Supplementary Figures PDF file - 114K, Includes sequence information on KSP and VEGF siRNAs, murine Hep3B tumor model data with ALN-VSP, ALN-VSP Phase I study design, KSP and VEGF mRNA levels in tumor cell lines and normal liver, and comparison of ALN-VSP PK data in cancer patients and non-human primates

Supplementary Methods and Legends PDF file - 72K, Includes detailed methods for 5' RACE assay and DCE-MRI scans, as well as figure legends for the six supplementary figures

Supplementary Tables PDF file - 67K, Includes data showing effect of ALN-VSP on spleen in non-human primates, tumor response data from the Phase I trial, and safety data (including adverse events and dose-limiting toxicities) from the Phase I trial

Background:Familial amyloid polyneuropathy (FAP) is a progressive, fatal disease caused by deposition of transthyretin (TTR). Patisiran is an investigational systemically administered lipid nanoparticle formulation of a small interfering RNA (siRNA) targeting wild-type and mutant TTR. A Phase 2 trial of patisiran in patients with FAP showed >80[percnt] sustained knockdown of serum TTR with a generally favorable safety profile. Methods: The Phase 2 open-label extension (OLE; NCT01961921) study of patisiran in patients with FAP was initiated in 2013. The primary objective is to evaluate the safety of intravenous patisiran 0.3 mg/kg administered q3w for 2 years. Secondary objectives evaluated every 6 months include assessment of TTR levels, mNIS+7 neurologic impairment score, and quality of life (QOL).Results: Twenty-seven patients were enrolled; median age 64 years (range: 29-77 years). Chronic dosing with patisiran has been generally well tolerated out to 21 months. Five patients experienced serious adverse events unrelated to study drug. Flushing and infusion-related reactions were observed in 22.2[percnt] and 18.5[percnt] of the patients, respectively; these were mild in severity, and did not result in any discontinuations. Sustained mean serum TTR lowering of approximately 80[percnt] for over 18 months was achieved, with mean maximal knockdown of 91[percnt]. Neurologic impairment scores were stable at 12 months with a mean change in mNIS+7 and NIS of -3.1 and 0.2 points, respectively; this compares favorably to the 10-18 point increase in neurologic impairment scores estimated at 12 months from prior FAP studies in a patient population with similar baseline NIS. Additional clinical measures, including EQ-5D QOL, remained unchanged at 12 months.Conclusion: Twelve-month data from this patisiran Phase 2 OLE study are consistent with the therapeutic hypothesis that TTR knockdown has the potential to halt neuropathy progression. As of Oct 2015, dosing continues in 26 patients; 18 -month results will be presented. Disclosure: Dr. Adams has received research support from Alnylam Pharmaceuticals. Dr. Coelho has received research support from Alnylam Pharmaceuticals. Dr. Conceicao has received research support from Alnylam Pharmaceuticals. Dr. Waddington Cruz has received research support from Alnylam Pharmaceuticals. Dr. Schmidt has received research support from Alnylam Pharmaceuticals. Dr. Juan has received research support from Alnylam Pharmaceuticals. Dr. Josep Campistol has received research support from Alnylam Pharmaceuticals. Dr. Pouget has nothing to disclose. Dr. Berk has received research support from Alnylam Pharmaceuticals. Dr. Falzone has received personal compensation for activities with Alnylam Pharmaceuticals. Dr. White has received personal compensation for activities with Alnylam Pharmaceuticals. Dr. Bettencourt has received personal compensation for activities with Alnylam Pharmaceuticals as an employee. Dr. Jeff Cehelsky has received personal compensation for activities with Alnylam Pharmaceuticals. Dr. Nochur has received personal compensation for activities with Alnylam Pharmaceuticals. Dr. Vaishnaw has received personal compensation for activities with Alnylam Pharmaceuticals. Dr. Gruis has received personal compensation from Alnylam Pharmaceuticals. Dr. Goolob has received personal compensation for activities with Alnylam Pharmaceuticals. Dr. Suhr has received research support from Alnylam Pharmaceuticals.

