Lentiviral Vectors: Safety Issues

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Lentiviral Vectors Safety Issues

• Daniel Takefman, Ph.D.
• Division of Cellular and Gene Therapies
• CBER, FDA

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Gammaretrovirus
env
Retroviridae
Lentivirus
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Lentivirus
Gammaretrovirus

• Cant transduce
• non-dividing cells

• Transduces
• non-dividing cells

• Efficient adaptation
• to SIN technology

• Inefficient 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

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Lentiviral Vector SystemsUnder Development

• Primate
• Human immunodeficiency virus (HIV)
• Simian immunodeficiency virus (SIV)
• Non-primate
• Feline immunodeficiency virus (FIV)
• Equine infectious anemia virus (EIAV)

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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

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Retroviral Recombination Lessons Learned From
Gammaretroviruses

• Homologous recombination can occur when two
  different RNAs 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

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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.

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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

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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

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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

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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

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How to Address Safety Concerns With Lentiviral
Vectors

• Vector design
• - incorporate features to decrease likelihood of
  recombination and mobilization
• 2. Safety testing during manufacture
• 3. Preclinical safety studies
• 4. Clinical monitoring

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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
  cis-acting elements of HIV

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1st Generation Vectors

• Limited homology between vector and helper
  sequences
• Separation of helper plasmids
• Still retains HIV accessory genes in the
  packaging plasmid

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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

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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

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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

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How to Address Safety Concerns With Lentiviral
Vectors

• Vector Design
• - incorporate features to decrease recombination
  and mobilization
• 2. Safety Testing during manufacture
• 3. Preclinical safety studies
• 4. Clinical Monitoring

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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?

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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

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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

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How to Address Safety Concerns With Lentiviral
Vectors

• Vector Design
• - incorporate features to decrease recombination
  and mobilization
• 2. Safety Testing during manufacture
• 3. Preclinical Safety Studies
• 4. Clinical Monitoring

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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

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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

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Lentiviral Safety Concerns Conclusions

• Recombination during manufacture
• Vector design
• Safety testing
• Recombination with wild type virus in HIV
  subjects
• Clinical monitoring

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Lentiviral Safety Concerns Conclusions

• Mobilization by wild type virus in HIV subjects
• In vitro assay
• Preclinical animal model
• Clinical monitoring