 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