RNAi Gene Silencing Applications Advance

Researchers from academia and industry will present the latest developments in the application of interference RNA (RNAi) as a tool for gene silencing at “RNAi2008 Functions and Applications of Non-coding RNAs” to be held in March in Oxford, U.K. The meeting will focus on advances in the field and current opportunities and challenges in developing RNAi-based strategies for use in understanding the molecular mechanisms underlying disease processes, identifying new drug targets, and developing therapeutic agents capable of silencing disease-related genes.

siRNA Therapeutics

“Refined Animal Models for Optimizing Delivery of Functional siRNAs to Skin,” is the title of the presentation to be given by Roger Kaspar, Ph.D., on behalf of TransDerm (www.transderm.org). The skin disorder on which the company is focusing its initial product development effort is pachyonychia congenital (PC), a rare autosomal dominant disease in which a mutation in the gene for keratin 6a (K6a) causes painful skin lesions to form. The K6a N171 mutation is a single-nucleotide replacement mutation in which an adenine is present in the mutant gene form in place of a cytosine, resulting in an amino acid change.

Dr. Kaspar, CEO of TransDerm, will describe how the company’s TD101 therapeutic small interfering RNA (siRNA) specifically targets the N171K mutant form of the gene without affecting the wild-type gene.

To assess the activity of TD101 and optimize a controlled-dose delivery system, TranDerm is developing transgenic mouse models. In collaboration with Christopher Contag, Ph.D., and colleagues in the molecular imaging program at Stanford University, the company is using molecular imaging in transgenic mice to demonstrate the effectiveness of siRNA knockdown of reporter genes.

Two in vivo imaging-based approaches for validating RNAi activity have yielded quantitative and qualitative evidence of gene expression inhibition. In one method, the researchers intradermally coinjected a plasmid expressing the firefly reporter gene luciferase with siRNA molecules targeting the reporter gene mRNA into mouse paws. The results demonstrated potent inhibition of luciferase expression. Northern blot analysis supported luciferase mRNA inhibition as the mechanism of action.

The second method involved the construction of a mutant K6a/luciferase bicistronic reporter construct that was codelivered into mouse skin with K6a mutant-specific siRNAs. In vivo bioluminescence-based imaging showed weak luciferase activity in the coinjected mouse skin compared to control animals.

The company plans to use human skin explants derived from PC patient biopsies grafted onto immunocompromised, nude mice as a test stage to determine whether the RNAi activity seen in mouse skin will translate to similar efficacy in human skin samples. This work, being carried out with collaborators at the Ciemat Institute in Madrid, Spain, is still in the early stages.

TransDerm’s TD101 product is an unmodified siRNA intended to be administered directly to the PC lesions. Although intradermal injection offers a reliable delivery method, it is not a particularly patient-friendly option, especially given the need for repeated administration of the drug to treat new lesions. TransDerm is experimenting with two alternative siRNA delivery methods.

One is a topical, lipid-based formulation called GeneCreme. Its main limitation at present is how to ensure reliable uptake and dosing. The other approach is called the Soluble Tip Microneedle Array. Each array is composed of a grid of dissolvable, hollow protrusions into which siRNAs can be loaded. The microneedles penetrate the outer layer of the skin, the stratum corneum, where the tips are dislodged and remain. Time-release dispersion of the siRNA occurs as the tips dissolve.

“An ideal application would be to embed the array in a type of Band-aid device that the patient would apply and push down on to deliver the drug,” says Dr. Kaspar.

siRNA drugs intended for systemic administration require chemical modification or some sort of protective packaging to prevent rapid enzymatic degradation in the bloodstream, which would compromise their ability to reach the intended target and to exert a therapeutic effect.

Jørgen Kjems, Ph.D., a professor in the department of molecular biology at Aarhus University in Denmark, part of the Interdisciplinary Nanoscience Center (iNANO; www.inano.dk), is experimenting with three-stranded siRNAs as a means of optimizing the ability to introduce chemical modifications without changing the molecules’ activity.

Dr. Kjems begins with a small, double-stranded RNA, keeps one strand whole, and cleaves the second nonfunctional strand into two pieces. The result is a short internally segmented interfering RNA (sisiRNA). His group has demonstrated that these three-stranded constructs are amenable to a greater number of chemical modifications. With a double-stranded RNAi molecule about 20% of the nucleotides can be modified without losing activity, 100% of the nucleotides in an sisiRNA molecule can undergo chemical modification, allowing for more options to improve their pharmacodynamic properties.

This finding created an opportunity for evaluating a large variety of chemical modifications. Dr. Kjems has shown that the difference between an unmodified siRNA and a fully modified sisiRNA may be as great as a 100-fold increase in stability in serum.

This triple-stranded approach offers an additional benefit related to RNAi uptake and activity. When an siRNA is taken up by a cell, an intracellular complex selects one of the two RNA strands to retain. With the sisiRNA, the uncleaved strand—the active strand—is preferentially retained.

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