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Vutrisiran (ALN-TTRsc02) is a liver-directed, investigational, small interfering ribonucleic acid drug for the treatment of transthyretin (TTR)-mediated amyloidosis. This phase I, randomized, single-blind, placebo-controlled, single ascending dose study evaluated the pharmacodynamics, pharmacokinetics, and safety profile of subcutaneously administered vutrisiran (5-300 mg) in healthy subjects (n = 80). Vutrisiran treatment achieved potent and sustained TTR reduction in a dose-dependent manner, with mean maximum TTR reduction of 57-97%, maintained for ≥ 90 days post dose. Vutrisiran was rapidly absorbed (peak plasma concentration 3-5 hours post dose), had a short plasma half-life (4.2-7.5 hours), and plasma concentrations increased in a dose-proportional manner. Pharmacodynamic and pharmacokinetic results were similar in Japanese and non-Japanese subjects. Vutrisiran had an acceptable safety profile; the most common treatment-related adverse event was mild, transient injection site reactions in four (6.7%) vutrisiran-treated subjects. The favorable pharmacokinetic, pharmacodynamic, and safety results observed here support vutrisiran's continued clinical development.

Purpose The Phase 3 ENDEAVOUR study evaluated revusiran, an investigational RNA interference therapeutic targeting hepatic transthyretin (TTR) production, for treating cardiomyopathy caused by hereditary transthyretin-mediated (hATTR) amyloidosis. Methods Patients with hATTR amyloidosis with cardiomyopathy were randomized 2:1 to receive subcutaneous daily revusiran 500 mg (n = 140) or placebo (n = 66) for 5 days over a week followed by weekly doses. Co-primary endpoints were 6-min walk test distance and serum TTR reduction. Results Revusiran treatment was stopped after a median of 6.71 months; the study Sponsor prematurely discontinued dosing due to an observed mortality imbalance between treatment arms. Eighteen (12.9%) patients on revusiran and 2 (3.0%) on placebo died during the on-treatment period. Most deaths in both treatment arms were adjudicated as cardiovascular due to heart failure (HF), consistent with the natural history of the disease. A post hoc safety investigation of patients treated with revusiran found that, at baseline, a greater proportion of those who died were ≥ 75 years and showed clinical evidence of more advanced HF compared with those who were alive throughout treatment. Revusiran pharmacokinetic exposures and TTR lowering did not show meaningful differences between patients who died and who were alive. Revusiran did not deleteriously affect echocardiographic parameters, cardiac biomarkers, or frequency of cardiovascular and HF hospitalization events. Conclusions Causes for the observed mortality imbalance associated with revusiran were thoroughly investigated and no clear causative mechanism could be identified. Although the results suggest similar progression of cardiac parameters in both treatment arms, a role for revusiran cannot be excluded. Clinical Trial Registration NCT02319005.

Hereditary transthyretin-mediated (hATTR) amyloidosis is an underdiagnosed, progressively debilitating disease caused by mutations in the transthyretin (TTR) gene. V122I, a common pathogenic TTR mutation, is found in 3–4% of individuals of African ancestry in the United States and has been associated with cardiomyopathy and heart failure. To better understand the phenotypic consequences of carrying V122I, we conducted a phenome-wide association study scanning 427 ICD diagnosis codes in UK Biobank participants of African ancestry (n = 6062). Significant associations were tested for replication in the Penn Medicine Biobank (n = 5737) and the Million Veteran Program (n = 82,382). V122I was significantly associated with polyneuropathy in the UK Biobank (odds ratio [OR] = 6.4, 95% confidence interval [CI] 2.6–15.6, p = 4.2 × 10−5), which was replicated in the Penn Medicine Biobank (OR = 1.6, 95% CI 1.2–2.4, p = 6.0 × 10–3) and Million Veteran Program (OR = 1.5, 95% CI 1.2–1.8, p = 1.8 × 10−4). Polyneuropathy prevalence among V122I carriers was 2.1%, 9.0%, and 4.8% in the UK Biobank, Penn Medicine Biobank, and Million Veteran Program, respectively. The cumulative incidence of common hATTR amyloidosis manifestations (carpal tunnel syndrome, polyneuropathy, cardiomyopathy, heart failure) was significantly enriched in V122I carriers compared with non-carriers (HR = 2.8, 95% CI 1.7–4.5, p = 2.6 × 10−5) in the UK Biobank, with 37.4% of V122I carriers having at least one of these manifestations by age 75. Our findings show that V122I carriers are at increased risk of polyneuropathy. These results also emphasize the underdiagnosis of disease in V122I carriers with a significant proportion of subjects showing phenotypic changes consistent with hATTR amyloidosis. Greater understanding of the manifestations associated with V122I is critical for earlier diagnosis and treatment.

