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Background: Primary hyperoxaluria type 1 (PH1) is a rare genetic disease caused by hepatic overproduction of oxalate that leads to kidney stones, nephrocalcinosis, kidney failure, and systemic oxalosis. Lumasiran, an investigational RNA interference (RNAi) therapeutic agent, reduces hepatic oxalate production by targeting glycolate oxidase. Methods: In this double-blind, phase 3 trial, we randomly assigned (in a 2:1 ratio) patients with PH1 who were 6 years of age or older to receive subcutaneous lumasiran or placebo for 6 months (with doses given at baseline and at months 1, 2, 3, and 6). The primary end point was the percent change in 24-hour urinary oxalate excretion from baseline to month 6 (mean percent change across months 3 through 6). Secondary end points included the percent change in the plasma oxalate level from baseline to month 6 (mean percent change across months 3 through 6) and the percentage of patients with 24-hour urinary oxalate excretion no higher than 1.5 times the upper limit of the normal range at month 6. Results: A total of 39 patients underwent randomization; 26 were assigned to the lumasiran group and 13 to the placebo group. The least-squares mean difference in the change in 24-hour urinary oxalate excretion (lumasiran minus placebo) was -53.5 percentage points (P

The materialistic properties of a cell largely depend on the biological activities of the cell. These properties are the indicators of the maintenance of homeostasis in the human body. Any abnormal alterations in these properties can affect the day-to-day physiological functions of a healthy human body. In the present paper, we inquest the materialistic properties of a tumor cell in comparison with normal healthy somatic cells. Later continued research targets to mathematically model the tumor-immune system compartmentally by coupling with the materialistic property equation for the tumor cells. At the end of the paper, we present a continuum model for the spread of tumors as a function of the elastic modulus of the tumor cells. The model fabricated is a novel model that takes into account both the role of elastic modulus decrease as well as the role of immune cells in anti-tumor activities. The models available in the literature do not consider the role of the elastic property of the tumor cells into account.

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.

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.

Background: 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. Methods: 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. Results: 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, P 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). Conclusions: 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. Clinical Trial Registration: URL: https://www.clinicaltrials.gov . Unique identifier: NCT01960348.

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.

A tumor is a condition of uncontrolled cell division in solid tissues such as an organ, bone, or muscle. After a sequence of mutations (changes in the genetic sequence), tumor cells normally develop and become progressively abnormal. These mutations in our environment are either hereditary or, more commonly, caused by carcinogens (cancer-causing substances). This abnormal condition of tumor growth evolves a series of mechanisms that can caricature the peripheral immune forbearance and hence escaping the tumoricidal attack by the immune cells. In the present research, we contemplate mocking the roles of various immune cells in tumor attack using the mathematical model formulated. Also, we study the constitution of these various cell densities, involved under our study, by employing drug interventions through diverse therapy techniques. The mathematical model formulated for our study prevails to be unique due to the exemplary composition: tumor cells, natural killers, dendritic cells, CD8+ cells, CD4+ cells, interleukin 2 cytokines (IL-2 cytokines), and drug interference term. The model established is analyzed mathematically for its stability using standard methods. The system of differential equations is solved using an ordinary differential equation solver, with the values of the parameters taken from pathologically tested sources. The cases of with and without drug interference have been analyzed using the graphical results obtained. Also, we intend to study the tumor cell density by changing the frequency of therapy employed. The results followed a hysteresis loop, adding uniqueness to our present work.

Background: Angiotensinogen (AGT) is the sole precursor of all angiotensin peptides and plays a key role in hypertension pathogenesis. We evaluated the effect of ALN-AGT01, a subcutaneous investigational RNAi therapeutic targeting hepatic AGT synthesis, on blood pressure in hypertensive patients. Methods: As part of a phase 1 program designed to assess the safety and tolerability of ALN-AGT01, we conducted a multicenter study randomizing patients aged 18-65 years with mild to moderate hypertension (mean seated systolic blood pressure [SBP] of >130 and ≤165 mmHg after washout of antihypertensive medication) 2:1 to ascending single doses of ALN-AGT01 or placebo. Change from baseline in BP at 8 weeks was measured by ambulatory BP monitoring (ABPM). We report interim results as of May 14, 2020. Results: Sixty patients (mean age 52 years, 45% female, mean baseline 24h SBP 139 +/- 7 mm Hg) were enrolled in ascending dose cohorts of 10 mg, 25 mg, 50 mg, 100 mg, or 200 mg. Dose-related reductions in serum AGT levels were observed (figure), with reductions >90% in the 100 and 200 mg dose cohorts. AGT remained durably reduced through 12 weeks after single dose administration. Concomitant reductions in BP from baseline were observed with AGT knockdown, with an over 10 mm Hg reduction of mean 24-hour SBP observed at Week 8 after single doses of 100 mg or 200 mg. No symptomatic hypotension, treatment-related serious adverse events, or clinically significant elevations in blood creatinine or potassium were seen. Conclusions: Single dose administration of ALN-AGT01 to hypertensive patients resulted in dose-related reductions in serum AGT and BP over 8 weeks without hypotension or other related serious adverse events. Durable AGT knockdown to 12 weeks supports further evaluation of once quarterly or potentially less frequent dose administration.

