DNA-directed RNA interference

DNA-directed RNA interference (ddRNAi) is a gene-silencing technique that utilizes DNA constructs to activate an animal cell’s endogenous RNA interference (RNAi) pathways. DNA constructs are designed to express self-complementary double-stranded RNAs, typically short-hairpin RNAs (shRNA), that once processed bring about silencing of a target gene or genes. Any RNA, including endogenous mRNAs or viral RNAs, can be silenced by designing constructs to express double-stranded RNA complementary to the desired mRNA target. DNA-directed RNA interference (ddRNAi) is a gene-silencing technique that utilizes DNA constructs to activate an animal cell’s endogenous RNA interference (RNAi) pathways. DNA constructs are designed to express self-complementary double-stranded RNAs, typically short-hairpin RNAs (shRNA), that once processed bring about silencing of a target gene or genes. Any RNA, including endogenous mRNAs or viral RNAs, can be silenced by designing constructs to express double-stranded RNA complementary to the desired mRNA target. This mechanism has great potential as a novel therapeutic to silence disease-causing genes. Proof-of-concept has been demonstrated across a range of disease models, including viral diseases such as HIV, hepatitis B or hepatitis C, or diseases associated with altered expression of endogenous genes such as drug-resistant lung cancer, neuropathic pain, advanced cancer and retinitis pigmentosa. As seen in Figure 1, a ddRNAi construct encoding an shRNA is packaged into a delivery vector or reagent tailored to target specific cells. Inside the cell, the DNA is transported to the nucleus where transcription machinery continually manufactures the encoded RNAs. The shRNA molecules are then processed by endogenous systems and enter the RNAi pathway and silence the desired genes. Unlike small interfering RNA (siRNA) therapeutics that turn over within a cell and consequently only silence genes transiently, DNA constructs are continually transcribed, replenishing the cellular ‘dose’ of shRNA, thereby enabling long-term silencing of targeted genes. The ddRNAi mechanism, therefore, offers the potential for ongoing clinical benefit with reduced medical intervention. Figure 2 illustrates the most common type of ddRNAi DNA construct, which is designed to express a shRNA. This consists of a promoter sequence, driving expression of sense and antisense sequences separated by a loop sequence, followed by a transcriptional terminator. The antisense species processed from the shRNA can bind to the target RNA and specify its degradation. shRNA constructs typically encode sense and antisense sequences of 20 – 30 nucleotides. Flexibility in construct design is possible: for example, the positions of sense and antisense sequences can be reversed, and other modifications and additions can alter intracellular shRNA processing. Moreover, a variety of promoter loop and terminator sequences can be used. A particularly useful variant is a multi-cassette (Figure 2b). Designed to express two or more shRNAs, they can target multiple sequences for degradation simultaneously. This is a particularly useful strategy for targeting viruses. Natural sequence variations can render a single shRNA-target site unrecognizable preventing RNA degradation. Multi-cassette constructs that target multiple sites within the same viral RNA circumvent this issue. Delivery of ddRNAi DNA constructs is simplified by the existence of a number of clinically-approved and well-characterized gene therapy vectors developed for the purpose. Delivery is a major challenge for RNAi-based therapeutics with new modifications and reagents continually being developed to optimize target cell delivery. Two broad strategies to facilitate delivery of DNA constructs to the desired cells are available: these use either viral vectors or one of a number of classes of transfection reagents. In vivo delivery of ddRNAi constructs has been demonstrated using a range of vectors and reagents with different routes of administration (ROA). ddRNAi constructs have also been successfully delivered into host cells ex vivo, and then transplanted back into the host.