For oligonucleotide therapeutics, chemical modifications of the sugar-phosphate backbone are frequently used to confer drug-like properties. Because 2′-deoxy-2′-fluoro (2′-F) nucleotides are not known to occur naturally, their safety profile was assessed when used in revusiran and ALN-TTRSC02, two short interfering RNAs (siRNAs), of the same sequence but different chemical modification pattern and metabolic stability, conjugated to an N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Exposure to 2′-F-monomer metabolites was low and transient in rats and humans. In vitro, 2′-F-nucleoside 5′-triphosphates were neither inhibitors nor preferred substrates for human polymerases, and no obligate or non-obligate chain termination was observed. Modest effects on cell viability and mitochondrial DNA were observed in vitro in a subset of cell types at high concentrations of 2′-F-nucleosides, typically not attained in vivo. No apparent functional impact on mitochondria and no significant accumulation of 2′-F-monomers were observed after weekly administration of two GalNAc–siRNA conjugates in rats for ∼2 years. Taken together, the results support the conclusion that 2′-F nucleotides can be safely applied for the design of metabolically stabilized therapeutic GalNAc–siRNAs with favorable potency and prolonged duration of activity allowing for low dose and infrequent dosing.

International audience; BACKGROUND AND OBJECTIVES: In the rare disease primary hyperoxaluria type 1, overproduction of oxalate by the liver causes kidney stones, nephrocalcinosis, kidney failure, and systemic oxalosis. Lumasiran, an RNA interference therapeutic, suppresses glycolate oxidase, reducing hepatic oxalate production. The objective of this first-in-human, randomized, placebo-controlled trial was to evaluate the safety, pharmacokinetic, and pharmacodynamic profiles of lumasiran in healthy participants and patients with primary hyperoxaluria type 1. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: This phase 1/2 study was conducted in two parts. In part A, healthy adults randomized 3:1 received a single subcutaneous dose of lumasiran or placebo in ascending dose groups (0.3-6 mg/kg). In part B, patients with primary hyperoxaluria type 1 randomized 3:1 received up to three doses of lumasiran or placebo in cohorts of 1 or 3 mg/kg monthly or 3 mg/kg quarterly. Patients initially assigned to placebo crossed over to lumasiran on day 85. The primary outcome was incidence of adverse events. Secondary outcomes included pharmacokinetic and pharmacodynamic parameters, including measures of oxalate in patients with primary hyperoxaluria type 1. Data were analyzed using descriptive statistics. RESULTS: Thirty-two healthy participants and 20 adult and pediatric patients with primary hyperoxaluria type 1 were enrolled. Lumasiran had an acceptable safety profile, with no serious adverse events or study discontinuations attributed to treatment. In part A, increases in mean plasma glycolate concentration, a measure of target engagement, were observed in healthy participants. In part B, patients with primary hyperoxaluria type 1 had a mean maximal reduction from baseline of 75% across dosing cohorts in 24-hour urinary oxalate excretion. All patients achieved urinary oxalate levels ≤1.5 times the upper limit of normal. CONCLUSIONS: Lumasiran had an acceptable safety profile and reduced urinary oxalate excretion in all patients with primary hyperoxaluria type 1 to near-normal levels. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Study of Lumasiran in Healthy Adults and Patients with Primary Hyperoxaluria Type 1, NCT02706886.

ObjectiveTo identify changes in the proteome associated with onset and progression of hereditary transthyretin-mediated (hATTR) amyloidosis, also known as ATTRv amyloidosis, we performed an observational, case-controlled study that compared proteomes of patients with ATTRv amyloidosis and healthy controls.MethodsPlasma levels of >1,000 proteins were measured in patients with ATTRv amyloidosis with polyneuropathy who received either placebo or patisiran in a Phase 3 study of patisiran (APOLLO), and in healthy controls. The effect of patisiran on the time profile of each protein was determined by linear mixed model at 0, 9, and 18 months. Neurofilament light chain (NfL) was further assessed with an orthogonal quantitative approach.ResultsLevels of 66 proteins were significantly changed with patisiran vs placebo, with NfL change most significant (p< 10−20). Analysis of changes in protein levels demonstrated that the proteome of patients treated with patisiran trended toward that of healthy controls at 18 months. Healthy controls' NfL levels were 4-fold lower than in patients with ATTRv amyloidosis with polyneuropathy (16.3 pg/mL vs 69.4 pg/mL, effect −53.1 pg/mL [95% confidence interval –60.5 to −45.9]). NfL levels at 18 months increased with placebo (99.5 pg/mL vs 63.2 pg/mL, effect 36.3 pg/mL [16.5–56.1]) and decreased with patisiran treatment (48.8 pg/mL vs 72.1 pg/mL, effect −23.3 pg/mL [–33.4 to −13.1]) from baseline. At 18 months, improvement in modified Neuropathy Impairment Score +7 score after patisiran treatment significantly correlated with reduced NfL (R= 0.43 [0.29–0.55]).ConclusionsFindings suggest that NfL may serve as a biomarker of nerve damage and polyneuropathy in ATTRv amyloidosis, enable earlier diagnosis of patients with ATTRv amyloidosis, and facilitate monitoring of disease progression.Classification of EvidenceThis study provides Class III evidence that NfL levels may enable earlier diagnosis of polyneuropathy in patients with ATTRv amyloidosis and facilitate monitoring of disease progression.

