Simultaneous Use of Two Retroviral Vectors in Human Gene Marking Trials: Feasibility and Potential Applications

Abstract

Two Moloney murine leukemia virus (Mo-MLV)-based neoR retroviral vectors—LNL6 and G1Na—were used to transduce various human tumor-infiltrating lymphocytes (TIL) populations. These groups included bulk CD8⁺- and CD4⁺-enriched TIL from human renal cell carcinomas and melanomas. Transduction efficiencies averaged 5% for single 4-hr supernatant infections. Integrated provirus could be detected for up to 4 weeks of in vitro culture. LNL6 provirus could be distinguished from G1Na provirus using specific polymerase chain reaction (PCR) primers. A single neomycin phosphotransferase (neoR) gene copy could be detected in 10⁵ TIL. Using quantitative PCR, the relative ratio of LNL6 to G1Na copies in the same sample could be determined even at low copy numbers. These preclinical studies demonstrate the feasibility of using two retroviral marking vectors in human gene therapy efforts.

Overview summary

Preclinical studies were conducted to determine the feasibility of simultaneously using two neoR retroviral vectors in human gene therapy marking trials. With specific PCR primers, cells transduced with the two vectors could be distinguished and their relative proportions determined even at low gene copy numbers. The use of two marking vectors may enhance the design and scientific yield of human gene marking and therapeutic trials.