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Home / Equine Research Bank / Hum Gene Ther Methods / Development of an equine-tropic replication-competent lentiv...
Human gene therapy methods2012; 23(5); 309-323; doi: 10.1089/hgtb.2012.102

Development of an equine-tropic replication-competent lentivirus assay for equine infectious anemia virus-based lentiviral vectors.

Abstract: The release of lentiviral vectors for clinical use requires the testing of vector material, production cells, and, if applicable, ex vivo-transduced cells for the presence of replication-competent lentivirus (RCL). Vectors derived from the nonprimate lentivirus equine infectious anemia virus (EIAV) have been directly administered to patients in several clinical trials, with no toxicity observed to date. Because EIAV does not replicate in human cells, and because putative RCLs derived from vector components within human vector production cells would most likely be human cell-tropic, we previously developed an RCL assay using amphotropic murine leukemia virus (MLV) as a surrogate positive control and human cells as RCL amplification/indicator cells. Here we report an additional RCL assay that tests for the presence of theoretical "equine-tropic" RCLs. This approach provides further assurance of safety by detecting putative RCLs with an equine cell-specific tropism that might not be efficiently amplified by the human cell-based RCL assay. We tested the ability of accessory gene-deficient EIAV mutant viruses to replicate in a highly permissive equine cell line to direct our choice of a suitable EIAV-derived positive control. In addition, we report for the first time the mathematical rationale for use of the Poisson distribution to calculate minimal infectious dose of positive control virus and for use in monitoring assay positive/spike control failures in accumulating data sets. No RCLs have been detected in Good Manufacturing Practice (GMP)-compliant RCL assays to date, further demonstrating that RCL formation is highly unlikely in contemporary minimal lentiviral vector systems.
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Publication Date: 2012-11-02 PubMed ID: 23121195PubMed Central: PMC3732122DOI: 10.1089/hgtb.2012.102Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research paper primarily deals with the development of a new testing procedure for replication-competent lentivirus (RCL) related to equine infectious anemia virus (EIAV), improving the safety of releasing lentiviral vectors for clinical use. The study also contributes a rationale, based on Poisson distribution, for determining minimum infectious doses and monitoring control failures in data collection.

Background

  • The safe application of lentiviral vectors in clinical practices necessitates rigorous testing method for replication-competent lentivirus (RCL), vector materials, and if applicable, ex vivo-transduced cells.
  • EIAV-based lentiviral vectors, which do not replicate in human cells, have been directly given to patients in several clinical trials with no apparent toxic effects. However, possible RCLs from human cell vector productions can most likely influence human cells, prompting the need for more versatile testing methods.

Development of the Assay

  • The researchers developed an RCL assay that checks for the theoretical ‘equine-tropic’ RCLs, offering an additional layer of safety by identifying potential RCLs that might not be amplified efficiently by human cell-based RCL assays.
  • To identify a suitable EIAV-derived control, they examined how accessory gene-deficient EIAV mutant viruses replicated in a highly permissive equine cell line.

Mathematical Rationale for Use of Poisson Distribution

  • The study introduces a mathematical basis for using the Poisson distribution to calculate the minimal infectious dose of the positive control virus.
  • The same mathematical logic also helps in monitoring the failure of assay positive/spike controls in accumulating data sets.

Findings and Conclusion

  • No RCLs have been detected in the Good Manufacturing Practice (GMP)-compliant RCL assays which have been analysed to date.
  • This reaffirms the belief that contemporary minimal lentiviral vector systems are highly unlikely to form RCLs, reinforcing the safety of these vector systems.

Cite This Article

APA
Farley DC, Bannister R, Leroux-Carlucci MA, Evans NE, Miskin JE, Mitrophanous KA. (2012). Development of an equine-tropic replication-competent lentivirus assay for equine infectious anemia virus-based lentiviral vectors. Hum Gene Ther Methods, 23(5), 309-323. https://doi.org/10.1089/hgtb.2012.102

Publication

Human gene therapy methods
ISSN: 1946-6544
NlmUniqueID: 101573202
Country: United States
Language: English
Volume: 23
Issue: 5
Pages: 309-323

Researcher Affiliations

Farley, Daniel C
  • Oxford BioMedica, Oxford OX4 4GA, United Kingdom. d.farley@oxfordbiomedica.co.uk
Bannister, Richard
    Leroux-Carlucci, Marie A
      Evans, Nerys E
        Miskin, James E
          Mitrophanous, Kyriacos A

            MeSH Terms

            • Animals
            • Biological Assay
            • Cell Line
            • Gene Order
            • Genetic Vectors / genetics
            • Horses
            • Humans
            • Infectious Anemia Virus, Equine / genetics
            • Infectious Anemia Virus, Equine / physiology
            • Leukemia Virus, Murine
            • Mice
            • Reproducibility of Results
            • Transduction, Genetic
            • Viral Tropism
            • Virus Replication

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            Citations

            This article has been cited 2 times.
            1. Farley DC, McCloskey L, Thorne BA, Tareen SU, Nicolai CJ, Campbell DJ, Bannister R, Stewart HJ, Pearson LJ, Moyer BJ, Robbins SH, Zielinski L, Kim T, Radcliffe PA, Mitrophanous KA, Gombotz WR, Miskin JE, Kelley-Clarke B. Development of a replication-competent lentivirus assay for dendritic cell-targeting lentiviral vectors. Mol Ther Methods Clin Dev 2015;2:15017.
              doi: 10.1038/mtm.2015.17pubmed: 26029728google scholar: lookup
            2. Farley D, Stockdale S, Moore-Kelly C, Miskin J, Reiser J, Mitrophanous K. Risks of replication-competent retro/lentivirus from associated vector systems: Is it time for a roadmap toward reduced testing?. Mol Ther Methods Clin Dev 2025 Dec 11;33(4):101601.
              doi: 10.1016/j.omtm.2025.101601pubmed: 41141960google scholar: lookup
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