Advancement of RNAi-based therapeutics depends on effective delivery to the site of protein synthesis. Although intravenously administered, multi-component delivery vehicles have enabled small interfering RNA (siRNA) delivery and progression into clinical development, advances of single-component, systemic siRNA delivery have been challenging. In pre-clinical models, attachment of a triantennary N -acetylgalactosamine (GalNAc) ligand to an siRNA mediates hepatocyte uptake via the asialoglycoprotein receptor enabling RNAi-mediated gene silencing. In this phase 1 study, we assessed translation of this delivery approach by evaluating the safety, tolerability, pharmacokinetics, and pharmacodynamics of a GalNAc-siRNA conjugate, revusiran, targeting transthyretin (TTR). Subjects received a placebo or ascending doses of revusiran subcutaneously ranging from 1.25–10 mg/kg in the single and 2.5–10 mg/kg in the multiple ascending dose phases. Revusiran was generally well tolerated, with transient, mild to moderate injection site reactions the most common treatment-emergent adverse events. Doses of 2.5–10 mg/kg revusiran elicited a significant reduction of serum TTR versus the placebo (p 0.01), with mean TTR reductions of approximately 90% observed with multiple dosing. These results demonstrate translation of this novel delivery platform, enabling clinical development of subcutaneously administered GalNAc-siRNAs for liver-based diseases.

Hereditary transthyretin-mediated (hATTR) amyloidosis is a rapidly progressive, multisystem disease that presents with cardiomyopathy or polyneuropathy. The APOLLO study assessed the efficacy and tolerability of patisiran in patients with hATTR amyloidosis. The effects of patisiran on cardiac structure and function in a prespecified subpopulation of patients with evidence of cardiac amyloid involvement at baseline were assessed.APOLLO was an international, randomized, double-blind, placebo-controlled phase 3 trial in patients with hATTR amyloidosis. Patients were randomized 2:1 to receive 0.3 mg/kg patisiran or placebo via intravenous infusion once every 3 weeks for 18 months. The prespecified cardiac subpopulation comprised patients with a baseline left ventricular wall thickness ≥13 mm and no history of hypertension or aortic valve disease. Prespecified exploratory cardiac end points included mean left ventricular wall thickness, global longitudinal strain, and N-terminal prohormone of brain natriuretic peptide. Cardiac parameters in the overall APOLLO patient population were also evaluated. A composite end point of cardiac hospitalizations and all-cause mortality was assessed in a post hoc analysis.In the cardiac subpopulation (n=126; 56% of total population), patisiran reduced mean left ventricular wall thickness (least-squares mean difference ± SEM: -0.9±0.4 mm, P=0.017), interventricular septal wall thickness, posterior wall thickness, and relative wall thickness at month 18 compared with placebo. Patisiran also led to increased end-diastolic volume (8.3±3.9 mL, P=0.036), decreased global longitudinal strain (-1.4±0.6%, P=0.015), and increased cardiac output (0.38±0.19 L/min, P=0.044) compared with placebo at month 18. Patisiran lowered N-terminal prohormone of brain natriuretic peptide at 9 and 18 months (at 18 months, ratio of fold-change patisiran/placebo 0.45, P0.001). A consistent effect on N-terminal prohormone of brain natriuretic peptide at 18 months was observed in the overall APOLLO patient population (n=225). Median follow-up duration was 18.7 months. The exposure-adjusted rates of cardiac hospitalizations and all-cause death were 18.7 and 10.1 per 100 patient-years in the placebo and patisiran groups, respectively (Andersen-Gill hazard ratio, 0.54; 95% CI, 0.28-1.01).Patisiran decreased mean left ventricular wall thickness, global longitudinal strain, N-terminal prohormone of brain natriuretic peptide, and adverse cardiac outcomes compared with placebo at month 18, suggesting that patisiran may halt or reverse the progression of the cardiac manifestations of hATTR amyloidosis.URL: https://www.clinicaltrials.gov . Unique identifier: NCT01960348.

