A comparison of multiple shRNA expression methods for combinatorial RNAi.

RNAi gene therapies for HIV-1 will likely need to employ multiple shRNAs to counter resistant strains. We evaluated 3 shRNA co-expression methods to determine their suitability for present use; multiple expression vectors, multiple expression cassettes and single transcripts comprised of several dsRNA units (aka domains) with each being designed to a different target. Though the multiple vector strategy was effective with 2 shRNAs, the increasing number of vectors required is a major shortcoming. With single transcript configurations we only saw adequate activity from 1 of 10 variants tested, the variants being comprised of 2 - 3 different target domains. Whilst single transcript configurations have the most advantages on paper, these configurations can not yet be rapidly and reliably re-configured for new targets. However, our multiple cassette combinations of 2, 3 and 4 (29 bp) shRNAs were all successful, with suitable activity maintained in all positions and net activities comparable to that of the corresponding single shRNAs. We conclude that the multiple cassette strategy is the most suitably developed for present use as it is easy to design, assemble, is directly compatible with pre-existing shRNA and can be easily expanded.

Cassette deletion in multiple shRNA lentiviral vectors for HIV-1 and its impact on treatment success.

BACKGROUND: Multiple short hairpin RNA (shRNA) gene therapy strategies are currently being investigated for treating viral diseases such as HIV-1. It is important to use several different shRNAs to prevent the emergence of treatment-resistant strains. However, there is evidence that repeated expression cassettes delivered via lentiviral vectors may be subject to recombination-mediated repeat deletion of 1 or more cassettes. RESULTS: The aim of this study was to determine the frequency of deletion for 2 to 6 repeated shRNA cassettes and mathematically model the outcomes of different frequencies of deletion in gene therapy scenarios. We created 500+ clonal cell lines and found deletion frequencies ranging from 2 to 36% for most combinations. While the central positions were the most frequently deleted, there was no obvious correlation between the frequency or extent of deletion and the number of cassettes per combination. We modeled the progression of infection using combinations of 6 shRNAs with varying degrees of deletion. Our in silico modeling indicated that if at least half of the transduced cells retained 4 or more shRNAs, the percentage of cells harboring multiple-shRNA resistant viral strains could be suppressed to < 0.1% after 13 years. This scenario afforded a similar protection to all transduced cells containing the full complement of 6 shRNAs. CONCLUSION: Deletion of repeated expression cassettes within lentiviral vectors of up to 6 shRNAs can be significant. However, our modeling showed that the deletion frequencies observed here for 6x shRNA combinations was low enough that the in vivo suppression of replication and escape mutants will likely still be effective.

Long-term survival and concomitant gene expression of ribozyme-transduced CD4+ T-lymphocytes in HIV-infected patients.

BACKGROUND: An anti-HIV-1 tat ribozyme, termed Rz2, has been shown to inhibit HIV-1 infection/replication and to decrease HIV-1-induced pathogenicity in T-lymphocyte cell lines and normal peripheral blood T-lymphocytes. We report here the results of a phase I gene transfer clinical trial using Rz2. METHODS: Apheresis was used to obtain a peripheral blood cell population from each of four HIV-negative donors. After enrichment for CD4+ T-lymphocytes, ex vivo expansion and genetic manipulation (approximately equal aliquots of the cells were transduced with the ribozyme-containing (RRz2) and the control (LNL6) retroviral vector), these cells were infused into the corresponding HIV-1-positive twin recipient. Marking was assessed over an initial 24-week period and in total over an approximate 4-year period. RESULTS: The gene transfer procedure was shown to be safe, and technically feasible. Both RRz2- and LNL6-gene-containing peripheral blood mononuclear cells (PBMC) were detected at all time points examined to 4 years. There was concomitant gene construct expression in the absence of the need for ex vivo peripheral blood cell stimulation and there was no evidence of immune elimination of the neoR T-lymphocytes nor of silencing of the Moloney murine leukemia virus long terminal repeat. CONCLUSIONS: The proof of principle results reported here demonstrate safety and feasibility of this type of gene transfer approach. While not specifically tested, T-lymphocytes containing an anti-HIV gene construct may impact on HIV-1 viral load and CD4+ T-lymphocyte count, potentially representing a new therapeutic modality for HIV-1 infection.