Lentiviral Vectors: Safety Issues

1. Lentiviral Vectors: Safety Issues Daniel Takefman, Ph.D. Division of Cellular and Gene Therapies CBER, FDA

2. gag pol LTR LTR rev tat vif gag LTR LTR vpu vpr env pol nef Gammaretrovirus env Retroviridae Lentivirus

3. Lentivirus Gammaretrovirus • Can’t 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

4. Lentiviral Vector SystemsUnder Development • Primate • Human immunodeficiency virus (HIV) • Simian immunodeficiency virus (SIV) • Non-primate • Feline immunodeficiency virus (FIV) • Equine infectious anemia virus (EIAV)

5. 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

6. 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

7. 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.

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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

14. 1st Generation Vectors • Limited homology between vector and helper sequences • Separation of helper plasmids • Still retains HIV accessory genes in the packaging plasmid

15. 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

16. 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

17. 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

18. 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

19. 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?

20. 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

21. 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 • Usefulfor VSV G detection

22. 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

23. 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

24. 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

25. Lentiviral Safety Concerns: Conclusions • Recombination during manufacture • Vector design • Safety testing • Recombination with wild type virus in HIV+ subjects • Clinical monitoring

26. Lentiviral Safety Concerns: Conclusions • Mobilization by wild type virus in HIV+ subjects • In vitro assay • Preclinical animal model • Clinical monitoring