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Analysis of partial recombinants in lentiviral vector preparations
Seraphin Kuate1, Michael P Marino, Jakob Reiser
1Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research , U.S. Food and Drug Administration, Bethesda, MD 20892.
Human Gene Therapy Methods
|December 26, 2013
Summary
A new drug selection assay quantifies partial recombinants, a safety concern in lentiviral vector production. Reducing sequence overlaps in vector constructs significantly decreased these partial recombinants, enhancing lentiviral vector safety.
Area of Science:
- Molecular Biology
- Virology
- Gene Therapy
Background:
- Replication-competent lentivirus (RCL) in lentiviral vectors poses safety risks for clinical use.
- RCL are thought to arise from rare partial or Psi-Gag recombinant vector genomes.
Purpose of the Study:
- To quantitatively assess the prevalence of partial recombinants in lentiviral vector preparations.
- To analyze the DNA sequences of these partial recombinants.
- To develop strategies for reducing partial recombinant formation.
Main Methods:
- Established a drug selection assay using a blasticidin resistance gene in a lentiviral packaging construct.
- Quantified drug-resistant colonies per million vector particles.
- Verified recombination events via PCR and DNA sequencing.
- Modified vector constructs by reducing sequence overlaps, including substituting the Rev response element (RRE).
Main Results:
- Detected up to 156 blasticidin-resistant colonies per 10^6 transducing vector particles.
- Confirmed recombination events through molecular analysis of resistant clones.
- Reducing sequence overlaps led to a sevenfold decrease in blasticidin-resistant colonies.
- Demonstrated diminished capacity for partial recombinant formation.
Conclusions:
- The developed drug selection assay effectively quantifies partial recombinants in lentiviral vectors.
- Minimizing sequence overlaps between vector and packaging constructs is a viable strategy to reduce partial recombinant formation.
- These findings contribute to improving the safety of lentiviral vectors for clinical applications.
