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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

Replication Competent Viruses Testing at the Indiana University Vector Production Facility

K. Cornetta, M.D.

disclosures
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.
timing of rcr rcl testing
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

general design of rcr assay
General Design of RCR Assay

Vector

Virus

Amplification Phase (3 weeks)

Indicator Phase

MSV

MLV

MSV

S+/L- Plaque Assay

cells used for amplification phase are envelope dependent
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.

retroviral production methods
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

rcr experience at the iu vpf
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
national gene vector laboratory program
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
move to lentiviral vectors
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?
lentiviral production methods
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

slide13

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?
rcl assay design
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

rational for indicator phase
Rational for Indicator Phase

The kinetics of a RCL is currently unknown

rational for indicator phase1
Rational for Indicator Phase

Potential to transfer sequences without true RCL

Sastry et al. Mol Therapy 8: 830-839, 2003

cell to vector ratio based in part on vector toxicity
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
slide18

RCL Testing of Anti-HIV Vector

rHIV7-shI-TAR-CCR5RZ vector

DiGiusto, D.L. et al. Sci. Transl. Med. 2, 36-43 (2010).

rcl method and performance
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

rcl method and performance1
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

rcl summary
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
rcl assay moving forward
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

detecting rcl in infused product
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%)
slide24

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