Patisiran, an investigational RNA interference therapeutic agent, specifically inhibits hepatic synthesis of transthyretin.In this phase 3 trial, we randomly assigned patients with hereditary transthyretin amyloidosis with polyneuropathy, in a 2:1 ratio, to receive intravenous patisiran (0.3 mg per kilogram of body weight) or placebo once every 3 weeks. The primary end point was the change from baseline in the modified Neuropathy Impairment Score+7 (mNIS+7; range, 0 to 304, with higher scores indicating more impairment) at 18 months. Other assessments included the Norfolk Quality of Life-Diabetic Neuropathy (Norfolk QOL-DN) questionnaire (range, -4 to 136, with higher scores indicating worse quality of life), 10-m walk test (with gait speed measured in meters per second), and modified body-mass index (modified BMI, defined as [weight in kilograms divided by square of height in meters]×albumin level in grams per liter; lower values indicated worse nutritional status).A total of 225 patients underwent randomization (148 to the patisiran group and 77 to the placebo group). The mean (±SD) mNIS+7 at baseline was 80.9±41.5 in the patisiran group and 74.6±37.0 in the placebo group; the least-squares mean (±SE) change from baseline was -6.0±1.7 versus 28.0±2.6 (difference, -34.0 points; P0.001) at 18 months. The mean (±SD) baseline Norfolk QOL-DN score was 59.6±28.2 in the patisiran group and 55.5±24.3 in the placebo group; the least-squares mean (±SE) change from baseline was -6.7±1.8 versus 14.4±2.7 (difference, -21.1 points; P0.001) at 18 months. Patisiran also showed an effect on gait speed and modified BMI. At 18 months, the least-squares mean change from baseline in gait speed was 0.08±0.02 m per second with patisiran versus -0.24±0.04 m per second with placebo (difference, 0.31 m per second; P0.001), and the least-squares mean change from baseline in the modified BMI was -3.7±9.6 versus -119.4±14.5 (difference, 115.7; P0.001). Approximately 20% of the patients who received patisiran and 10% of those who received placebo had mild or moderate infusion-related reactions; the overall incidence and types of adverse events were similar in the two groups.In this trial, patisiran improved multiple clinical manifestations of hereditary transthyretin amyloidosis. (Funded by Alnylam Pharmaceuticals; APOLLO ClinicalTrials.gov number, NCT01960348 .).

BACKGROUND: Current hemophilia treatment involves frequent intravenous infusions of clotting factors, which is associated with variable hemostatic protection, a high treatment burden, and a risk of the development of inhibitory alloantibodies. Fitusiran, an investigational RNA interference (RNAi) therapy that targets antithrombin (encoded by SERPINC1), is in development to address these and other limitations. METHODS: In this phase 1 dose-escalation study, we enrolled 4 healthy volunteers and 25 participants with moderate or severe hemophilia A or B who did not have inhibitory alloantibodies. Healthy volunteers received a single subcutaneous injection of fitusiran (at a dose of 0.03 mg per kilogram of body weight) or placebo. The participants with hemophilia received three injections of fitusiran administered either once weekly (at a dose of 0.015, 0.045, or 0.075 mg per kilogram) or once monthly (at a dose of 0.225, 0.45, 0.9, or 1.8 mg per kilogram or a fixed dose of 80 mg). The study objectives were to assess the pharmacokinetic and pharmacodynamic characteristics and safety of fitusiran. RESULTS: No thromboembolic events were observed during the study. The most common adverse events were mild injection-site reactions. Plasma levels of fitusiran increased in a dose-dependent manner and showed no accumulation with repeated administration. The monthly regimen induced a dose-dependent mean maximum antithrombin reduction of 70 to 89% from baseline. A reduction in the antithrombin level of more than 75% from baseline resulted in median peak thrombin values at the lower end of the range observed in healthy participants. CONCLUSIONS: Once-monthly subcutaneous administration of fitusiran resulted in dose-dependent lowering of the antithrombin level and increased thrombin generation in participants with hemophilia A or B who did not have inhibitory alloantibodies. (Funded by Alnylam Pharmaceuticals; ClinicalTrials.gov number, NCT02035605 .).

Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to LDL receptors, leading to their degradation. Genetics studies have shown that loss-of-function mutations in PCSK9 result in reduced plasma LDL cholesterol and decreased risk of coronary heart disease. We aimed to investigate the safety and efficacy of ALN-PCS, a small interfering RNA that inhibits PCSK9 synthesis, in healthy volunteers with raised cholesterol who were not on lipid-lowering treatment.We did a randomised, single-blind, placebo-controlled, phase 1 dose-escalation study in healthy adult volunteers with serum LDL cholesterol of 3·00 mmol/L or higher. Participants were randomly assigned in a 3:1 ratio by computer algorithm to receive one dose of intravenous ALN-PCS (with doses ranging from 0·015 to 0·400 mg/kg) or placebo. The primary endpoint was safety and tolerability of ALN-PCS. Secondary endpoints were the pharmacokinetic characteristics of ALN-PCS and its pharmacodynamic effects on PCSK9 and LDL cholesterol. Study participants were masked to treatment assignment. Analysis was per protocol and we used ANCOVA to analyse pharmacodynamic endpoint data. This trial is registered with ClinicalTrials.gov, number NCT01437059.Of 32 participants, 24 were randomly allocated to receive a single dose of ALN-PCS (0·015 mg/kg [n=3], 0·045 mg/kg [n=3], 0·090 mg/kg [n=3], 0·150 mg/kg [n=3], 0·250 mg/kg [n=6], or 0·400 mg/kg [n=6]) and eight to placebo. The proportions of patients affected by treatment-emergent adverse events were similar in the ALN-PCS and placebo groups (19 [79%] vs seven [88%]). ALN-PCS was rapidly distributed, with peak concentration and area under the curve (0 to last measurement) increasing in a roughly dose-proportional way across the dose range tested. In the group given 0·400 mg/kg of ALN-PCS, treatment resulted in a mean 70% reduction in circulating PCSK9 plasma protein (p0·0001) and a mean 40% reduction in LDL cholesterol from baseline relative to placebo (p0·0001).Our results suggest that inhibition of PCSK9 synthesis by RNA interference (RNAi) provides a potentially safe mechanism to reduce LDL cholesterol concentration in healthy individuals with raised cholesterol. These results support the further assessment of ALN-PCS in patients with hypercholesterolaemia, including those being treated with statins. This study is the first to show an RNAi drug being used to affect a clinically validated endpoint (ie, LDL cholesterol) in human beings.Alnylam Pharmaceuticals.