Familial amyloid polyneuropathy (FAP) is a progressive disease. Patisiran is an investigational small interfering RNA (siRNA) targeting TTR. The primary objective of the Phase 2 study is to evaluate the safety of 0.3 mg/kg patisiran administered intravenously once every 3 weeks. Twenty-seven patients were enrolled; the mean duration of treatment was 7 months (range 3–12), with 282 doses administered (median of 11 doses/patient). Chronic dosing with patisiran has been generally well tolerated. Two patients experienced serious adverse events regarded as being unrelated to study drug. Infusion-related reactions were observed in 14.8% of the patients, were mild in severity, and did not result in any discontinuations. Sustained TTR lowering of at least 80% was achieved based on serial TTR measurements for over 9 months, with further nadir of up to 89.6% between doses. Neurologic impairment scores were stable after 6 months of treatment with patisiran. A mean decrease from baseline in mNIS+7 of 0.95 points (N=19) observed in this study compared favorably to the estimated increase of 7–10 points in mNIS+7 at 6 months from prior FAP studies in a patient population with similar baseline NIS values. Dosing continues in all patients, and 12–month results will be presented.

Background Familial Amyloid Polyneuropathy (FAP) is a progressive disease caused by deposition of transthyretin (TTR). Patisiran is an investigational, small interfering RNA (siRNA) inhibiting TTR. This abstract highlights patisiran9s long-term safety. Methods Phase 2 OLE study to evaluate patisiran9s safety. Patisiran9s effect on serum TTR levels, impact on neuropathy impairment scores and QOL were assessed. Results 27 patients with FAP enrolled; median age 64 years. Patisiran was generally well tolerated out to 23-months. Five patients experienced SAEs (unrelated) including one discontinuation (gastroesophageal cancer); patient subsequently died. Flushing (25.9%) and infusion-related reactions (18.5%) were mild in severity; no discontinuations resulted. Approximately 80% sustained mean serum TTR lowering resulted with a mean nadir of up to 93% between doses. Among the 20 evaluable patients, neuropathy impairment scores were stable through 18-months; mean change in mNIS+7 and NIS of 1.7 and 4.2 points, respectively. This compares favorably to 17–26 point mNIS+7/NIS increase estimated at 18-months from prior FAP studies. Stabilization of QOL measures and improvement of distal thigh sweat gland nerve fiber density observed. Conclusion Data demonstrates that 18-months of patisiran administration was generally well tolerated, resulted in sustained mean serum TTR lowering, supporting the hypothesis that TTR knockdown potentially halts neuropathy progression.

