Small activating RNAs (saRNAs) are double-stranded RNA molecules that induce gene expression at the transcriptional level, a phenomenon known as RNA activation (RNAa). This contrasts with the gene silencing typically associated with small interfering RNAs (siRNAs) in RNA interference. saRNAs offer a novel approach to upregulate genes of therapeutic interest, and have progressed to clinical trials.
saRNAs, typically 19 nucleotides in length with 2-nucleotide overhangs (similar to siRNAs),[1] mediate RNAa through the RNA-induced transcriptional activation (RITA) complex. This complex includes Argonaute 2 (AGO2), RNA helicase A (RHA), and CTR9 (a component of the PAF1 complex).[2] The RITA complex facilitates the transition of RNA polymerase II from a paused to an elongating state at the target gene's promoter, leading to increased transcription. (For a more detailed explanation of the mechanism, see RNA activation.)
saRNA Design and Use
Designing effective saRNAs involves careful consideration of several factors. Unlike siRNAs, which primarily target mRNA sequences for degradation, saRNAs target promoter regions, and their efficacy is highly dependent on the specific target location.[3][4] Proximity to the transcription start site (TSS), sequence context, and local chromatin state are critical determinants of activation versus silencing outcomes.[5][6][3]
Key considerations for saRNA design include:
Target Site Selection: saRNAs typically target promoter regions, often within a few hundred base pairs upstream of the TSS. Computational tools and empirical testing are used to identify optimal target sites.[3][5]
Sequence Specificity: The saRNA sequence must be carefully designed to ensure specific binding to the target promoter and avoid off-target effects.
Chemical Modifications: Chemical modifications, similar to those used in siRNA therapeutics, can be incorporated to enhance saRNA stability, reduce off-target effects, and improve delivery.[7][8]
Delivery: Efficient delivery to the target tissue and into the cell nucleus is crucial for saRNA activity. Delivery methods include lipid nanoparticles (LNPs), GalNAc conjugates, lipid conjugates and other approaches.[9][10]
A set of guidelines for designing saRNAs has been published [3] and an online resource for saRNAs has been developed to integrate experimentally verified saRNAs and proteins involved.[11]
Therapeutic Applications
saRNAs represent a promising therapeutic modality for diseases where increasing the expression of a specific gene is beneficial. This approach is particularly attractive for targeting genes considered "undruggable" by conventional small molecule or antibody-based therapies.[12][13]
Cancer: A major focus of saRNA research has been on reactivating tumor suppressor genes that are silenced in cancer cells. Examples include:
p21: RAG-01, an saRNA targeting p21, has received FDA approval for Phase I trials for non-muscle invasive bladder cancer (NMIBC).[14][15][16]
C/EBP-α: MTL-CEBPA, the first saRNA drug candidate, targets C/EBP-α and has shown efficacy in hepatocellular carcinoma (HCC) in Phase II clinical trials.[17][18]
LHPP: saRNAs targeting LHPP have shown preclinical efficacy in HCC.[19]
PTPRO: saRNAs have been used to overcome trastuzumab resistance in HER2-positive breast cancer by reactivating PTPRO.[20]
CDH13 saRNAs have been used to upregulate CDH13 expression in CML cells overcoming imatinib-resistance[21]
Acute Lung Injury: saRNAs targeting CEBPA have shown potential in preclinical models of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) by reducing inflammation.[22]
Cardiovascular Diseases: saRNAs have been used to activate genes like βII spectrin to improve cardiac function in preclinical models.[23]
Metabolic Disorders: saRNAs targeting SIRT1 have shown potential for reversing metabolic syndrome in preclinical studies.[24]
Proliferative Vitreoretinopathy (PVR): A saRNA designed to upregulate the p21 gene has shown therapeutic efficacy in a rabbit model of proliferative vitreoretinopathy.[25]
Neurodegenerative Diseases: saRNAs have been explored for modulating gene expression in diseases like Alzheimer's disease, for example, targeting BACE2.[26]
Clinical Progress
saRNA based therapeutics have advanced from preclinical studies to human clinical trials.
MTL-CEBPA: Developed by MiNA Therapeutics, MTL-CEBPA is the first saRNA drug candidate to enter clinical trials. It targets the C/EBP-α gene. A Phase I trial in patients with advanced HCC showed an acceptable safety profile and anticancer efficacy. Phase II trials are ongoing (NCT04710641).[17][18]
RAG-01: Developed by Ractigen Therapeutics, RAG-01 is an saRNA designed to activate the p21 tumor suppressor gene. It has received FDA approval for Phase I trials for the treatment of non-muscle invasive bladder cancer (NMIBC).[14] A Phase I trial is underway in Australia (NCT06351904).[12]
Challenges and Future Directions
While saRNAs hold significant therapeutic promise, challenges remain:
Delivery: Efficient and targeted delivery to the nucleus of target cells remains a major hurdle. Advances in delivery technologies, such as improved lipid nanoparticles, novel conjugates and targeted extrahepatic delivery, are crucial.[10]
Off-target Effects: Ensuring sequence specificity and minimizing unintended effects on other genes is essential.
Durability of Effect: While RNAa effects are generally more durable than RNAi, long-term efficacy and potential for repeated dosing need further investigation.
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