Last week, Alnylam Pharmaceuticals reported that its researchers were able to use intravenously delivered siRNAs to knock down in rats the gene encoding apolipoprotein B, a protein essential for the formation of low-density lipoproteins and associated with cholesterol metabolism (see RNAi News, 11/12/2004).
In press releases and conference calls with investors and the media, the company touted the publication of these data in the journal Nature as a first. But statements from an industry analyst, as well as an executive from a rival RNAi-based drugs developer, are calling Alnylam’s claim into question.
Alnylam’s claims of being the first to publish in vivo data demonstrating that “RNAi via a clinically relevant systemic delivery is able to significantly lower the expression of a clinically relevant target … are false, given that there are published articles fitting this exact description,” Jonathan Aschoff, an analyst with Brean Murray, wrote in a research report last week.
Specific claims from Alnylam include one from Hans-Peter Vornlocher, lead author of the Nature paper and vice president of research at Alnylam’s European division, who said in a statement dated Nov. 10 that “we have meaningfully advanced the field of RNAi in this first ever demonstration of RNAi mediated by systemically delivered siRNAs, providing tangible evidence of the broad potential for RNAi therapeutics.”
Additionally, on that same day during a conference call discussing the company’s third-quarter financial results, Alnylam President and CEO John Maraganore said that “we are extremely proud of [the paper] … since it is the first peer-reviewed and published data demonstrating RNAi-mediated gene silencing in mammals by a method that potentially can be applied to systemic RNAi therapeutics for human disease.”
In his research note, Aschoff cites a paper by Stephanie Filleur and colleagues at the French National Center for Scientific Research (CNRS) published in Cancer Research on July 15, 2003, describing the use of “low doses of siRNA administered by a systemic route [in mice via the tail vein, or directly into tumor tissue, to] penetrate into tumors and control the expression of target genes to produce phenotypic effects.”
According to the paper, “systemic in vivo administration of crude anti-VEGF siRNA reduced the growth of unaltered fibrosarcoma tumor cells [in mice], and when the anti-VEGF siRNA was expressed from tumor cells themselves, such inhibition was synergistic with the inhibitory effects derived from … the secretion [of the anti-angiogenic molecule thrombospondin-1] … by the tumor cells.
“Anti-VEGF siRNA delayed the emergence of TSP1-resistant tumors and strikingly reduced their subsequent growth rate,” the paper states.
Florence Cabon, a corresponding author on the Cancer Research paper, noted in an e-mail to RNAi News that in the study “very low doses (125µg/kg/day, 400 times less than in the Alnylam study) of siRNA allowed 70 [percent] reduction in VEGF synthesis and impaired tumor growth. No vectorization [agents] such as cholesterol or liposomes were used in our study; siRNA were only diluted in saline.”
Aschoff added in his research note that in addition to the Cancer Research paper, “Sirna has presented data several times in recent past ... describing the significant knockdown of intended clinically relevant targets such as hepatitis B virus and VEGF-R1.”
Aschoff told RNAi News that he does not hold shares of Sirna, but that Brean Murray does have an investment banking relationship with the company.
Alnylam spokeswoman Kathryn Morris told RNAi News in an e-mail this week that Vornlocher has said that “the reasons that [the Nature paper] is a first are: in vivo silencing of an endogenous gene using a clinically relevant route of administration with confirmation of RNAi-mediated cleavage of mRNA using 5’ RACE.”
Morris added in the e-mail that “all other in vivo reports, to the best of our knowledge, used local delivery (which we also think will work), a gene therapy approach, or non-clinically relevant methods such as hydrodynamic injection or transfection reagents.”
Alnylam’s position was further supported by the Beckman Research Institute’s John Rossi.
Rossi told RNAi News in an e-mail this week that the Cancer Research paper, “although showing in vivo efficacy of an unmodified, but liposome-delivered siRNA, was not as thorough as the study by the Alnylam group, which showed tissue distribution of the siRNA and actual evidence of RNAi mediated cleavage.
“The [Cancer Research] manuscript by Filleur et al., although tantalizing, uses a somewhat artificial system of implanted tumors with a luciferase target or measured tumor volume in nude mice, [and] did not show knockdown at the level of RNA in vivo as did the Alnylam study, which targeted an endogenous transcript and demonstrated RNA knockdown,” Rossi wrote in his e-mail.
Sirna COO Nassim Usman countered in an e-mail to RNAi News that “with the modifier ... the confirmation of cleavage ... [Alnylam’s claim] is technically true. However, endogenous gene silencing without confirmation of cleavage, but with molecular RNA and protein knockdown, has been published,” he noted, citing the Cancer Research paper and various scientific meetings.
Usman added in his e-mail that the “detection of the cleavage products is a very positive aspect of the paper, confirming [that] siRNA [had] mediated target destruction. On the other hand, the dose was extremely high — 50 mg/kg is not a clinically viable dose — and there was no dose response. On balance, the [Nature] paper advances the siRNA field and points to the promise of siRNA-based therapeutics becoming a reality,” he concluded.