Background: Familial amyloid polyneuropathy (FAP) is a progressive, fatal disease caused by deposition of transthyretin (TTR). Patisiran is an investigational systemically administered lipid nanoparticle formulation of a small interfering RNA (siRNA) targeting wild-type and mutant TTR. A Phase 2 trial of patisiran in FAP patients showed >80[percnt] sustained knockdown of serum TTR with a generally favorable safety profile. Methods: The Phase 2 open-label extension (OLE) study of patisiran in FAP patients was initiated in 2013. The primary objective is to evaluate the safety of intravenous patisiran 0.3 mg/kg administered q3w. Secondary objectives include assessment of TTR levels, mNIS+7 neurologic impairment score, and quality of life (QOL). Results: Twenty-seven patients were enrolled; the mean duration of treatment was 7 months (range 3-12), with 282 doses administered (median of 11 doses/patient). Chronic dosing has been generally well tolerated. Two patients experienced serious adverse events regarded as being unrelated to study drug. Mild infusion-related reactions that did not cause discontinuation were observed in 14.8[percnt] of the patients. No clinically significant changes in liver function, renal function or hematological parameters were observed. Sustained TTR lowering of at least 80[percnt] for over 9 months was achieved, with nadir of up to 89.6[percnt] between doses. Neurologic impairment was stable after 6 months of patisiran in patients with or without concurrent TTR tetramer stabilizers. A mean decrease from baseline in mNIS+7 of 0.95 points (N=19) observed in this study compared favorably to the estimated increase of 7-10 points in mNIS+7 at 6 months from prior studies. Stabilization of QOL, neurologic and cardiac assessments was also observed. Conclusion: The 6-month data from the ongoing patisiran Phase 2 OLE study show preliminary evidence of safety and disease stabilization in FAP patients. Dosing continues and 12-month data will be presented. This study is supported by Alnylam Pharmaceuticals. Disclosure: Dr. Coelho has nothing to disclose. Dr. Suhr has nothing to disclose. Dr. Conceicao has received personal compensation for activities with GLG Councils. Dr. Waddington Cruz has nothing to disclose. Dr. Schmidt has nothing to disclose. Dr. Juan has nothing to disclose. Dr. Campistol has received personal compensation for activities with Wyeth Pharmaceuticals, Astellas Pharma, Roche, and Novartis as a consultant. Dr. Pouget has nothing to disclose. Dr. Berk has received personal compensation for activities with Banyu Pharmaceutical Co, Ltd. as a lecturer. Dr. Falzone has nothing to disclose. Dr. White has nothing to disclose. Dr. Bettencourt has nothing to disclose. Dr. Cehelsky holds stock and/or stock options. Dr. Nochur holds stock and/or stock options. Dr. Vaishnaw has nothing to disclose. Dr. Gollob has nothing to disclose. Dr. Adams has received personal compensation for activities with Pfizer and ALNYLAM as a speaker and/or a consultant.

Phase 2 open-label extension study (ole) of patisiran, an investigational sIRNA investigational agent for familial amyloid polyneuropathy (fap)

Familial Amyloidotic Polyneuropathy (FAP) is a rare, inherited, progressively debilitating disease with a high unmet medical need. The purpose of this analysis is to assess the impact of FAP on healthcare resource utilization, quality of life, employment status, and activities of daily living (ADLs). A Phase 2 open-label extension study of patisiran in FAP patients was utilized to collect patient-reported outcomes, including EQ-5D, Rasch-built Overall Disability Scale (R-ODS), and a healthcare resource utilization questionnaire. The study included 27 patients, 18 males and 9 females, 29-77 years of age. Baseline data are presented for 14 patients with a Polyneuropathy Disability (PND) Score I and 13 patients with a PND Score ≥ II. Characterized by FAP Stage, 24 patients are FAP Stage 1 and 3 patients are FAP Stage 2. Two patients (PND Score ≥ II) reported a total of six hospitalizations due to FAP in the past 12 months, each for 3 or more nights in duration. Mean EQ-5D scores were 0.82 (PND Score I) and 0.74 (PND Score ≥ II). Patients reported their perceived health status on the EQ-VAS with mean scores of 75 (PND Score I) and 60 (PND Score ≥ II). Ten patients (8/10 PND Score ≥ II) reported they cannot work because of FAP. Patients also reported inability to perform various ADLs. Most commonly, 77% of patients with PND Score ≥ II cannot stand for hours (14% in PND Score I) and 69% cannot run (21% in PND Score I). FAP patients experience considerable burden of illness early in the course of disease and this burden increases with disease progression. The factors described will be influential in the development of a comprehensive FAP cost-consequence analysis. Additional parameters may also be needed to fully capture the totality of burden.

