Self-delivering RNAi compounds as therapeutic agents in the central nervous system to enhance axonal regeneration after injury.

The therapeutic use of RNAi has grown but often faces several hurdles related to delivery systems, compound stability, immune activation, and on-target/off-tissue effects. Self-delivering RNAi (sdRNA) molecules do not require delivery agents or excipients. Here we demonstrate the ability of sdRNA to reduce the expression of PTEN (phosphatase and tensin homolog) to stimulate regenerative axon regrowth in the injured adult CNS. PTEN-targeting sdRNA compounds were tested for efficacy in vivo by intravitreal injection after adult rat optic nerve injury. We describe critical steps in lead compound generation through the optimization of nucleotide modifications, enhancements for stability in biological matrices, and screening for off-target immunostimulatory activity. The data show that PTEN expression in vivo can be reduced using sdRNA and this enhances regeneration in adult CNS neurons after injury, raising the possibility that this method could be utilized for other clinically relevant nervous system indications.

Diphencyprone Treatment of Alopecia Areata: Postulated Mechanism of Action and Prospects for Therapeutic Synergy with RNA Interference.

Diphencyprone (DPCP) is a potent topical sensitizing agent that has been used since the late 1970s by physicians for the treatment of alopecia areata (AA), viral warts (human papillomavirus) and cutaneous metastases of melanoma. Although to date the compound is not approved as a drug by the FDA or EMA, physicians have continued to use DPCP because of its proven effects in these dermatological conditions. The use of the drug has been highly variable because of differences in compounding, and as a result, the literature reports vary widely in the concentrations used for sensitization and challenge treatment with DPCP. The efficacy of DPCP has generally been ascribed to immunological reactions by the host. Inducing inflammation with a contact sensitizer is counterintuitive to treating AA, an autoimmune disorder. We have hypothesized that the body's attempt to downregulate the inflammation caused by the contact sensitizer may also ameliorate AA. Studies using microarray and miRNA profiling may provide information about how DPCP induces inflammation in human skin at different times. Gene targets and microRNAs identified through these data may be modulated by an RNA interference approach to enhance DPCP efficacy and response rates. In addition, this approach may result in the discovery and development of drugs that are more potent and selective for the treatment of AA.

Novel hydrophobically modified asymmetric RNAi compounds (sd-rxRNA) demonstrate robust efficacy in the eye.

PURPOSE: The major challenges of developing an RNAi therapeutic include efficient delivery to and entry into the cell type of interest. Conventional ("naked" and chemically stabilized) small interfering RNAs (siRNAs) have been used in the eye in the past but they demonstrated limited clinical efficacy. Here we investigated a recently developed class of small, hydrophobic, asymmetric RNAi compounds. These compounds, termed "self-delivering rxRNAs" (sd-rxRNA(®)), are extensively modified, have a small duplex region of <15 base pairs, contain a fully phosphorothioated single-stranded tail, and readily enter cells and tissues without the requirement for a delivery vehicle. METHODS: We compared sd-rxRNA compounds with stabilized siRNAs in vitro (in ARPE-19 cells) and in vivo (intravitreal injection in mouse and rabbit eyes). Specifically, we investigated the retinal uptake, distribution, efficacy, and preliminary safety of sd-rxRNAs. RESULTS: Treatment with sd-rxRNAs resulted in uniform cellular uptake and full retina penetration in both animal models while no detectable cellular uptake was observed with stabilized siRNAs either in vitro or in vivo. Further, both in vitro and in vivo delivery (without any transfection reagent or formulation) resulted in a significant reduction of the targeted mRNA levels, which lasted 14-21 days in vivo. Retinal morphology and function were unaltered following a single administration of sd-rxRNAs. CONCLUSION: These data support the potential of developing sd-rxRNAs as a therapeutic for ocular disease.

Potent and systematic RNAi mediated silencing with single oligonucleotide compounds.

RNA interference (RNAi) has been established as an important tool for functional genomics studies and has great promise as a therapeutic intervention for human diseases. In mammalian cells, RNAi is conventionally induced by 19-27-bp RNA duplexes generated by hybridization of two complementary oligonucleotide strands (oligos). Here we describe a novel class of RNAi molecules composed of a single 25-28-nucleotide (nt) oligo. The oligo has a 16-nt mRNA targeting region, followed by an additional 8-10 nt to enable self-dimerization into a partially complementary duplex. Analysis of numerous diverse structures demonstrates that molecules composed of two short helices separated by a loop can efficiently enter and activate the RNA-induced silencing complex (RISC). This finding enables the design of highly effective single-oligo compounds for any mRNA target.

Modified dsRNAs that are not processed by Dicer maintain potency and are incorporated into the RISC.

Chemical modification of RNA duplexes can provide practical advantages for RNA interference (RNAi) triggering molecules including increased stability, safety and specificity. The impact of nucleotide modifications on Dicer processing, RISC loading and RNAi-mediated mRNA cleavage was investigated with duplexes >or=25 bp in length. It is known that dsRNAs >or=25 bp are processed by Dicer to create classic 19-bp siRNAs with 3'-end overhangs. We demonstrate that the presence of minimal modification configurations on longer RNA duplexes can block Dicer processing and result in the loading of the full-length guide strand into RISC with resultant mRNA cleavage at a defined site. These longer, modified duplexes can be highly potent gene silencers, with EC50s in the picomolar concentration range, demonstrating that Dicer processing is not required for incorporation into RISC or potent target silencing.

The kinetic mechanism of the human bifunctional enzyme ATIC (5-amino-4-imidazolecarboxamide ribonucleotide transformylase/inosine 5'-monophosphate cyclohydrolase). A surprising lack of substrate channeling.

5-Amino-4-imidazolecarboxamide ribonucleotide transformylase/IMP cyclohydrolase (ATIC) is a bifunctional protein possessing two enzymatic activities that sequentially catalyze the last two steps in the pathway for de novo synthesis of inosine 5'-monophosphate. This bifunctional enzyme is of particular interest because of its potential as a chemotherapeutic target. Furthermore, these two catalytic activities reside on the same protein throughout all of nature, raising the question of whether there is some kinetic advantage to the bifunctionality. Rapid chemical quench, stopped-flow absorbance, and steady-state kinetic techniques were used to elucidate the complete kinetic mechanism of human ATIC. The kinetic simulation program KINSIM was used to model the kinetic data obtained in this study. The detailed kinetic analysis, in combination with kinetic simulations, provided the following key features of the enzyme reaction pathway. 1) The rate-limiting step in the overall reaction (2.9 +/- 0.4 s(-1)) is likely the release of tetrahydrofolate from the formyltransferase active site or a conformational change associated with tetrahydrofolate release. 2) The rate of the reverse transformylase reaction (6.7 s(-1)) is approximately 2-3-fold faster than the forward rate (2.9 s(-1)), whereas the cyclohydrolase reaction is essentially unidirectional in the forward sense. The cyclohydrolase reaction thus draws the overall bifunctional reaction toward the production of inosine monophosphate. 3) There was no kinetic evidence of substrate channeling of the intermediate, the formylaminoimidazole carboxamide ribonucleotide, between the formyltransferase and the cyclohydrolase active sites.