The continued absence of an effective vaccine and current limitations of conventional HIV-1 drug treatments compel the development of alternative approaches. Stem cell strategies involving modified hematopoietic stem cells (HSC) offer the potential for multi-year to lifetime protection from the virus, because the modified HSC continually regenerate and because T cells and macrophage derived from these HSC express protective transgenes. Interest in stem cell strategies has increased following the apparent cure of an HIV+ patient who received a bone marrow transplant from a ?32 (CCR5-/-) donor. This patient has subsequently been off ART and free of virus for a number of years. A current frontier in stem cell therapies for HIV-1 is to introduce protective genetic modifications into human CD34 HSC for eventual transplantation into patients. Small, non-coding antiviral RNAs, such as aptamers, ribozymes and siRNA, are especially attractive cargo to deliver to these cells, in part because of their potency and non-immunogenicity. Small animal models such as Rag-hu immunodeficient mice support human T cells differentiation and are highly valuable for evaluating in vivo efficacy of candidate therapeutic transgene cargos. The long term objective of our work is to optimize aptamers for anti-HIV therapies, particularly aptamers that bind HIV-1 reverse transcriptase (RT). Aptamers that bind RT with high affinity inhibit the enzyme in vitro by competing with primer/template (p/t) for access to RT. Importantly they also suppress the virus in cell-based assays. In recent years we have defined the sensitivity of several aptamer classes to RT amino acid sequence variation, identified previously-unrecognized structural classes of RNA and single-stranded DNA aptamers, defined the molecular interfaces for several aptamer-RT complexes, developed and implemented a bioinformatics platform for evaluating high-throughput sequencing (HTS) data from aptamer selections, developed new platforms for intracellular expression of RNA aptamers and demonstrated in vitro antiviral efficacy of RNA aptamers expressed from within those platforms. Building from these strong foundations and on substantial new data obtained since its original submission, this revised collaborative proposal seeks to move validated aptamers into an established Rag-hu mouse model of HSC immune reconstitution and to strengthen the aptamer developmental pipeline. Interestingly, there has been no careful evaluation of the emergence of aptamer resistant in replicating viruses. To address its potential impact on antiviral therapeutic strategies, we will thoroughly evaluate HIV's ability to evolve resistance to expressed aptamers both in cell culture and in vivo, in addition to exploring several routes to overcome resistance.
The proposed study has both translational and basic science significance. It will evaluate the suitability of existing aptamers and aptamer-expressing platforms for use in a clinically relevant animal model of anti- HIV-1 gene therapy, in addition to providing new aptamers and aptamer combinations with increased efficacy and reduced susceptibility to resistance. It will also deepen our understanding of the biochemical and cellular parameters that govern antiviral effectiveness of these aptamers. Completion of this phase of the project will position us to make meaningful plans for advancing toward clinical application.
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