A NOVEL METHOD FOR THE DIRECT QUANTIFICATION OF GENE-TRANSFER INTO CELLS USING PCR IN-SITU

There are several limitations to current methods for the detection of target genes following gene transfer. We report a novel PCR in situ pr ocedure which overcomes many of these and permits the direct quantific ation of gene transfer in individual cells, PCR amplification of a pro viral specific nucleotide sequence in target cells was followed in sit u hybridization using fluorescent probes complementary to different re gions of the amplicon. Many of the problems previously encountered usi ng in situ PCR, particularly the generation of false positive results and extracellular leakage of PCR products, were overcome by modificati ons of existing protocols. Positive cells were readily identified by f luorescence microscopy and a high sensitivity specificity and correlat ion coefficient were demonstrated in mixing experiments using varying proportions of known provirus positive and negative cells. The method was applied successfully to identify low numbers of gene-modified hema topoietic cells in clinical specimens in a trial of retrovirus-mediate d gene transfer into blood forming stem cells. This approach is simple and reliable, has the potential for use in a variety of gene therapy applications and may become the method of choice for the assessment of gene transfer efficacy.