Background: Transthyretin-mediated amyloidosis is an inherited, progressively debilitating disease caused by mutations in the transthyretin gene. This study evaluated the safety, tolerability, pharmacokinetics, and pharmacodynamics of multiple doses of patisiran (ALN-TTR02), a small interfering RNA encapsulated within lipid nanoparticles, in patients with transthyretin-mediated familial amyloid polyneuropathy (FAP). Methods: In this phase II study, patients with FAP were administered 2 intravenous infusions of patisiran at one of the following doses: 0.01 (n = 4), 0.05 (n = 3), 0.15 (n = 3), or 0.3 (n = 7) mg/kg every 4 weeks (Q4W), or 0.3 mg/kg (n = 12) every 3 weeks (Q3W). Results: Of 29 patients in the intent-to-treat population, 26 completed the study. Administration of patisiran led to rapid, dose-dependent, and durable knockdown of transthyretin, with the maximum effect seen with patisiran 0.3 mg/kg; levels of mutant and wild-type transthyretin were reduced to a similar extent in Val30Met patients. A mean level of knockdown exceeding 85 % after the second dose, with maximum knockdown of 96 %, was observed for the Q3W dose. The most common treatment-related adverse event (AE) was mild-to-moderate infusion-related reactions in 10.3 % of patients. Four serious AEs (SAEs) were reported in 1 patient administered 0.3 mg/kg Q3W (urinary tract infection, sepsis, nausea, vomiting), and 1 patient administered 0.3 mg/kg Q4W had 1 SAE (extravasation-related cellulitis). Conclusions: Patisiran was generally well tolerated and resulted in significant dose-dependent knockdown of transthyretin protein in patients with FAP. Patisiran 0.3 mg/kg Q3W is currently in phase III development., All authors were responsible for reviewing and interpreting the data. The authors received editorial support from Adelphi Communications Ltd. Biostatistical analyses were conducted by Veristat LLC, Holliston, MA, USA. The authors acknowledge the support of the following co-investigators: Mercedes Uson, Carles Montala, and Cristina Descals (Hospital Son Llatzer, Palma de Mallorca, Spain), and Cecile Cauquil and Marie Theaudin (Univ Paris-Sud, Le Kremlin-Bicetre, Paris, France). The authors also acknowledge contributions from Dorothee Lamann (Universitatsklinikum Munster, Munster, Germany) who provided technical support and Vanessa Benito who was the Study Coordinator at the Hospital Son Llatzer, Palma de Mallorca, Spain. This study was sponsored by Alnylam Pharmaceuticals, Inc.

We studied preexisting respiratory syncytial virus (RSV)-specific serum and nasal antibodies and their correlation with infectivity, viral dynamics, and disease severity in a human experimental infection model. Higher preinoculation serum neutralizing antibody titers and nasal immunoglobulin (Ig) A predicted lower infectivity and lower measures of viral replication. However, once individuals were infected, no significant protective effect of preexisting antibodies was seen. Lack of correlation between serum and mucosal antibodies was observed, implying that they are independent co-correlates of protection against RSV infection. We suggest that protection from RSV infection is a function of a complex interplay between mucosal and serum humoral immune responses.

Background Alefacept, human LFA-3/IgG 1 fusion protein, selectively reduces memory-effector (CD45RO + ) T cells, a source of the pathogenic mediators of psoriasis. Objective To evaluate the effect of alefacept on immune function, T-cell-dependent humoral responses to a neoantigen (φX174) and recall antigen (tetanus toxoid) were assessed. Methods Patients with psoriasis were randomized to the control group or to receive alefacept (7.5 mg intravenously weekly for 12 weeks). The alefacept group received φX174 immunizations at weeks 6, 12, 20, and 26 and tetanus toxoid at week 21; control subjects received φX174 at weeks 6 and 12 and tetanus at week 10. Results Mean anti-φX174 titers were comparable in both groups. There was no difference in the percentage of responders (anti-φX174 IgG ≥30% of the total anti-φX174) between the alefacept group and the control group (86% and 82%, respectively; P = .73). The percentage of patients with anti-tetanus toxoid titer increases ≥2 times baseline also was similar (alefacept, 89%; control 91%). Conclusion A single 12-week course of alefacept did not impair primary or secondary antibody responses to a neoantigen or memory responses to a recall antigen. The selective immunomodulatory effect of alefacept against a potentially pathogenic T-cell subset is associated with maintenance of a significant aspect of immune function (antibody response) to fight infection and respond to vaccinations.

