Restriction Endonuclease-Mediated Selective Polymerase Chain Reaction : A Novel Assay for the Detection of K-ras Mutations in Clinical Samples

The enriched polymerase chain reaction (PCR) assay has been used extensively in the detection of ras gene mutations in many types of human malignancies. Although it is very sensitive, it has a number of features that limit its use in the routine diagnostic laboratory. The aim of this study was to develop a novel enriched PCR strategy, in which the concurrent activity of the restriction enzyme BstNI and Taq polymerase allowed the amplification of mutant K-ras while inhibiting the formation of wild-type product. This restriction endonuclease-mediated selective PCR assay uses three sets of primers, together with BstNI, in the reaction mix, and the amplification products are analyzed by gel electrophoresis. The reliability of the restriction endonuclease-mediated selective PCR assay to detect activated K-ras was determined in a variety of clinical samples, including 139 fresh colorectal carcinomas and 113 paraffin-embedded blocks from 80 separate tumors of the colon and rectum, pancreas, breast, or kidney. Codon 12 mutations of the K-ras oncogene were identified in DNA from both fresh and paraffin-embedded tumors in a rapid, sensitive, and reproducible manner. Mutations were detected in 33 (24%) of the fresh colorectal cancers and 16 (20%) of the paraffin-embedded tumors. These results were 97% concordant in cases in which paraffin blocks and fresh specimens from the same tumor were available for analysis. We conclude that restriction endonuclease-mediated selective PCR is a sensitive, rapid, and robust assay for the detection of point mutations in a variety of clinical samples. Importantly, there is no need for manipulation of the sample once the PCR has been set up, and therefore, the chance of contamination is significantly reduced. In contrast to previous assays, restriction endonuclease-mediated selective PCR is not labor intensive, and its format is suitable for use in routine diagnostic laboratory

Phase 2 gene therapy trial of an anti-HIV ribozyme in autologous CD34+ cells.

Gene transfer has potential as a once-only treatment that reduces viral load, preserves the immune system and avoids lifetime highly active antiretroviral therapy. This study, which is to our knowledge the first randomized, double-blind, placebo-controlled, phase 2 cell-delivered gene transfer clinical trial, was conducted in 74 HIV-1-infected adults who received a tat-vpr-specific anti-HIV ribozyme (OZ1) or placebo delivered in autologous CD34+ hematopoietic progenitor cells. There were no OZ1-related adverse events. There was no statistically significant difference in viral load between the OZ1 and placebo group at the primary end point (average at weeks 47 and 48), but time-weighted areas under the curve from weeks 40-48 and 40-100 were significantly lower in the OZ1 group. Throughout the 100 weeks, CD4+ lymphocyte counts were higher in the OZ1 group. This study indicates that cell-delivered gene transfer is safe and biologically active in individuals with HIV and can be developed as a conventional therapeutic product

Phase 2 gene therapy trial of an anti-HIV ribozyme in autologous CD34 + cells

Gene transfer has potential as a once-only treatment that reduces viral load, preserves the immune system and avoids lifetime highly active antiretroviral therapy. This study, which is to our knowledge the first randomized, double-blind, placebo-controlled, phase 2 cell-delivered gene transfer clinical trial, was conducted in 74 HIV-1-infected adults who received a tat-vpr-specific anti-HIV ribozyme (OZ1) or placebo delivered in autologous CD34+ hematopoietic progenitor cells. There were no OZ1-related adverse events. There was no statistically significant difference in viral load between the OZ1 and placebo group at the primary end point (average at weeks 47 and 48), but time-weighted areas under the curve from weeks 40-48 and 40-100 were significantly lower in the OZ1 group. Throughout the 100 weeks, CD4+ lymphocyte counts were higher in the OZ1 group. This study indicates that cell-delivered gene transfer is safe and biologically active in individuals with HIV and can be developed as a conventional therapeutic product