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By David A. Bumcrot, Ph.D.

December 15, 2004 | Breakthroughs in understanding RNA's extensive role in essential cellular processes have opened up the potential for a whole new class of drugs based on RNAi. The double-stranded RNA molecules that mediate RNAi are known as short interfering RNA (siRNA) molecules, and are able to sequence-specifically inhibit expression of genes. RNAi drugs have the potential to be more selective and, as a result, more effective and less toxic than traditional drugs.

In contrast to the extensive lead optimization steps required in small molecule and protein drug discovery, RNAi drug candidates can be identified using bioinformatics to select sequences complementary to the target mRNA. The process of choosing an RNAi-based drug candidate may simply involve the synthesis and testing of a relatively small number siRNAs, incorporating chemical modifications to confer stability and to direct these molecules to the appropriate tissues and cells, and/or mixing these siRNAs with appropriate delivery agents to achieve the same goals.


Mystery Solved 
RNA interference, or RNAi, is a naturally occurring mechanism within cells for selectively silencing and regulating specific genes that is potentially the basis for a new class of therapeutic products. Since many diseases are caused by the inappropriate activity of specific genes, the ability to silence and regulate such genes selectively through RNAi could provide a means to treat a wide range of human diseases.

In the process of gene silencing by RNAi, double stranded RNA (dsRNA) specifically degrades mRNAs that are complementary to one of the two strands. The mechanism of gene silencing by dsRNAs was first observed in C. elegans (Fire et al., 1998). The specific form of dsRNA mediating RNAi, the siRNAs, were first isolated from plants by Hamilton and Baulcombe (Hamilton & Baulcombe, 1999). A host of discoveries have been recently made on a chemically similar class of short RNA duplexes called microRNAs (miRNAs). Synthetic duplex RNA has been shown to silence genes in a host of systems. Leading researchers have further shown that RNAi is most effective in mammalian cells following the introduction of siRNAs that are 21-25 base pairs in length (Elbashir et al., 2001).

Within cells, specific factors incorporate siRNAs and miRNAs into RNA-induced silencing complexes (RISCs) that can recognize and destroy target mRNAs. The RISC complex, activated by ATP, unwinds the duplex formed by the siRNA or miRNA strands. One of the strands of the duplex is complementary to a particular mRNA to be regulated (Hutvagher & Zamore, 2002). The unwound strand guides the endonucleolytic cleavage of the target mRNA at a specific position (Schwarz et al., 2002). Thus, the specificity of the technology results from the Watson-Crick base pairing of the RNAi oligonucleotide with the target mRNA.


Potential Benefits of RNAi-Based Therapies 
Therapies based upon RNAi are likely to have a number of inherent and fundamental benefits:

  • High specificity - with the built-in accuracy of Watson-Crick base pairings, RNAi-based therapies can specifically inhibit production of a single protein;
  • Opportunity for rapid early-stage drug development - RNAi-based therapy development can readily leverage documented gene sequence data.
  • Potential for a safety profile superior to conventional drugs because RNAi-based therapies mimic a natural process.

This strong combination of inherent benefits underscores the exciting potential that surrounds the emerging field of therapeutic use of RNAi. A number of unmet patient needs can potentially be addressed with drugs that prospectively deliver a high therapeutic index. Furthermore, RNAi provides the foundation for a breakthrough product engine that will be able to rapidly generate novel therapeutics, as scientists apply RNAi's elegant mechanism of action to well-documented human gene sequence data.


Applying RNAi to Development of Therapies 
While virtually all diseases are fundamentally gene-based, RNAi will likely be most applicable in areas where proteins are incorrectly over-expressed. RNAi products can be divided into two broad classes based on the locality of therapeutic action:

  • Direct RNAi, RNAi therapeutics that will be administered directly at sites of diseases, which can be applied, for example to treat ocular, central nervous system, and respiratory diseases, and
  • Systemic RNAi, RNAi therapeutics that travel through the bloodstream and can be used to treat a broad range of diseases, including oncologic, metabolic, and autoimmune diseases.

