RNA Directed Strategies for the Treatment of HIV-1 Infection

Many strategies for HIV-1 treatment are being tested clinically. Since there are still limited options for patients affected by HIV-1, we have proposed two different RNA based strategies to expand these options. We chose RNA directed strategies due to efficacy and advantages in regards to potential cell-type delivery systems and viral vector production.;Our first strategy was to use aptamers targeting HIV-1 gp120 protein to carry mRNA into the cell. The following document provides proof of principle studies delivering reporter mRNA cargo to gp120 presenting cells using the aptamer. Unfortunately, we failed to obtain robust protein expression due to poor serum stability of the mRNA and endosomal trapping upon cellular uptake. Instead of proceeding with aptamer mediated delivery, we switched to liposomal delivery using commercially available lipid reagents. Further proof-of-concept studies were conducted to track mRNA upon systemic delivery and to edit cells in vivo using cre recombinase encoding mRNA. By obtaining recombination in vivo using this approach, we believe that other approaches for gene editing using systemically delivered mRNA are feasible.;Genome editing is a powerful way to obtain a permanent gene deletion, addition, or inversion by the introduction of a molecule capable of DNA cleavage. Multiple forms of genome editing are available; including cre recombinase induced genetic recombination and the clustered regularly interspaced short palindromic repeats (CRISPR)/ cas9 system. To represent a more clinically relevant strategy for HIV-1 treatment, we have used the CRISPR/cas9 system to eliminate HIV viral protein production from cell lines and primary cells with a lentiviral vector delivery. Current limitations in regards to delivery prohibited us from using an mRNA approach as the nucleic acid material encoding gene editing enzymes, but the lentivirus system was extremely successful. Using mRNA is still advantageous to limit potential off-target effects, but further development of delivery systems for nucleic acid delivery of gene editing enzymes is necessary.