Interim results from phase ii trial of aln-ttr02, a novel RNAi therapeutic for the treatment of familial amyloidotic polyneuropathy

BACKGROUND:Transthyretin amyloidosis is caused by the deposition of hepatocyte-derived transthyretin amyloid in peripheral nerves and the heart. A therapeutic approach mediated by RNA interference (RNAi) could reduce the production of transthyretin.METHODS:We identified a potent antitransthyretin small interfering RNA, which was encapsulated in two distinct first- and second-generation formulations of lipid nanoparticles, generating ALN-TTR01 and ALN-TTR02, respectively. Each formulation was studied in a single-dose, placebo-controlled phase 1 trial to assess safety and effect on transthyretin levels. We first evaluated ALN-TTR01 (at doses of 0.01 to 1.0 mg per kilogram of body weight) in 32 patients with transthyretin amyloidosis and then evaluated ALN-TTR02 (at doses of 0.01 to 0.5 mg per kilogram) in 17 healthy volunteers.RESULTS:Rapid, dose-dependent, and durable lowering of transthyretin levels was observed in the two trials. At a dose of 1.0 mg per kilogram, ALN-TTR01 suppressed transthyretin, with a mean reduction at day 7 of 38%, as compared with placebo (P=0.01); levels of mutant and nonmutant forms of transthyretin were lowered to a similar extent. For ALN-TTR02, the mean reductions in transthyretin levels at doses of 0.15 to 0.3 mg per kilogram ranged from 82.3 to 86.8%, with reductions of 56.6 to 67.1% at 28 days (P<0.001 for all comparisons). These reductions were shown to be RNAi-mediated. Mild-to-moderate infusion-related reactions occurred in 20.8% and 7.7% of participants receiving ALN-TTR01 and ALN-TTR02, respectively.CONCLUSIONS:ALN-TTR01 and ALN-TTR02 suppressed the production of both mutant and nonmutant forms of transthyretin, establishing proof of concept for RNAi therapy targeting messenger RNA transcribed from a disease-causing gene. (Funded by Alnylam Pharmaceuticals; ClinicalTrials.gov numbers, NCT01148953 and NCT01559077.).

Abstract RNA interference (RNAi) is a potent and specific mechanism for regulating gene expression. Harnessing RNAi to silence genes involved in disease holds promise for the development of a new class of therapeutics. Delivery is key to realizing the potential of RNAi, and lipid nanoparticles (LNP) have proved effective in delivery of siRNAs to the liver and to tumors in animals. To examine the activity and safety of LNP-formulated siRNAs in humans, we initiated a trial of ALN-VSP, an LNP formulation of siRNAs targeting VEGF and kinesin spindle protein (KSP), in patients with cancer. Here, we show detection of drug in tumor biopsies, siRNA-mediated mRNA cleavage in the liver, pharmacodynamics suggestive of target downregulation, and antitumor activity, including complete regression of liver metastases in endometrial cancer. In addition, we show that biweekly intravenous administration of ALN-VSP was safe and well tolerated. These data provide proof-of-concept for RNAi therapeutics in humans and form the basis for further development in cancer. Significance: The findings in this report show safety, pharmacokinetics, RNAi mechanism of action, and clinical activity with a novel first-in-class LNP-formulated RNAi therapeutic in patients with cancer. The ability to harness RNAi to facilitate specific multitargeting, as well as increase the number of druggable targets, has important implications for future drug development in oncology. Cancer Discov; 3(4); 406–17. ©2012 AACR. This article is highlighted in the In This Issue feature, p. 363

Transthyretin (TTR) cardiac amyloidosis is caused by the deposition of liver-derived mutant and/or wild-type TTR in the myocardium, leading to heart failure and death. The hereditary form, known as familial amyloidotic cardiomyopathy (FAC), is most frequently associated with the Val122Ile mutation in older African-American and Afro-Caribbean males and is estimated to affect at least 40,000 people worldwide. Treatment options are limited, and include medical management of heart failure symptoms, as well as heart transplantation in a small number of patients young enough to undergo this procedure. ALN-TTRsc, which is being developed to treat FAC, is a subcutaneously administered RNA interference (RNAi) therapeutic. It is comprised of a small interfering RNA (siRNA) targeting both mutant and wild-type TTR mRNA conjugated to a N-acetylgalactosamine (GalNAc) ligand that enables receptor-mediated delivery to the liver via the asialoglycoprotein receptor expressed on hepatocytes. In non-human primates, repeat-dose subcutaneous administration of ALN-TTRsc resulted in potent and sustained suppression of serum TTR protein, with approximately 80% reduction observed at doses as low as 2.5 mg/kg. In single- and multi-dose pre-clinical safety studies, ALN-TTRsc was found to be generally safe and well tolerated at doses as high as 300 mg/kg in non-human primates. In March 2013, a Phase I, randomized, double-blind, placebo-controlled, single and multi-dose, dose escalation study was initiated in normal healthy volunteers with an anticipated enrollment of up to 40 subjects to evaluate the safety, tolerability, pharmacokinetics and pharmacodynamics of subcutaneously administered ALN-TTRsc before proceeding to further development in FAC. In the multi-dose phase, subjects receive 5 daily doses as induction followed by 5 additional weekly maintenance doses. The pharmacodynamic effect of ALN-TTRsc is followed through serial measurements of serum TTR. In this presentation, we will provide an update on the results of this ongoing first-in-humans trial.