For both Direct RNAi and Systemic RNAi therapeutics, identification of appropriate drug candidates can be more straightforward than it is for small-molecule or protein drugs. The process of choosing an RNAi drug candidate involves a series of steps:

  1. Selecting a relatively small number of siRNAs as a screening set for synthesis and testing using a combination of applied bioinformatics and empiricism to maximize potency and selectivity. At this point in the process, the sequences of prospective candidates are best selected so as to confirm specificity while being intelligently chosen to cross-react to homologues in traditional toxicology species. In a representative program that specifically involves targeting the angiogenic factor VEGF, bioinformatics reduced the number of desirable drug candidates from approximately 400 to 115;
  2. Making select chemical modifications to confer additional stability at critical places in the siRNA drug candidates. Considerable progress has been made in the antisense and ribozyme fields towards chemical stabilization of RNA (Cook 2001; Manoharan 1999) and this work provides a valuable starting place for RNAi. At this juncture, additional tests to verify potency and select the most stable candidate often yield a clear "best candidate"; and
  3. Finally, depending in large part on whether the RNAi drug candidate is to be a Direct RNAi or Systemic RNAi therapeutic, an appropriate conjugate or delivery formulation that aids in delivery of the RNAi drug candidate to the cells can be selected. There are two broad chemical strategies for enabling effective and efficient RNAi drug delivery. Conjugation of small molecules to the appropriate sites of RNA drugs can dramatically enhance protein-binding properties and also improve their cell permeation (Manoharan, 2002). Alternatively, the RNA drugs can be formulated with a variety of synthetic delivery systems equipped with tissue or cell specific targeting functionalities to achieve broad in vivo tissue distribution and cellular permeation. The cost and safety of these formulations must also be considered.

With the breakthrough potential of RNAi, there comes an understandably significant interest in rapidly delivering drug candidates to the clinic. Not surprisingly, RNAi faces the typical challenges of novel drug development, and successfully developing and commercializing RNAi therapeutics will take time and considerable effort. However, with a relatively straightforward drug candidate identification process, it is easy to understand why many view RNAi as one of the most promising new frontiers in drug discovery.

David A. Bumcrot is director, preclinical, at Alnylam Pharmaceuticals. 


Additional Reading 

Bartel, DP. "MicroRNAs: Genomics, Biogenesis, Mechanism, and Function" Cell 116, 281-297, January 23, 2004.

Cook, PD. "Medicinal chemistry of antisense oligonucleotides." Antisense Drug Technology, Marcel Dekker NY; 29-56; 2001.

Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. "Duplexes of 21-nucleotide RNAs mediate RNA interference in mammalian cell culture." Nature 411, 494-498; 2001.

Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans." Nature 391, 806-11; 1998.

Hamilton AJ & Baulcombe DC. "A species of small antisense RNA in posttranscriptional gene silencing in plants." Science 286, 950-952; 1999.

Hutvagher G & Zamore PD. "RNAi: Nature Abhors a Double Strand." Current Opinion in Genetics & Development 12, 225-232; 2002

Manoharan, M. "2'-Carbohydrate modifications in antisense oligonucleotide therapy: importance of conformation, configuration and conjugation" Biochimica et Biophysica Acta (1999), 1489(1), 117-130.

Manoharan M. "Oligonucleotide conjugates as potential antisense drugs with improved uptake, biodistribution, targeted delivery, and mechanism of action." Antisense & Nucleic Acid Drug Development 12, 103-128; 2002.

McManus MT & Sharp PA. "Gene Silencing in Mammals by Small Interfering RNAs." Nature Reviews Genetics 3, 737-747; 2002.

Schwarz DS, Hutvagner G, Haley B & Zamore PD. "Evidence that siRNAs Function as Guides, Not Primers, in the Drosophila and Human RNAi Pathways." Molecular Cell 10, 537-548; 2002.

Tuschl T & Borkhardt A. "Small Interfering RNAs: A Revolutionary Tool for the Analysis of Gene Function and Gene Therapy." Molecular Interventions 2, 158-167; 2002.

Tuschl T. "RNA Interference and Small Interfering RNAs." ChemBioChem 2, 239-245; 2001.

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