In silico modeling indicates the development of HIV-1 resistance to multiple shRNA gene therapy differs to standard antiretroviral therapy.

BACKGROUND: Gene therapy has the potential to counter problems that still hamper standard HIV antiretroviral therapy, such as toxicity, patient adherence and the development of resistance. RNA interference can suppress HIV replication as a gene therapeutic via expressed short hairpin RNAs (shRNAs). It is now clear that multiple shRNAs will likely be required to suppress infection and prevent the emergence of resistant virus. RESULTS: We have developed the first biologically relevant stochastic model in which multiple shRNAs are introduced into CD34+ hematopoietic stem cells. This model has been used to track the production of gene-containing CD4+ T cells, the degree of HIV infection, and the development of HIV resistance in lymphoid tissue for 13 years. In this model, we found that at least four active shRNAs were required to suppress HIV infection/replication effectively and prevent the development of resistance. The inhibition of incoming virus was shown to be critical for effective treatment. The low potential for resistance development that we found is largely due to a pool of replicating wild-type HIV that is maintained in non-gene containing CD4+ T cells. This wild-type HIV effectively out-competes emerging viral strains, maintaining the viral status quo. CONCLUSIONS: The presence of a group of cells that lack the gene therapeutic and is available for infection by wild-type virus appears to mitigate the development of resistance observed with systemic antiretroviral therapy.

96 shRNAs designed for maximal coverage of HIV-1 variants.

BACKGROUND: The RNA interference (RNAi) pathway is a mechanism of gene-suppression with potential gene therapy applications for treating viral disease such as HIV-1. The most suitable inducer of RNAi for this application is short hairpin RNA (shRNA) although it is limited to suppressing a single target. A successful anti-HIV-1 therapy will require combinations of multiple highly active, highly conserved shRNAs to adequately counter the emergence of resistant strains. RESULTS: We calculated the percentage conservations of 8, 846 unique 19 nucleotide HIV-1 targets amongst 37, 949 HIV-1 gene sequence fragments containing 24.8 million 19 mers. We developed a novel method of determining conservation in 'profile' sets of 5 overlapping 19 mer sequences (covering 23 nucleotides in total) to ensure that the intended conservation of each shRNA would be unaffected by possible variations in shRNA processing. Ninety six of the top ranking targets from 22 regions were selected based on conservation profiles, predicted activities, targets and specific nucleotide inclusion/exclusion criteria. We constructed 53 shRNAs with 20 bp stems and 43 shRNAs with 21 bp stems which we tested and ranked using fluorescent reporter and HIV-1 expression assays. Average suppressive activities ranged from 71 - 75%, with 65 hairpins classed as highly active (> 75% activity). Overall we found little difference in activities from minor changes in stem length (20 cf. 21), or between neighboring targets differing by a single nucleotide in start position. However, there were several exceptions which suggest that all sequences, irrespective of similarities in target site or design, may be useful candidates. We encountered technical limitations with GFP reporter assays when the target domain was long and or when the distance between the target site and fusion junction was large. Assay performance was improved by dividing large targets into several shorter domains. CONCLUSION: In summary, our novel selection process resulted in a large panel of highly active shRNAs spanning the HIV-1 genome, representing excellent candidates for use in multiple shRNA gene therapies. Our core selection method ensuring maximal conservation in the processed product(s) is also widely applicable to other shRNA applications.

An infinitely expandable cloning strategy plus repeat-proof PCR for working with multiple shRNA.

Vector construction with restriction enzymes (REs) typically involves the ligation of a digested donor fragment (insert) to a reciprocally digested recipient fragment (vector backbone). Creating a suitable cloning plan becomes increasingly difficult for complex strategies requiring repeated insertions such as constructing multiple short hairpin RNA (shRNA) expression vectors for RNA interference (RNAi) studies. The problem lies in the reduced availability of suitable RE recognition sites with an increasing number of cloning events and or vector size. This report details a technically simple, directional cloning solution using REs with compatible cohesive ends that are repeatedly destroyed and simultaneously re-introduced with each round of cloning. Donor fragments can be made by PCR or sub-cloned from pre-existing vectors and inserted ad infinitum in any combination. The design incorporates several cloning cores in order to be compatible with as many donor sequences as possible. We show that joining sub-combinations made in parallel is more time-efficient than sequential construction (of one cassette at a time) for any combination of 4 or more insertions. Screening for the successful construction of combinations using Taq polymerase based PCR became increasingly difficult with increasing number of repeated sequence elements. A Pfu polymerase based PCR was developed and successfully used to amplify combinations of up to eleven consecutive hairpin expression cassettes. The identified PCR conditions can be beneficial to others working with multiple shRNA or other repeated sequences, and the infinitely expandable cloning strategy serves as a general solution applicable to many cloning scenarios.