Certification assays for HIV-1-based vectors: Frequent passage of gag sequences without evidence of replication-competent viruses

Lakshmi Sastry, Yi Xu, Terry Johnson, Kunal Desai, David Rissing, Jonathan Marsh, Kenneth Cornetta

Research output: Contribution to journalArticle

50 Citations (Scopus)

Abstract

A principal concern regarding the safety of HIV-1-based vectors is replication-competent lentivirus (RCL). We have developed two PCR assays for detecting RCL; the first detects recombination between gag regions in the transfer vector and the packaging construct (sensitivity of detection ∼10-100 copies of target sequence). The second assay uses real-time PCR to detect vesicular stomatitis virus glycoprotein (VSVG) envelope DNA (sensitivity ∼5-50 VSVG sequences). In an attempt to amplify any RCL, test vectors were used to transduce C8166 and 293 cells, which were then screened weekly for 3 weeks. Psi-gag recombinants were routinely detected (20 of 21 analyses) in four transductions using the RRL-CMV-GFP vector. In contrast, VSVG sequences were detected only once in 21 analyses. Interestingly, p24 levels (as measured by ELISA) were occasionally detectable after 3 weeks of culture. To determine if a true RCL was present, 21-day cell-free medium was used to transcluce naïve cells. No evidence of psi-gag or VSVG transfer was detected, indicating that the recombination events were insufficient to reconstitute a true RCL. These findings have important implications for the design and safety of HIV-1-based vectors intended for clinical applications.

Original languageEnglish (US)
Pages (from-to)830-839
Number of pages10
JournalMolecular Therapy
Volume8
Issue number5
DOIs
StatePublished - Nov 2003

Fingerprint

Lentivirus
Certification
Virus Replication
Vesicular Stomatitis
HIV-1
Glycoproteins
Viruses
Genetic Recombination
Safety
Product Packaging
Real-Time Polymerase Chain Reaction
Enzyme-Linked Immunosorbent Assay
Polymerase Chain Reaction
DNA

Keywords

  • Lentivirus
  • p24 ELISA
  • PCR
  • Psi-gag
  • RCL
  • Recombination
  • Safety
  • VSVG

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Certification assays for HIV-1-based vectors : Frequent passage of gag sequences without evidence of replication-competent viruses. / Sastry, Lakshmi; Xu, Yi; Johnson, Terry; Desai, Kunal; Rissing, David; Marsh, Jonathan; Cornetta, Kenneth.

In: Molecular Therapy, Vol. 8, No. 5, 11.2003, p. 830-839.

Research output: Contribution to journalArticle

Sastry, Lakshmi ; Xu, Yi ; Johnson, Terry ; Desai, Kunal ; Rissing, David ; Marsh, Jonathan ; Cornetta, Kenneth. / Certification assays for HIV-1-based vectors : Frequent passage of gag sequences without evidence of replication-competent viruses. In: Molecular Therapy. 2003 ; Vol. 8, No. 5. pp. 830-839.
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