Alnylam Pharmaceuticals, Inc., a leading RNAi therapeutics company, today presented new clinical data from its ALN-VSP Phase I liver cancer trial in a presentation at the Dana-Farber Cancer Institute in Boston. Results from molecular analysis of human biopsy samples showed achievement of pharmacologically relevant siRNA drug levels in tissues. In addition, using a highly precise polymerase chain reaction (PCR)-based technique known as 5'- rapid amplification of cDNA ends (5'-RACE), analysis of human tissue samples showed proof of RNAi-mediated target mRNA cleavage, and thus RNAi in man with the systemically delivered RNAi therapeutic. These results provide significant human proof of concept for Alnylam's efforts in advancing RNAi therapeutics to patients.
"These data provide conclusive evidence that RNAi can be harnessed in man and, as such, represent a notable and important milestone in the advancement of RNAi therapeutics as a potential new class of medicines," said Phillip Sharp, Ph.D., Institute Professor, The Koch Institute for Integrative Cancer Research, MIT, and Chairman of Alnylam Scientific Advisory Board and Alnylam Director. "I applaud Alnylam scientists, clinicians, and their collaborators for reaching this important achievement and I look forward to the results of their continued efforts."
Post-treatment tumor biopsies from eight patients were analyzed for ALN-VSP drug levels. All biopsy samples were obtained on a voluntary basis using a CT-guided procedure. Five of these biopsy samples were obtained from tumor in the liver and three were taken from tumor located outside the liver in patients receiving doses of ALN-VSP ranging from 0.4 to 1.25 mg/kg. The two siRNAs targeting VEGF and KSP that comprise ALN-VSP were detected in almost all of these biopsy samples at concentrations ranging from 0.3 to 142 ng/g tissue. These levels of siRNA are pharmacologically relevant since in pre-clinical studies with systemically delivered siRNAs, a tissue level of 1 ng/g has been shown to be associated with 50% target gene silencing (Landesman et al., Silence, 1:16, 2010).
RNAi is an endogenous cellular enzymatic process whereby siRNAs mediate sequence-dependent cleavage of target mRNAs; cleavage of the target mRNA is highly precise, occurring exactly 10 nucleotide positions from the 5'-end of the siRNA antisense strand. In order to establish RNAi-mediated effects in vivo, 5'RACE has been established as a robust method for detecting the specific cleavage products of siRNA pharmacology in pre-clinical animal models (Soutschek et al., Nature, 432: 173-8, 2004; Zimmermann et al., Nature, 441: 111-4, 2006). As a PCR-based technique, 5'RACE is not a quantitative measurement; a positive result simply confirms that there is evidence of RNAi-mediated target mRNA cleavage.
As currently analyzed, three patients had biopsies that were of sufficient quality to permit blinded 5' RACE analysis for the VEGF target mRNA following the first dose of ALN-VSP; additional analyses are ongoing. All three biopsy samples were from the 0.4 mg/kg dose group, and post-treatment biopsy samples were comprised of 80% to 100% normal liver. In two patients whose post-treatment biopsies were performed two days after dosing, the 5' RACE assay combined with deep sequencing showed that approximately 27% and 29% of all VEGF-derived mRNA fragments corresponded exactly to the predicted RNAi-mediated cleavage product based on the VEGF siRNA sequence. By contrast, a pre-dose biopsy available for one of those patients contained only approximately 1% predicted VEGF cleavage product, and analysis of banked normal liver and tumor samples from untreated patients showed a background level of only 0.1% to 0.7%. Compared to these low background levels, the amount of predicted VEGF cleavage product in the two post-treatment biopsies was highly statistically significant (p<0.0001). In the third patient at 0.4 mg/kg whose post-treatment biopsy was obtained 7 days post-dose, there was no detectable increase in the predicted VEGF cleavage product compared to the pre-dose biopsy. These positive 5' RACE data from human biopsies provide clear evidence for RNAi in man following systemic administration of lipid nanoparticle (LNP)-formulated siRNA.
"Achievement of these results is very important for Alnylam's overall RNAi therapeutics efforts, and has many implications. First, we have been able to quantify significant human tissue levels of siRNAs at concentrations that exceed our 1 ng/g target level that is associated with effective target gene silencing in pre-clinical animal models. More importantly, we have demonstrated proof of RNAi mechanism in man with LNP delivery of siRNAs, an important milestone in overall human translation of RNAi therapeutics," said John Maraganore, Ph.D., Chief Executive Officer of Alnylam. "As this systemic delivery platform is employed in our pipeline of clinical and development-stage programs, we believe that we have now made a major leap forward in advancing RNAi therapeutics to patients."