Transcript Document 7521162

Lentiviral Vectors:
Safety Issues
Daniel Takefman, Ph.D.
Division of Cellular and Gene Therapies
CBER, FDA
Gammaretrovirus
LTR
gag
pol
LTR
env
Retroviridae
Lentivirus
tat
vif
LTR
rev
gag
LTR
vpu
vpr
pol
env
nef
Gammaretrovirus
•Can’t transduce
Lentivirus
non-dividing cells
• Transduces
non-dividing cells
•Inefficient adaptation
to SIN technology
• Efficient adaptation
to SIN technology
• Integration into host chromosome
• No viral genes expressed in
target cells
•Potential for recombination resulting
in replicating virus with possible pathogenicity
Lentiviral Vector Systems
Under Development
• Primate
– Human immunodeficiency virus (HIV)
– Simian immunodeficiency virus (SIV)
• Non-primate
– Feline immunodeficiency virus (FIV)
– Equine infectious anemia virus (EIAV)
Safety Concerns Specific to
Lentiviral Vectors
• Recombination during manufacture may
generate a replication-competent lentivirus
(RCL)
- HIV a known human pathogen
- vesicular stomatitis virus (VSV G) envelope
broadens tropism
• Recombination with wild type virus in HIV+
subjects
• Mobilization of lentiviral vector by wild type
virus
Retroviral Recombination: Lessons
Learned From Gammaretroviruses
• Homologous recombination can occur when
two different RNA’s are packaged into one
virion
• Result of reverse transcriptase (RT) template
switching (strand transfer)
– Temin, H., et al., PNAS 90(15):6900-3
• Same mechanism shown to occur with HIV
RT in vitro as well
– Wu, W., et al., J Biol Chem 270(1):325-32
Retroviral Recombination: lessons
learned from Gammaretroviral vectors
• Immune suppressed Rhesus monkeys exposed to
bone marrow cells transduced with a preparation of
RCR positive retroviral vector
– 3/10 developed lymphomas, died within 200 days
– Donahue, R.E., et al., J. Exp. Med. 176: p. 1125-1135.
– Monkeys had sequences identified as recombinants
between vector and helper or between vector and
endogenous sequences.
– Vanin, E.F., et al., J. Virology. 68(7): p. 4241-4250.
– Purcell, D.F.J., et al., J. Virology. 70(2): p. 887-897.
Retroviral Recombination: lessons
learned from Gammaretroviruses
• Non-homologous recombination occurs at a
rate approximately 100-1000-fold lower than
homologous recombination
• Reduction in homology between vector and
helper sequences will lower likelihood of
recombination
– as little as 10 base pairs of nucleotide identity
between packaging and vector sequences were
sufficient to allow for RCR generation
• Otto, E., et al., Hum Gene Ther 5(5):567-75
Retroviral Recombination: lessons
learned from Gammaretroviruses
• Splitting helper sequences into more
than one plasmid (i.e., separation of env
and gag-pol) is likely to decrease the
incidence of RCR generation
Vector Mobilization
• An additional concern with the use of lentiviral
vectors in HIV-positive subjects
• Occurs when vector genome is packaged by
a wild-type HIV-1 present in the same cell
• Same mechanisms that allow helper
sequences to package vector genomes
Vector Mobilization
• Advantage
– mobilization of a vector designed to inhibit or
prevent HIV replication or pathogenesis has been
argued to enhance the therapeutic effect
• Disadvantage
– vector spread beyond the intended target tissue
may have safety consequences
– co-packaging of wt-type HIV RNA and vector RNA
may result in recombination
How to Address Safety Concerns
With Lentiviral Vectors
1. Vector design
- incorporate features to decrease
likelihood of recombination and
mobilization
2. Safety testing during manufacture
3. Preclinical safety studies
4. Clinical monitoring
1st Generation Lentivirus Vectors
Transient transfection of three plasmids in 293T :
Packaging plasmid:
all HIV viral genes, except env
Envelope plasmid:
G envelope glycoprotein of vesicular stomatitis
virus (VSV G)
HIV transfer vector:
gene or cDNA of interest and the minimal cisacting elements of HIV
1st Generation Vectors
• Limited homology between vector and helper
sequences
• Separation of helper plasmids
• Still retains HIV accessory genes in the
packaging plasmid
2nd Generation Vectors
Elimination of accessory genes from packaging
plasmid
• No effect on vector titer
• Retains property of transduction of many
dividing and non-dividing cells
• Increased safety margin
3rd Generation Vectors
Self-inactivating (SIN) vectors
• Deletion in the enhancer region of the 3’ U3 of the
long terminal repeat (LTR)
• Results in a transcriptionally inactive vector that can
not be converted into a full length RNA
• Reduces likelihood of RCL regeneration
• Hampers mobilization by wild-type HIV
• May reduce risk of tumorigenesis via promoter
insertion
Other Vector Developments
• Splitting the helper sequences onto three
separate plasmids by:
– Expressing rev on a separate plasmid
– Separation of gag-pol coding region onto
two plasmids
• Development of stable packaging cell lines
based on 3rd generation technology
• Non-HIV vectors: EIAV, SIV, FIV
How to Address Safety Concerns
With Lentiviral Vectors
1. Vector Design
- incorporate features to decrease
recombination and mobilization
2. Safety Testing during manufacture
3. Preclinical safety studies
4. Clinical Monitoring
Detection of Replication
Competent Lentivirus (RCL)
Detection of RCL by infectivity assay
• Several passages on permissive cell line
• Endpoint assay for viral sequence (p24 or
RT) or transgene sequence
• Positive control?
Detection of Helper Sequences
Functional assay
• Tat transfer
– Tat-transactivation of an LTR-reporter
gene construct
• Test for recombination intermediates
– To be discussed this afternoon by Dr.
Kappes
• Mol Ther 1(2):47-55
Detection of Helper Sequences
Detection of helper sequences in a vector
product lot or transduced cells by PCR assay
• Can be very sensitive, not the most
biologically relevant assay
• Useful for VSV G detection
How to Address Safety Concerns
With Lentiviral Vectors
1. Vector Design
- incorporate features to decrease
recombination and mobilization
2. Safety Testing during manufacture
3. Preclinical Safety Studies
4. Clinical Monitoring
Use of animal models to
assess safety
• Studies to assess mobilization and
recombination with wild type HIV are difficult.
• Appropriate animal model?
– HIV replicates, but is non-pathogenic in
Chimpanzees
– Macaque model appropriate for SIV vector
– Murine model limited due to blocks in HIV
replication
– SCID mouse models can serve as “in vivo test
tube”, but replication still limited to human cells
Clinical Monitoring
• Assay for RCL
– How best to do this in HIV+ subject?
• Assay for recombination with wild type
HIV
• Assay for changes in patient wt HIV
Lentiviral Safety Concerns:
Conclusions
• Recombination during manufacture
– Vector design
– Safety testing
• Recombination with wild type virus in
HIV+ subjects
– Clinical monitoring
Lentiviral Safety Concerns:
Conclusions
• Mobilization by wild type virus in HIV+
subjects
– In vitro assay
– Preclinical animal model
– Clinical monitoring