Lymphomas were studied in kindreds with autoimmune lymphoproliferative syndrome (ALPS; Canale-Smith syndrome), a disorder of lymphocyte homeostasis usually associated with germline Fas mutations. Fas (CD95/APO-1) is a cell surface receptor that initiates programmed cell death, or apoptosis, of activated lymphocytes. Lymphoma phenotype was determined by immunohistochemistry, frequency of CD3(+)CD4(-)CD8(-) T-cell-receptor alpha/beta cells by flow cytometry, nucleotide sequences of the gene encoding Fas (APT1, TNFRSF6), and the percentage of lymphocytes undergoing apoptosis in vitro. Of 223 members of 39 families, 130 individuals possessed heterozygous germline Fas mutations. Eleven B-cell and T-cell lymphomas of diverse types developed in 10 individuals with mutations in 8 families, up to 48 years after lymphoproliferation was first documented. Their risk of non-Hodgkin and Hodgkin lymphomas, respectively, was 14 and 51 times greater than expected (each P

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

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

The Canale-Smith syndrome is a childhood disorder characterized by lymphadenopathy and autoimmunity. The similarity between this syndrome and that in mice with the lymphoproliferation (lpr) phenotype or the generalized-lymphoproliferative-disease (gld) phenotype led us to investigate whether it too is caused by mutations of the Fas gene (lpr mice) or the Fas ligand (gld mice), which regulate apoptosis in lymphocytes.We studied four patients with the syndrome and their families. T-lymphocyte phenotypes were analyzed, and the susceptibility of activated T cells to Fas-mediated apoptosis in vitro was determined. Mutations of Fas were sought by nucleotide-sequence analysis.Patients with the Canale-Smith syndrome had increased numbers of circulating double-negative T cells (20 percent) and profoundly impaired apoptosis of activated T cells incubated with an anti-Fas antibody. Three novel Fas mutations were identified, all of which were heterozygous and predicted to impair signal transduction by Fas. Autoimmune manifestations of the disease, such as hemolytic anemia and thrombocytopenia, persisted into adolescence. Two patients followed into adulthood had intermittent lymphadenopathy, which diminished over time. Neoplasms developed in both, and one died of hepatocellular carcinoma at the age of 43.Patients with the Canale-Smith syndrome have mutations in Fas, which implicates this gene in the accumulation of lymphocytes and the autoimmunity characteristic of the syndrome.

Respiratory syncytial virus (RSV) is the leading cause of childhood lower respiratory infection, yet viable therapies are lacking. Two major challenges have stalled antiviral development: ethical difficulties in performing pediatric proof-of-concept studies and the prevailing concept that the disease is immune-mediated rather than being driven by viral load.The development of a human experimental wild-type RSV infection model to address these challenges.Healthy volunteers (n = 35), in five cohorts, received increasing quantities (3.0-5.4 log plaque-forming units/person) of wild-type RSV-A intranasally.Overall, 77% of volunteers consistently shed virus. Infection rate, viral loads, disease severity, and safety were similar between cohorts and were unrelated to quantity of RSV received. Symptoms began near the time of initial viral detection, peaked in severity near when viral load peaked, and subsided as viral loads (measured by real-time polymerase chain reaction) slowly declined. Viral loads correlated significantly with intranasal proinflammatory cytokine concentrations (IL-6 and IL-8). Increased viral load correlated consistently with increases in multiple different disease measurements (symptoms, physical examination, and amount of nasal mucus).Viral load appears to drive disease manifestations in humans with RSV infection. The observed parallel viral and disease kinetics support a potential clinical benefit of RSV antivirals. This reproducible model facilitates the development of future RSV therapeutics.

Small interfering RNAs (siRNAs) work through RNA interference (RNAi), the natural RNA inhibitory pathway, to down-regulate protein production by inhibiting targeted mRNA in a sequence-specific manner. ALN-RSV01 is an siRNA directed against the mRNA encoding the N-protein of respiratory syncytial virus (RSV) that exhibits specific in vitro and in vivo anti-RSV activity. The results of two safety and tolerability studies with ALN-RSV01 involving 101 healthy adults (65 active, 36 placebo, single- and multiple dose, observer-blind, randomized dose-escalation) are described. Intranasal administration of ALN-RSV01 was well tolerated over a dose range up through 150mg as a single dose and for five daily doses. Adverse events were similar in frequency and severity to placebo (normal saline) and were transient, mild to moderate, with no dose-dependent trend. The frequency or severity of adverse events did not increase with increasing ALN-RSV01 exposure. All subjects completed all treatments and assessments with no early withdrawals or serious adverse events. Physical examinations, vital signs, ECGs and laboratory tests were normal. Systemic bioavailability of ALN-RSV01 was minimal. ALN-RSV01 appears safe and well tolerated when delivered intranasally and is a promising therapeutic candidate for further clinical development.

