RNA interference (RNAi) is a technique that utilizes double-stranded RNA molecules to regulate the expression of protein-coding genes. The therapeutic applications of this have yet to be fully explored, but their potential in organ transplantation has accumulated recent interest.
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Also known as post-transcriptional gene silencing, RNAi is a conserved biological process of defense seen in prokaryotic and eukaryotic organisms. By exogenously introducing double-stranded molecules of RNA to the cell, it is possible to selectively silence target genes. The RNA molecules introduced are cut into fragments of small interfering RNA (siRNA) by the Dicer, a ribonuclease that will cut RNA such that its termini have distinct features; these features aid in recognition and integration with the RNA-induced silencing complex (RISC).
Once the bound in this complex, the strands of RNA are separated and the “passenger” strand is cleaved. The single strand of RNA remaining in the complex acts as a guide for binding the RISC to the target messenger RNA (mRNA). After homology-dependent recognition of the target, the RISC can cleave the mRNA and hence inhibit translation.
Since its discovery in 1998, RNAi technology has garnered the Nobel Prize in Medicine, and its clinical applications continue to gain interest. The first FDA-approved RNAi therapy was some 20 years after its discovery, in 2018, for a rare form of amyloidosis. Successful applications of RNAi have continued to arise, in an expanding number of disease areas. But, the clinical applications of RNAi have often been limited by delivery and target-site accumulation.
Due to the high potential of siRNAs for degradation, as well as their short half-life of several minutes to an hour, delivery systems have had to be developed to improve stability and targeting. To overcome the limitations of delivery, chemical modifications can be made directly to siRNA molecules, or delivery systems such as nanoparticle and lipid vesicles can be used. By altering the chemical structure of siRNA it is possible to improve half-life, protect against nucleases, as well as aid in transport and targeting. This strategy does come with limitations, as the RNAi machinery is only capable of accommodating moderate alterations to chemical structure.
Gene delivery systems are another commonly used strategy, with liposomes being the most extensively explored platform. Additional nanocarriers that have been developed include polymeric nanoparticles, metallic core nanoparticles, dendrimers, and polymeric micelles. These platforms are often used for the delivery of drug molecules; they are an attractive option due to the stability they offer, as well as their capacity for surface modifications. By modifying the surface of the delivery system, it is possible to enhance transport and targeting.
Clinical applications of RNAi
The therapeutic potential of RNAi has continued to gain attention, with a particular interest in its potential to improve outcomes for organ transplant patients. Organ transplants carry an unavoidable risk of ischemic-reperfusion injury (IRI) and graft rejection. Several preclinical studies using RNAi to improve outcomes for transplant patients have been performed; as of 2019, RNAi was reported to be used during liver, heart, kidney, and lung machine perfusions before transplant.
By targeting genes critical to mediating post-reperfusion injury and inflammation, as well as those that regulate apoptosis, it is possible to reduce the oxidative stress and inflammatory response to the transplant. There is also potential to reduce the risk of graft rejection after transplantation by modulating immune tolerance with siRNA gene silencing. For patients with end-stage renal disease, a kidney transplant could offer improved quality of life and reduced mortality, compared to dialysis. Unfortunately, donor's kidneys are in short supply, and organ IRI or rejection poses a severe risk to those who can receive a transplant.
The kidney is a good target for RNAi treatments due to its ability to rapidly uptake siRNA. Caspase 3 and the activation of the complement system have been associated with kidney IRI. By utilizing RNAi silencing tools to control the expression of these genes, it is possible to reduce apoptosis and inflammation, as well as improve renal function and survival in transplantation models (in pigs, mice, and rats).
Modulating the expression of multiple genes simultaneously with siRNA treatments has also been shown to be efficacious, with the targeting of complement 3, RelB, and Fas shown to reduce IRI as well as improve kidney function and graft survival. For organ transplant patients, they will likely receive RNAi treatment through ex vivo machine perfusion. This allows the siRNA solution to be directly delivered to the target site and prevents degradation, filtration, as well as off-target effects in the recipient. Treatment with RNAi is generally well tolerated and has promising results for improving outcomes, however, studies demonstrating this are limited.
The therapeutic applications of RNAi are considered to be in their infancy, with many avenues left to explore. But for organ transplant patients, RNAi therapeutics is a tangible reality that could vastly improve outcomes.
- National Center for Biotechnology Information. RNA Interference (RNAi). [Online] U.S. National Library of Medicine. Available at:
- https://www.ncbi.nlm.nih.gov/probe/docs/techrnai/ (Accessed on 30 September 2021)
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- Brüggenwirth I.MA, Martins, PN. RNA interference therapeutics in organ transplantation: The dawn of a new era. Am J Transplant. 2020; 20: 931– 941. doi.org/10.1111/ajt.15689
- Tonelli, M., et al. (2011), Systematic Review: Kidney Transplantation Compared With Dialysis in Clinically Relevant Outcomes. American Journal of Transplantation, 11: 2093-2109. https://doi.org/10.1111/j.1600-6143.2011.03686.x