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Replication Competent Viruses Testing at the Indiana University Vector Production Facility

Replication Competent Viruses Testing at the Indiana University Vector Production Facility . K. Cornetta, M.D. Disclosures. Director of the Indiana University Vector Production which focuses on the production of Retroviral and Lentiviral Vectors (Phase I/II) for academic investigators

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Replication Competent Viruses Testing at the Indiana University Vector Production Facility

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  1. Replication Competent Viruses Testing at the Indiana University Vector Production Facility K. Cornetta, M.D.

  2. Disclosures • Director of the Indiana University Vector Production which focuses on the production of Retroviral and Lentiviral Vectors (Phase I/II) for academic investigators • Co-Founder of Rimedion • Own Stock in Amgen and Starbucks • Research and contract funding through the NIH and USDA • Subcontract on an SBIR awarded to Maxcyte Inc.

  3. Timing of RCR/RCL Testing Testing Long-term Follow-up Master Cell Bank Patient Ex Vivo Transduced Cells Final Product And EOP Cells Biologic Assays ?? qPCR

  4. Options for RCL Testing

  5. General Design of RCR Assay Vector Virus Amplification Phase (3 weeks) Indicator Phase MSV MLV MSV S+/L- Plaque Assay

  6. Cells used for amplification phase are envelope dependent 1Reeves et al. Human Gene Therapy 13:1783-1790, 2002. 2Cornetta et al. Human Gene Therapy 4, 579-588, 1993 3Chen et al. Human Gene Therapy 12:61-70, 2001. 4Duffy et al. Preclinica May/June:53-59, 2003.

  7. Retroviral Production Methods • Packaging Cell Line • Well characterized line that allows sequential harvests • Retroviral vectors generally not concentrated • Most commonly use retroviral envelopes • Derived from single cell clones to 100 vial MCB then expansion for production • Expansion allows time for recombination and RCR development env gag/pol Vector

  8. RCR Experience at the IU VPF • 5 Master Cell Banks Failed Sterility / Mycoplasma • None were generated in IU VPF • 4 MCB or Final Products Failed due to RCR • 2/2 + PA317 • 2/7 + GPE+Am12 • > 17 PG13 have passed • In the past 5 years failures due to: • Rearrangement of Vector (2) • Low titer from new producer cell line

  9. National Gene Vector Laboratory Program • Repository of gene therapy reagents • Houses a searchable database of gene therapy Pharm/Tox studies • Archives GLP, GMP or patient samples so investigators can comply with FDA requirements • Performs insertion site analysis by LM/LAM-PCR • Performs RCR or RCL testing by qPCR to comply with post-trial monitoring requirements

  10. Post-trial Monitoring for RCR

  11. Move to Lentiviral Vectors • Potential efficiency and safety profile. • Present new challenges for RCL detection • RCL has not been detected with current vectors • RCL structure is not known • Contribution of HERV sequences?

  12. Lentiviral Production Methods • Transient • No clone selection saving months in production time • Concern of reproducibility • Large plasmid requirements • Product generally concentrated • Less cell expansion which may decrease recombination frequency Rev Vector env Gag/pol HEK293T cells

  13. The challenge of VSV-G envelope • VSV-G env causes cell fusion • Limits the number of end-of-production cells • Are the EOP cells relevant?

  14. RCL Assay Design Vector RCL • Amplify with C8166 cells • Highly infectable • Amplify to high titer Amplification Phase (3 weeks) Indicator Phase 7 days Assay by PCR and ELISA

  15. Rational for Indicator Phase The kinetics of a RCL is currently unknown

  16. Rational for Indicator Phase Potential to transfer sequences without true RCL Sastry et al. Mol Therapy 8: 830-839, 2003

  17. Cell to Vector Ratio based in part on vector toxicity • For RCL assay we dilute vector to a concentration of 1000 ng/mL • Ratio of 5 x 106 C8166 cells per mL of test article • Purification may improve toxicity profile • Currently challenging when testing vectors > 20 liter scale

  18. RCL Testing of Anti-HIV Vector rHIV7-shI-TAR-CCR5RZ vector DiGiusto, D.L. et al. Sci. Transl. Med. 2, 36-43 (2010).

  19. RCL Method and Performance Acceptance Criteria p24 PCR Negative Control Media 0/3 + 0/3 + • Performed 13 assays under GMP • Amplification Phase virus detection • Negative controls - 0/39 by p24, 0/30 psi-gag • Positive control – 33/39 by p24, 30/30 by psi-gag • Indicator Phase virus detection • Negative controls – 0/36 by p24 and 1/33 by psi-gag • Positive controls – 46/60 by p24 and 39/50 by psi-gag Positive Control 0.5 IU Initial Assay 1/3 + 1/3 + IP Negative Control Media 0/3 + 0/3 + IP Positive Control 0.5 IU 1/5 + 1/5 + Amplification Phase Indicator Phase

  20. RCL Method and Performance Acceptance Criteria p24 PCR • Performed 17 assays under GMP • Amplification Phase virus detection • Negative controls – 0/54 by p24, 0/51 psi-gag • Positive control – 54/54 by p24, 51/51 by psi-gag • Inhibition controls • All met acceptance criteria Negative Control Media 0/3 + 0/3 + Positive Control 5 IU Modified Assay 1/3 + 1/3 + Inhibition Control 50 IU + Vector 2/3 + 2/3 + Indicator Phase Amplification Phase

  21. RCL Summary • Material generated in 6 different GMP facilities (20% generated at IU) • 16 Vector Products • 17 End-of-Production Cells • 7 cell lines • Analyzed • 1.12 x 107 ng of p24 (1.3 x 1014 virions) • 1.8 x 109 cells • No evidence of RCL

  22. RCL Assay Moving Forward • Re-evaluate the toxicity as product is purified • Can we decrease the cell to vector ratio and maintain sensitivity? • Should the procedure be different for anti-HIV-1 vectors? • Validating alternative envelopes. • Is p24 sufficient / is psi-gag needed? • Transgene effects? Still at the point of qualifying RCL assay on a case by case basis

  23. Detecting RCL in infused product • PCR is likely to give false positive • Biologic assay take 6 weeks and is expensive • Amplification kinetics of primary cells unknown • How much is gained? • Consider about 90% of vector available after testing • Currently testing 5% of final product for RCL • If you used the entire lot in a single patient and tested 1% of transduced cells you are adding 0.9% of final product analyzed (testing 5.9%)

  24. Department of Medical and Molecular Genetics IU- VPF Lisa Duffy Daniela Bischof, PhD Troy Hawkins, PhD Clara Hazelgrove Sue Koop Jing Yao Lina Sego Mikhaila Douglas Alisha Auberry Aaron Ernstberger Aparna Jasti Lorraine Matheson Lilith Reeves Erol Cetinok Collaborators Larry Couture and David Hsu, City of Hope Phil Zoltick Richard Morgan, Steve Feldman, and Steve Rosenberg, NIH, NCI Support by NHLBI, HHSN26820074820 and PO1 HL53586 (Dinauer) NCRR P40 RR024928 NCI N02-RC-67002 Lilly Endowment: Indiana Genomics Initiative

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