3062 Background: ALN-VSP02 is an RNA interference (RNAi) therapeutic comprised of lipid nanoparticle-formulated small interfering RNAs targeting vascular endothelial growth factor (VEGF)-A and kinesin spindle protein (KSP). In a phase 1 trial, ALN-VSP02 administered as an iv infusion q2 wks was well-tolerated and showed evidence of anti-VEGF pharmacology and antitumor activity. Methods: Patients treated on the phase I trial with stable disease (SD) or better after 4 months (8 doses) were eligible to continue on an extension study until disease progression. Main objectives included continued evaluation of safety/tolerability and assessment of disease response. Results: Seven of 37 patients (18.9%) evaluable for response went onto the extension study, including 1 of 7 (14.2%) at 0.4 mg/kg, 2 of 5 (40%) at 0.7 mg/kg, and 4 of 11 (36.3%) at 1.0 mg/kg. All had progressed after one or more prior therapies. Tumor types included head and neck squamous cell carcinoma, angiosarcoma, endometrial cancer, renal cell carcinoma (RCC, N=2), and pancreatic neuroendocrine tumor (PNET, N=2). At the time of enrollment, 6 had SD and one (endometrial cancer with multiple liver metastases) had an unconfirmed partial response (PR). The average length of time on treatment (including phase I and extension studies) was 9.5 months (range 5-19). As of January 2012, 3 patients remain on study, including the endometrial cancer patient with an ongoing PR who has had >80% tumor regression after 19 months of treatment at 0.7 mg/kg and two patients with RCC and PNET with continued SD after nearly 1 year of treatment at 1.0 mg/kg. The other patients with RCC and PNET at 1.0 mg/kg with SD came off after 8.5 and 5.5 months, respectively, for adverse events that included fatigue or elevated alkaline phosphatase. A decrease in spleen volume, likely an on-target effect and not associated with any adverse events, occurred to a greater degree on the extension study than on the phase I trial and was most pronounced in patients receiving ≥ 12 doses. Conclusions: ALN-VSP02 has preliminary activity against endometrial cancer, RCC and PNET and a favorable safety profile that permits chronic dosing. Phase II trials are warranted in these and other VEGF-overexpressing tumors.

