Applications of the polymerase chain reaction in retroviral-mediated gene transfer and the analysis of gene-marked human TIL cells

Abstract

The polymerase chain reaction (PCR), a widely used new technology, was applied to several aspects of retroviral-mediated gene transfer. Ten oligonucleotide primer pairs were analyzed for their ability to amplify specific regions of a retroviral vector, including the long terminal repeat (LTR), and a NeoR selectable marker gene. By using the appropriate oligonucleotide primers, cells transduced by retroviral vectors could readily be detected and analyzed for deletions in proviral sequences by PCR, without Southern blotting. In combination with a simplified RNA isolation/reverse transcription protocol, an approximate titer of vector producer cell lines could be estimated by PCR in a single day, eliminating the need for time-consuming transductions and biological selection. Quantification of data obtained from PCR dilution experiment indicates that, under appropriate conditions, amplification is linear with respect to the amount of input DNA, permitting estimations of gene dosage in unknown samples. Specific PCR procedures have been developed as part of a protocol involving the transfer of retroviral-marked human tumor infiltrating lymphocytes (TIL) to cancer patients undergoing TIL cell cancer therapy. To augment biological safety testing methods, PCR has been used to detect the presence of possible amphotropic helper virus genomes at a sensitivity of one marked cell in 10(5) unmarked cells. The further use of PCR technology in the TIL cell human gene transfer protocol is demonstrated by the ability to detect small numbers of transduced TIL cells in the presence of hundreds of thousands of untransduced TIL.