Artificial capillary culture: expansion and retroviral transduction of CD4+ T-lymphocytes for clinical application

Abstract

An artificial capillary culture/transduction technique has readily attainable from 5 × 10 7 CD8-depleted lymphocytes. been developed for application in a phase I gene therapyIn addition, a sensitive and reliable quantitative competitive clinical trial for HIV. The trial protocol involves isolation of PCR method was developed to assess the levels of trans-CD4 + T-lymphocytes from a genetically matched HIV negaduction before infusion into the recipient. The transduction tive twin, retroviral transduction of equal numbers of cells data suggest that the efficiency of retroviral transduction with the ribozyme therapeutic and control genes, and was affected by the presence of inhibitory factors present expansion in Cellmax artificial capillary modules. Precliniin the virus preparations or generated as a result of the cal studies showed transduction efficiencies in the range transduction process itself. It is hypothesised that the of 3-30%, with preferential expansion of CD4+ lymphomethod of transduction could significantly affect the extent cytes over a culture period of 10-14 days. Over this time of this inhibition, and thus impact on clinical efficacy of period, an average yield of 1.7 × 10 9 lymphocytes was retrovirus mediated gene therapy.

UBC9 Autosumoylation Negatively Regulates Sumoylation of Septins in Saccharomyces cerevisiae

“…To create gene deletion mutants, the cells were transformed with PCR products bearing a selection marker (KanMX or HphMX) flanked by sequences complementary to the genes to be deleted. To generate ubc9 mutant strains, the LEU2 plasmids encoding mutated Ubc9 were transformed to the UBC9 shuffle strain whose genomic UBC9 gene was replaced by the HphMX marker through PCR-mediated gene replacement (32,33) and complemented by a centromeric URA3 plasmid encoding wildtype Ubc9. The URA3 plasmid encoding wild-type Ubc9 was then evicted from the cells by selecting the transformed cells on plates containing 5-fluoroorotic acid (34).…”

“…Analysis-Yeast total extracts were prepared according to a TCA precipitation method (32). Protein separation by SDS-PAGE and Western blotting were performed according to standard procedures.…”

Ribozymes in gene therapy of HIV-1

“…Other relatively recent advances have involved the reduction of retroviral packaging cell culture temperature to 32 o C, using concentrated virus (69) and the use of a recombinant fibronectin fragment (CH296) (70). These modifications act to increase the effective multiplicity of infection, and increase amphotropic viral transduction efficiency (71,72). Adeno-associated virus (AAV), is another viral system being developed as a gene delivery vector that has been shown to be capable of efficient insertion of anti-HIV-1 genes into hematopoietic cells (73).…”

“…This allowed monitoring of the survival of anti-HIV-1 ribozymeexpressing cells. Cell survival is monitored by simultaneously detecting ribozyme and control vector DNA sequences in peripheral blood using a semiquantitative PCR procedure (71). In order to examine the ability of the CD34+ stem cells to repopulate multiple cell lineages, PCR is performed on bone marrow, and purified cell populations.…”

Current develoments and future prospects for HIV gene therapy using interfering RNA-based strategies

“…Another phase I clinical trial was set up using CD4+ cells from uninfected identical twins (226,227). Healthy CD4+ PBLs from the uninfected twins were transduced with a single copy retroviral vector lacking or expressing a hammerhead ribozyme targeted against the HIV-1 tat coding region (under control of 5' LTR promoter) (160,192).…”