141. Combinatorial Aptamer Transcripts (CATs) for HIV RT Suppression

Abstract

The Human Immunodeficiency Virus (HIV) Reverse Transcriptase (RT) is a DNA polymerase encoded by the viral genome and it is preferentially targeted by current therapeutics due to its critical role in the viral life cycle. High affinity RNA aptamers that bind RT out-compete viral genome for access to the active site and thereby inhibit replication. Aptamer binding to RT during viral formation is believed to drive aptamer encapsidation into the budding virus, which leads to significantly reduced infectivity. We reasoned that co-transcribing multiple aptamer modules as combinatorial transcripts would increase avidity and packaging, resulting in greater net viral suppression. To test this hypothesis, we built a series of Combinatorial Aptamer Transcripts (CATs) carrying multiple, co-transcribed aptamer modules and compared their inhibitory capabilities as a function of valency. Specifically, aptamers from two different structural types (representing (6/5)AL and UCAA structural motifs) were combined into homodimeric and homotrimeric CATs.Two designs were explored for increasing valency; in the first we generated threads of homo-aptamer modules. In general, transcripts with more modules exhibited moderately increased RT inhibition in Primer Extension Assays and increased net binding affinity in Electrophoretic Mobility Shift Assays (EMSAs). The EMSAs do not show evidence of binding multiple RT per transcript, and preliminary cell-based assays do not indicate that this approach to multivalency improves net viral suppression. In contrast, an alternative design appears to be more promising, in which individual aptamer modules are incorporated into a stable3-Way Junction (3WJ) to increase modularity. The 3WJ structural core is expected both to isolate the individual aptamer structures from each other (and thereby reduce misfolding) and to allow sufficient separation to prevent steric hindrance in the binding of multiple RT. Other design features simplify the operational requirements of replacing individual modules with other aptamers or with libraries of aptamers. Initial data based on this design are encouraging.Combinatorial transcripts are expected to block evolutionary escape by forcing the virus to acquire multiple simultaneous mutations, especially for designs that include hetero-multimers. It is well established that combinations of small molecule drugs or of siRNA improves their targeting, delivery, and potency, while reducing their susceptibility to escape mutations. By demonstrating the benefits of multimerization we will inform downstream utilization of CATs as a therapeutic design in the treatment of HIV.