Gene Inhibition Therapy

Therapeutic Triggers of RNAi

Therapeutic triggers of RNAi can be used as therapeutic strategies.

In this video, we will review the therapeutic triggers of RNAi and how these have been harnessed as therapeutic strategies.

Therapeutic triggers of RNAi include synthetic siRNAs or transgenes expressing hairpin-based inhibitory RNAs such as short hairpin RNAs (or shRNAs) and artificial microRNAs13,18.

Let’s briefly talk about synthetic siRNAs. Synthetic siRNAs are synthesized as double-stranded RNAs, 21 to 23 base pairs long, that have two 3-prime overhangs that are 2 nucleotides long13,21,34.

For therapeutic purposes, synthetic siRNAs can be artificially introduced into cells, where they are processed by the endogenous RNAi pathway as portrayed in Section 2. siRNAs associate with RISC and are processed further within the siRNA–RISC complex, leading to cleavage of the target mRNA13,21.

A limitation to the use of synthetic siRNAs is their transient nature, with intracellular siRNA concentrations declining very rapidly, thus requiring repetitive dosing4,18. Synthetic siRNAs have been investigated in several diseases including polyneuropathy and amyotrophic lateral sclerosis, among many others22,35. As an example, in August 2018, a transthyretin-directed siRNA was approved in the United States for the treatment of polyneuropathy of hereditary transthyretin-mediated amyloidosis, a genetic disorder caused by dysfunctional transthyretin protein in the liver36,37. However, as synthetic siRNAs do not fall within the scope of gene therapy, they will not be discussed further in this video9-11.

shRNAs are hairpin structures that contain two complementary RNA sequences 19 to 22 base pairs long. These are linked by a short loop of 4 to 11 nucleotides and have one 3-prime overhang that is 2 nucleotides long38,39. The first generation of shRNAs are similar in structure to the naturally occurring pre-microRNA39,40.

shRNA complementary DNA (or cDNA) can be delivered into the cell via a viral vector or plasmid, which allows for prolonged gene inhibition38,41,42. shRNAs are usually transcribed by polymerase III, or sometimes polymerase II, to form the hairpin structures; the transcribed product is then directly transported into the cytoplasm where the loop is cleaved to form siRNA18,39,43. siRNAs then associate with RISC for mRNA cleavage activity in the same way as described in the endogenous pathway43. As shRNAs are generally designed to have perfect complementarity to mRNA sites, shRNAs cause gene inhibition via direct cleavage of the target mRNA at the site of complementarity39,43.

Now let’s talk about artificial microRNAs, a third type of therapeutic double-stranded RNA that can be used to trigger RNAi1. Artificial microRNAs are cellular microRNAs that have been modified to create partial complementarity to a target mRNA18. They are hairpin structures containing two complementary 19 to 25 base pair RNA sequences with interspaced mismatches, and a 5-prime and a 3-prime overhang18,21,44.

Artificial microRNAs can be introduced into the cells via an expression cassette containing the artificial microRNA cDNA or in a delivery vector such as an adeno-associated virus18. They can be transcribed by RNA polymerase II or III promoters, which are also included in the expression cassette31,45. Therapeutic strategies using artificial microRNAs aim to achieve the same biological functions as the endogenous microRNAs, and follow the natural microRNA signaling pathway21. Artificial microRNAs associate with RISC and are processed further to lead to inhibition of the target mRNA21. Because they have partial complementarity to the target mRNA, artificial microRNAs can induce gene inhibition via translational repression, mRNA degradation, or mRNA cleavage18,21,46. This summarizes key therapeutic triggers of RNAi. The next section will discuss key differences between shRNA and artificial microRNA.

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