3025 Background: ALN-VSP02 is a RNA interference (RNAi) therapeutic comprised of lipid nanoparticle-formulated small interfering RNAs (siRNAs) targeting the expression of vascular endothelial growth factor (VEGF)-A and kinesin spindle protein (KSP). Methods: A multi-center, open label, phase I dose-escalation trial of ALN-VSP02 administered as a 15-minute iv infusion q2 wks was initiated in patients (pts) with advanced solid tumors and at least one measurable liver lesion. Main objectives included evaluation of safety/tolerability and assessment of PK/PD. Results: Thirty-one pts were enrolled across 7 dose levels (0.1-1.5 mg/kg); median age 57 yrs, all with multiple prior therapies. A total of 140 doses were administered, mean of 4.5 (range 1-17). Treatment was generally well-tolerated, with no dose-dependent trends in clinical or laboratory adverse events. One on-study death (liver failure in a pt with near complete replacement of the liver by tumor) deemed possibly related to treatment occurred at 0.7 mg/kg. Low-grade acute infusion reactions occurred in 10% of pts and were managed with slowing of infusion. Dose-limiting toxicities at doses >0.7 mg/kg included 1 episode each of reversible grade 3 thrombocytopenia (1.25 mg/kg) and hypokalemia (1.5 mg/kg). Plasma PK showed dose-proportional AUC and Cmax. Post-treatment biopsies from 10 pts (7 liver and 3 extrahepatic tumors) showed pharmacologically relevant concentrations (0.3-142 ng/g tissue) of both siRNAs. Molecular evidence of RNAi-mediated VEGF mRNA cleavage was shown in liver (n=2 at 0.4 mg/kg) and in an extrahepatic tumor (ovarian cancer at 1.25 mg/kg) through use of the 5’ RACE assay on tumor biopsies. Additional evidence for an anti-VEGF effect with ALN-VSP02 included a decrease in Ktrans of at least 40% by DCE-MRI in 56% of evaluable pts. Among 27 pts evaluable for response, 8.3% (1 of 12) at doses ≤ 0.4 mg/kg had stable disease (SD) for at least 2 mo compared to 46.6% (7/15) with SD (n=6) or PR (n=1, endometrial cancer with liver metastases) at doses ≥ 0.7 mg/kg. Conclusions: ALN-VSP02 is well-tolerated and has antitumor activity. Pharmacodynamic data are consistent with an anti-VEGF effect, and 1.25 mg/kg q2wks is the recommended phase II dose.

3042 Background: Malignancies involving the liver represent a significant unmet medical need. ALN-VSP02 is a novel RNA interference (RNAi) therapeutic comprised of lipid nanoparticle-formulated small interfering RNAs (siRNAs) targeting the expression of vascular endothelial growth factor (VEGF)-A and kinesin spindle protein (KSP). Methods: A multicenter, open label, phase 1 dose escalation trial of ALN-VSP02 administered as a 15-minute iv infusion every two weeks was initiated in March 2009. Patients with advanced solid tumors are eligible if they have at least one measurable liver lesion and adequate liver function. The study uses a 3+3 design with 8 potential dose levels: 0.1, 0.2, 0.4, 0.7, 1.0, 1.25, 1.5, and 1.7 mg/kg. The primary objective is evaluation of safety and tolerability. Secondary objectives include assessment of PK and pharmacodynamic (PD) activity through DCE-MRI, biomarkers of angiogenesis, tumor biopsies, and response rate. Results: As of December 2009, 12 patients (most with colorectal cancer) have been treated on the first 4 dose levels. Forty-one doses have been administered; 0.1-0.4 mg/kg ALN-VSP02 was well- tolerated, with no hepatotoxicity. The only significant adverse event (AE) was a grade 2 infusion reaction in one patient at 0.4 mg/kg that responded to slowing of the infusion. At 0.7 mg/kg, a patient with pancreatic neuroendocrine tumor extensively involving both lobes of the liver died of hepatic failure following the second dose; this was deemed possibly related to study drug. Two additional patients treated at 0.7 mg/kg did not exhibit hepatotoxicity or any other significant AEs. PK showed Cmax and AUC that were dose proportional with no accumulation. Serial DCE-MRI scans performed 2-9 days after the first dose on 8 of the 12 patients showed a ≥40% decline in Ktrans in 12 of 15 liver tumors (80%) evaluated. This decline in blood flow was associated with extensive tumor necrosis in the patient with the neuroendocrine tumor. Conclusions: ALN-VSP02 was well-tolerated by the majority of patients across the first 4 dose levels. DCE-MRI results show preliminary evidence of an anti-VEGF effect. Accrual is continuing, and additional safety and PD data will be forthcoming. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Alnylam Pharmaceuticals Alnylam Pharmaceuticals Alnylam Pharmaceuticals Alnylam Pharmaceuticals