Alefacept, human LFA-3/IgG1 fusion protein, is currently under clinical development for the treatment of chronic plaque psoriasis and other T cell mediated disorders. This recombinant protein binds CD2 on T cells and Fc gamma RIII on accessory cells (e.g., natural killer cells, macrophages), inhibiting T cell activation/proliferation and inducing selective T cell apoptosis. These effects are associated with selective reductions in memory-effector (CD4+ CD45RO+ and CD8+ CD45RO+) T cells. Two open-label studies were conducted in healthy male volunteers to evaluate the pharmacokinetics, biologic activity, and tolerability of a single dose of alefacept when administered as a 0.15 mg/kg 30-sec i.v. bolus (n = 12), 0.04 mg/kg intramuscular (i.m.) injection (n = 8), or 0.04 mg/kg 30-min intravenous (i.v.) infusion (n = 8). i.v. infusion produced a higher Cmax (0.96 +/- 0.26 mcg/ml vs. 0.36 +/- 0.19 mcg/ml) and a shorter Tmax (2.8 +/- 1.9 hr vs. 86 +/- 60 hr) when compared to i.m. injection. Based on AUC0-last and AUC0-infenity values, the relative bioavailability of i.m. to i.v. infusion was approximately 60%. After absorption from the i.m. injection was complete, the rate of alefacept elimination from the serum appeared consistent with the i.v. infusion half-life (approximately 12 days). Biologic activity was demonstrated by transient reductions in absolute number of CD2+ lymphocytes, with notable specificity for memory T-cell subsets. Alefacept was well tolerated; the most common adverse effects were headache, pharyngitis, rash, and myalgia. IM administration was not associated with significant local reactions. Results of these studies support i.v. bolus or i.m. administration of alefacept. An i.m. dose of approximately 150 to 200% of the i.v. dose is an appropriate and convenient alternative to i.v. administration.

Objective CD40–CD40 ligand (CD40L) interactions play a significant role in the production of autoantibodies and tissue injury in lupus nephritis. We performed an open-label, multiple-dose study to evaluate the safety, efficacy, and pharmacokinetics of BG9588, a humanized anti-CD40L antibody, in patients with proliferative lupus nephritis. The primary outcome measure was 50% reduction in proteinuria without worsening of renal function. Methods Twenty-eight patients with active proliferative lupus nephritis were scheduled to receive 20 mg/kg of BG9588 at biweekly intervals for the first 3 doses and at monthly intervals for 4 additional doses. Safety evaluations were performed on all patients. Eighteen patients receiving at least 3 doses were evaluated for efficacy. Results The study was terminated prematurely because of thromboembolic events occurring in patients in this and other BG9588 protocols (2 myocardial infarctions in this study). Of the 18 patients for whom efficacy could be evaluated, 2 had a 50% reduction in proteinuria without worsening of renal function. Mean reductions of 38.9% (P < 0.005), 50.1% (P < 0.005), and 25.3% (P < 0.05) in anti–double-stranded DNA (anti-dsDNA) antibody titers were observed at 1, 2, and 3 months, respectively, after the last treatment. There was a significant increase in serum C3 concentrations at 1 month after the last dose (P < 0.005), and hematuria disappeared in all 5 patients with significant hematuria at baseline. There were no statistically significant reductions in lymphocyte count or serum immunoglobulin, anticardiolipin antibody, or rubella IgG antibody concentrations after therapy. Conclusion A short course of BG9588 treatment in patients with proliferative lupus nephritis reduces anti-dsDNA antibodies, increases C3 concentrations, and decreases hematuria, suggesting that the drug has immunomodulatory action. Additional studies will be needed to evaluate its long-term effects.

Objective To determine the immunologic effects of anti–CD40 ligand (anti-CD40L) therapy in 5 patients with systemic lupus erythematosus nephritis who participated in an open-label study of a humanized anti-CD40L monoclonal antibody. Methods Serum and peripheral blood mononuclear cells were obtained before, during, and after treatment, and the frequency of Ig and anti-DNA antibody–secreting B cells was analyzed by enzyme-linked immunospot assay and by analysis of Epstein-Barr virus (EBV)–transformed B cell lines. To determine the effect of treatment on somatic mutation of Ig genes, reverse transcriptase–polymerase chain reaction was performed on messenger RNA from 4 patients, using primers specific for the DP-47 heavy chain gene and for IgG. Finally, B cell phenotype was investigated using flow cytometry. Results Even a brief period of treatment with anti-CD40L markedly reduced the frequency of IgG and IgG anti-DNA antibody–producing B cells, and these changes persisted for several months after cessation of treatment. To confirm these findings, EBV-transformed B cell lines were screened from each of 3 patients, and a 10-fold decrease in anti-DNA antibody–secreting cell lines was found after treatment in all 3 patients. Few differences in mutation patterns were observed before and after treatment; however, the frequency of germline-encoded DP-47 sequences was significantly increased before treatment and normalized following treatment. Flow cytometric analysis of B cells revealed expansion of a CD27−/IgD− B cell subset in some of the patients, which did not change with treatment. Conclusion These are the first mechanistic studies of the effect of anti-CD40L therapy in human autoimmune disease. The results suggest that further studies of CD40L blockade are warranted.

