RNA Interference Therapies for an HIV-1 Functional Cure

Abstract

HIV-1 drug therapies can prevent disease progression but cannot eliminate HIV-1 viruses from an infected individual. While there is hope that elimination of HIV-1 can be achieved, several approaches to reach a functional cure (control of HIV-1 replication in the absence of drug therapy) are also under investigation. One of these approaches is the transplant of HIV-1 resistant cells expressing anti-HIV-1 RNAs, proteins or peptides. Small RNAs that use RNA interference pathways to target HIV-1 replication have emerged as competitive candidates for cell transplant therapy and have been included in all gene combinations that have so far entered clinical trials. Here, we review RNA interference pathways in mammalian cells and the design of therapeutic small RNAs that use these pathways to target pathogenic RNA sequences. Studies that have been performed to identify anti-HIV-1 RNA interference therapeutics are also reviewed and perspectives on their use in combination gene therapy to functionally cure HIV-1 infection are provided.

Keywords: HIV-1; RNA interference; cell transplant; functional cure; micro RNA; small/short hairpin RNA; small/short interfering RNA.

Figure 1

RNA interference (RNAi) defense pathway. The general steps of the RNAi defense pathway are illustrated: (1) Double stranded RNAs (dsRNA) are produced from pathogens such as viruses, satellite RNAs and retrotransposons. (2) Regions of pathogenic dsRNAs are recognized by a complex including the Dicer enzyme, which processes these regions into small interfering RNAs (siRNA). (3) siRNAs are then loaded into the RNA-induced silencing complex (RISC). (4) The passenger strand is removed from the RISC. (5) The guide strand directs the RISC to its complementary target in the pathogenic RNA. (6) The target sequence is cleaved by an Argonaute (Ago) protein in the RISC.

Figure 2

Human micro RNA (miRNA) pathway. The general steps of the human miRNA pathway are illustrated starting with expression of a primary miRNA (Pri-miRNA) from a cell’s chromosomes (bottom, left). The Pri-miRNA is recognized by the endonuclease Drosha in complex with the RNA binding protein DiGeorge syndrome chromosomal region 8 (DGCR8). Drosha cleaves the Pri-miRNA into a precursor miRNA (Pre-miRNA) with a 3′ overhang. The Pre-miRNA is then exported to the cytoplasm by the Exportin 5 transporter complex and recognized by the endonuclease Dicer in complex with the Trans-activation response RNA binding protein (TRBP). Dicer cleaves the loop off of the Pre-miRNA to generate a mature miRNA. Following recruitment of additional proteins, including an Argonaute (Ago) protein, the passenger strand of the miRNA is removed and the miRISC complex targets complementary sequences in mRNA transcripts for translational repression or targeted degradation (bottom, right).

Figure 3

Canonical and Dicer substrate siRNA designs. On the top left, the typical or canonical siRNA design of 19 base pairs with two-nucleotide overhangs on the ends is illustrated. To ensure that the intended guide strand (red) is selected by the RNA induced silencing complex (RISC), siRNAs should have higher G/C content at the 3′ end of the intended guide strand and higher A/U content at its 5′ end, in particular for the terminal nucleotides. On the top right, Dicer substrate siRNA designs of 25 to 30 base pairs are illustrated. They have been designed both in symmetrical two-nucleotide overhang formats and with a 5′ blunt end (NN) on the intended guide strand. Both canonical and Dicer substrate siRNAs use the RISC to cleave their target RNA (green arrow). By first recruiting the Dicer enzyme complex, Dicer substrate siRNAs may improve loading of siRNAs into the RISC and improve preferential selection of the guide strand.

Figure 4

Therapeutic short hairpin RNAs (shRNAs). (A) shRNAs with 19 to 30 base pair (bp) stems and variable loop sequences can be transcribed from an integrated gene or transfected plasmid DNA using RNA polymerase III (Pol III) promoters. Transcription starts from the 5′ end of the intended passenger strand and terminates with two or more Us transcribed from the RNA Pol III termination signal (five or more As) at the 3′ end of the intended guide strand. The shRNA is transported out of the nucleus by Exportin 5, and the loop is cleaved off by the Dicer enzyme complex. Proteins of the RNA induced silencing complex (RISC) help direct Ago2 to cleave and remove the passenger strand and subsequently to cleave target RNA sequences complementary to the guide strand; (B) an example of a long or extended shRNA is illustrated. Sequential guide strands with the same or different targets can be incorporated with up to three active guide strands being generated by Dicer processing; (C) an Ago-shRNA design with a 17 to 19 bp stem and a 3 to 6 nucleotide loop is illustrated. Unlike standard shRNAs, the intended guide strand is located at the 5′ end of the transcript and the intended passenger strand is on the 3′ end. In this format the shRNA is too small to be cleaved by Dicer. Instead it is bound by Ago2, which cleaves the intended passenger strand. Although the details are not fully elucidated, the 3′ end is thought to be adenylated, followed by 3′ end trimming by the poly(A)-specific ribonuclease (PARN). The released guide strand can then direct Ago2 to cleave a target RNA. The RISC in this case may have a different composition compared to that used by a standard shRNA design.