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