Objective To investigate whether alefacept (a fully human lymphocyte function–associated antigen 3 [LFA-3]/IgG1 fusion protein that blocks the LFA-3/CD2 interaction) is able to reduce the signs and symptoms of joint inflammation in patients with active psoriatic arthritis (PsA). Methods Eleven patients with active PsA were treated with alefacept for 12 weeks in an open-label and explorative study. Clinical joint assessment and laboratory assessments were performed at baseline and after 4, 9, 12, and 16 weeks of treatment. Serial synovial tissue (ST) biopsy specimens from an inflamed index joint (knee, ankle, wrist, or metacarpophalangeal joint) were obtained by arthroscopy at baseline and after 4 and 12 weeks of treatment. Results At the completion of treatment, 6 of 11 patients (55%) fulfilled the Disease Activity Score (DAS) response criteria. Nine patients (82%) fulfilled the DAS response criteria at any point during the study. There was a statistically significant reduction in CD4+ lymphocytes (P < 0.05), CD8+ lymphocytes (P = 0.05), and CD68+ macrophages (P < 0.02) in the ST after 12 weeks of treatment compared with baseline. The ST and peripheral blood of those patients fulfilling the DAS response criteria contained more CD45RO+ cells at baseline and displayed a significant reduction in these cells compared with nonresponding patients. Conclusion The changes in ST, together with the improvement in clinical joint scores, after treatment with alefacept support the hypothesis that T cell activation plays an important role in this chronic inflammatory disease. Furthermore, since alefacept, a T cell–specific agent, led to decreased macrophage infiltration, the data indicate that T cells are highly involved in synovial inflammation in PsA.

To determine the clinical spectrum of disease in humans with mutations in the CD95 (Fas/ APO-1) receptor and to obtain mechanistic insight into the different clinical phenotypes observed.Clinical information for each of the index cases, first-degree relatives, and any family members reported to have Canale-Smith syndrome (or another autoimmune disease) was gathered by direct interview, chart review, and verification of data by the physician or pathologist concerned. Apoptosis of activated T or B lymphocytes was induced by agonistic anti-CD95 antibodies and quantified by a cell death assay (propidium iodide staining in the subdiploid peak) or cell viability assay (alamar blue or 3H-thymidine incorporation).Evaluation of an additional 8 probands with novel heterozygous CD95 mutations revealed hypergammaglobulinemia and immune-mediated cytopenias in all patients, as well as urticarial rash, oral ulceration, lymphopenia, and peripheral neuropathy in some individuals. One patient (P4) had systemic lupus erythematosus (SLE) characterized by a World Health Organization class V lupus nephropathy, a recurrent, reversible multifocal central nervous system disorder, high-titer antiphospholipid autoantibodies, and autoimmune cytopenias. In the P4 pedigree, the father had reduced T and B cell apoptosis associated with a CD95 mutation, whereas an independent B cell apoptotic defect was demonstrated in maternal family members who did not have a CD95 mutation. Three cases of B cell lymphoma occurred in carriers of the CD95 mutation.CD95 mutations are associated with loss of regulation of B lymphocytes, which predisposes to systemic autoimmunity including SLE. The P4 family provides a model of the complex genetic and functional interactions that are required for the development of a lupus-like syndrome.

Objective. To describe a new kindred with C1q deficiency and to identify the molecular lesions responsible for complete functional C1q deficiency in this and 2 other previously described kindreds. Methods. The A-, B-, and C-chain genes of C1q were amplified by polymerase chain reaction, cloned, and sequenced. The DNA sequence was checked for mutations. Results. Patient 1 had a homozygous G-to-A change at codon 6 of the C chain, causing an amino acid change from Gly to Arg. Patient 2 had a homozygous deletion of a C nucleotide at codon 43 of the C-chain, causing a frame shift, leading to a premature stop codon at codon 108. Patient 3 had a homozygous C-to-T mutation at amino acid position 41 of the C chain, resulting in a premature stop codon. Conclusion. In the homozygous state, the mutations are sufficient to cause complete deficiency of C1q. The mutation in patient 1 has been previously reported in a patient of different ethnic origin. A survey of a series of 158 DNA samples from patients with systemic lupus erythematosus showed no other examples of this mutant allele.

Heterozygous mutations of the receptor CD95 (Fas/Apo-1) are associated with defective lymphocyte apoptosis and a clinical disease characterized by lymphadenopathy, splenomegaly, and systemic autoimmunity. From our cohort of 11 families, we studied eight patients to define the mechanisms responsible for defective CD95-mediated apoptosis. Mutations in and around the death domain of CD95 had a dominant–negative effect that was explained by interference with the recruitment of the signal adapter protein, FADD, to the death domain. The intracellular domain (ICD) mutations were associated with a highly penetrant Canale-Smith syndrome (CSS) phenotype and an autosomal dominant inheritance pattern. In contrast, mutations affecting the CD95 extracellular domain (ECD) resulted in failure of extracellular expression of the mutant protein or impaired binding to CD95 ligand. They did not have a dominant–negative effect. In each of the families with an ECD mutation, only a single individual was affected. These observations were consistent with differing mechanisms of action and modes of inheritance of ICD and ECD mutations, suggesting that individuals with an ECD mutation may require additional defect(s) for expression of CSS.