By Doug Macron
RXi Pharmaceuticals this week announced the release of preclinical data demonstrating that its self-delivering RNAi compounds are efficiently taken up by a variety of cells without the need for a delivery vehicle, and are effective in vivo when administered either systemically or locally.
The company also unveiled data showing that a novel RNAi agent, derived from a single-stranded oligo, can knock down a target on a level equal to standard duplex RNAs. The data were presented at Keystone Symposia's RNA Silencing meeting last week.
While RXi had been focusing much of its effort as of late on an oral siRNA technology, a company official last year indicated that the firm was also making strides with the so-called self-delivering rxRNA molecules, which were acquired from Advirna (see RNAi News, 7/30/2009 & 10/1/2009).
This summer, RXi's then-CEO Tod Woolf told RNAi News that the self-delivering technology involves a combination of four undisclosed chemistries that facilitate the delivery of the molecules into the cytoplasm of a cell, but noted that the company had not yet determined the optimal configuration of those chemistries.
In a poster presented at Keystone, RXi showed how a panel of novel base, sugar, and backbone chemical modifications were screened "in order to determine configurations [that] favor self delivery.
"The simultaneous presence of multiple chemistries is required to achieve efficient cellular uptake in the absence of a delivery vehicle," and the screening revealed a combination of three parameters that resulted in the effective self delivery of the RNAi compounds to HeLa cells, the company added.
RXi also noted that the modification type, extent, and position play a key role in the efficient cellular uptake of the sd-rxRNAs, and that the size of the compounds can have a "major impact on efficacy." While sd-rxRNAs less than 15 base pairs in length were highly effective in silencing a target gene, increasing the length to 21 base pairs led to a "significant loss of efficacy," demonstrating that "reduction in oligonucleotide content is essential for efficient cellular uptake and efficacy."
When looking at delivery to different cell types, RXi found that its sd-rxRNAs were active in a variety of cell lines including difficult-to-transfect ones such as SH-SY5Y cells.
For in vivo applications, RXi's data showed that 50 mg/kg intravenous doses of the molecules, administered to mice once a day for three consecutive days, led to "robust" silencing of the target in the animals' livers.
At Keystone, RXi also presented data on intradermal delivery of the sd-rxRNAs to mice, which it said demonstrated the "rapid and efficient intracellular uptake [of the molecules] into dermal cells and significant reduction of targeted mRNA."
According to the company, efficacy studies were conducted in the skin of mice and rats. "Statistically significant silencing of the targeted mRNA [was] observed with intradermal injections of the … sd-rxRNA" compared with control oligos and saline."
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The data also showed that the silencing activity lasted for at least 48 hours after administration, and that similar levels of silencing were observed with either one or two intradermal doses of the sd-rxRNAs.
RXi noted that local delivery of RNAi agents opens "broad opportunities for therapeutic development in tissues where local administration is an option," including compromised skin, inflammation sites, mucosa, the lung, and the eye.
RXi also released new data on single-oligo RNAi technology it has dubbed rxRNAsolo, which the company said has the potential "to further reduce off-target effects and manufacturing costs, and may thus offer additional advantages for use in research and therapeutic applications" when compared to traditional RNAi molecules.
Interest in single-stranded RNAi molecules has been growing recently. Last April, Alnylam Pharmaceuticals and Isis Pharmaceuticals agreed to collaborate on the development of Isis' own ssRNAi technology (see RNAi News, 4/30/2009).
In October, struggling New Zealand firm Genesis Research and Development spun out its nascent ssRNAi technology to a new company called Solirna BioSciences (see RNAi News, 10/29/2009).
Getting in on the game, RXi last week presented data at Keystone detailing the development of molecules, derived from a single, short oligo sequence, "with silencing potency equal to conventional RNAi triggers."
According to a poster presentation, "it is currently perceived that two components are required to activate the RNAi machinery: the double-stranded nucleic acid motif, which is required for recognition by RNA-induced silencing complex components, and the guide strand, which serves as the mRNA binding co-factor in the activated RISC."
Taking these features into account, RXi said it developed novel molecules that have at least 16 bases complementary to a target mRNA, as well as a 3' extension that allows efficient self annealing, and prove to be highly active.
"To our surprise, the new single-stranded molecules yielded potent RNAi compounds with activity comparable to more traditional double-stranded RNAi triggers," the poster states. "Further analysis, however, revealed that the newly synthesized molecules can form a homo-duplex in addition to the monomer hairpin, and that it is the homo-duplex that is predominantly responsible for the gene-silencing activity."
Overall, the rxRNAsolo molecule consists of a homodimer of a single oligo as short as 27 nucleotides, according to RXi. The guide portion, which is 18 to 19 nucleotides long, is complementary to the target gene. The 5' and 3' portions, which can potentially be as few as 8 nucleotides long, are inverted palindromes, which are required for the formation of the homodimer.
RXi noted that the rxRNAsolo design is also amenable to a dual-targeting RNAi configuration that allows the knockdown of two targets.