IntroductionHereditary transthyretin-mediated (hATTR) amyloidosis is an underdiagnosed, progressively debilitating disease caused by mutations in the transthyretin (TTR) gene. The V122I variant, one of the most common pathogenic TTR mutations, is found in 3-4% of Black individuals, and has been associated with cardiomyopathy.MethodsTo better understand the phenotypic consequences of carrying V122I, we conducted a phenome-wide association study scanning 427 ICD diagnosis codes for association with this variant in Black participants of the UK Biobank (n= 6,062). Significant associations were tested for replication in the Penn Medicine Biobank (n= 5,737) and the Million Veteran Program (n= 82,382).ResultsOur analyses discovered a significant association between V122I and polyneuropathy diagnosis (odds ratio = 6.4, 95% confidence interval [CI] = 2.6 to 15.6, P = 4.2 × 10−5) in the UK Biobank,which was replicated in the Penn Medicine Biobank (p=6.0×10−3)) and Million Veteran Program (P= 1.8×10−4)). Polyneuropathy prevalence among V122I carriers was 2.1–9.0% across biobanks. The cumulative incidence of common hATTR amyloidosis manifestations (carpal tunnel syndrome, polyneuropathy, cardiomyopathy, heart failure) was significantly enriched in V122I carriers versus non-carriers (hazard ratio = 2.8, 95% CI = 1.7–4.5, P = 2.6 × 10−5) in the UK Biobank;37.4% of V122I carriers having a diagnosis of any one of these manifestations by age 75.ConclusionsOur findings show that, although the V122I variant is known to be associated with cardiomyopathy, carriers are also at significantly increased risk of developing polyneuropathy. These results also emphasize the underdiagnosis of disease in V122I carriers with a significant proportion of subjects showing phenotypic changes consistent with hATTR. Greater understanding of the manifestations associated with V122I is critical for earlier diagnosis and treatment.

Background Hereditary transthyretin-mediated (hATTR) amyloidosis is a progressive, life-threatening disease caused by mutations in the transthyretin (TTR) gene. hATTR amyloidosis phenotypes can vary by patient and mutation. The V122I (Val122Ile; p.V142I) variant is one of the most common pathogenic TTR mutations, is primarily found in people of West African descent and has historically been associated with cardiomyopathy (CM). Purpose To characterize the cumulative incidence of diagnoses frequently seen with hATTR amyloidosis in V122I carriers and non-carriers in the UK Biobank (UKBB) and the Penn Medicine BioBank (PMBB). Methods UKBB and PMBB are prospective studies with ∼500,000 and ∼60,000 subjects, respectively. Clinical presentations frequently seen with hATTR amyloidosis were assessed using ICD10 diagnosis codes: G62–polyneuropathy (PN), I50 or I098–heart failure (HF), G560–carpal tunnel syndrome (CTS), I42–CM, and E85–amyloidosis. The cumulative incidence of diagnoses was estimated using Kaplan-Meier curves. Time to first hATTR amyloidosis-related diagnosis was compared between V122I carriers and non-carriers using Cox proportional hazards regression, controlling for age, sex, smoking, and genetic ancestry. Results Of the 6,062 unrelated black participants in the UKBB, 243 were V122I carriers. Only 0.8% of V122I carriers had a formal diagnosis of hATTR amyloidosis. V122I carriers were significantly more likely to have a PN diagnosis than non-carriers (p=6.35x10–5), a finding which was replicated in the PMBB. Of the ICD10 codes assessed, Cox proportional hazards regression revealed a significant association between V122I genotype and time to first diagnosis (p=2.6x10–5), with 11.1% of V122I carriers having at least one diagnosis during follow-up versus 4.9% of non-carriers. The calculated population attributable risk showed an excess risk of 16.7% for a PN diagnosis, 6.5% for HF, 4.1% for CTS, and 2.4% for CM in V122I carriers. The cumulative incidence of any hATTR amyloidosis-related diagnosis among V122I carriers by age 65 was 11.9% (95% CI=3.1–19.8%). The incidence increased to 37.4% (95% CI=20.5–50.7%) by age 75, which was significantly higher than non-carriers (13.8%, 95% CI=11.6–16%). Additionally, an assessment of the cumulative incidence of each diagnosis separately revealed that PN became more prevalent at younger ages, while HF and CM became more prevalent at older ages. Conclusions V122I carriers were significantly more likely to receive diagnoses frequently seen with hATTR amyloidosis than non-carriers. Although these diagnoses may not all be attributed to amyloidosis, the small fraction of patients diagnosed with hATTR amyloidosis suggests a potential underdiagnosis of disease. The V122I mutation has historically been associated with a cardiac phenotype, yet these data also suggest an increased incidence of PN. Increased vigilance for the mixed phenotype may lead to earlier diagnoses and treatment of V122I carriers. Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Alnylam Pharmaceuticals

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.

Lung cancer still seems to be a very common cause of death among the people all over the world. This paper proposes a method which is a coalition of two statistical methods Photo Metric and Quality Metric enacted for lung cancer analysis and diagnosis. Suggested methodology is established on the basis of identified statistical and mathematical parameters exploring the light variation and quality features of the lung image to carry out the evaluation for cancerous and non-cancerous types. The distinguishing Photo Metric parameters are Brightness, Luminance, Contrast and LC index, whereas the Quality Metric parameters are VIF, MSE, VSNR and PSNR. These eight identified parameters are applied one by one on the cancerous and noncancerous lung images using image processing technique using MATLAB to get an individual parameter statistical range through number of iterations in real time. If the test image falls in the particular calculated statistical range for cancerous lung images, then the test image is infected or else normal. But practically some of the values of the statistical range for cancerous and noncancerous lungs overlaps due to which the final decision is taken by an image classifier. The ANN is used as an image classifier to sort out this problem resulting in an efficient lung cancer diagnosis. The microscopic lung images are used to implement the proposed methodology. The performance analysis of each identified parameter is the heart of this paper. The authentication of the proposed methods is tested successfully using Standard Diagnostic Test.

In this paper, an average informational parameter based approach for lung cancer detection and diagnosis has been proposed. Suggested methodology is established on average information parameters by utilizing image processing tools for lung cancer investigation. The real issue with the lung cancer diseases is the time constraint for physical diagnosis that expands the death possibilities. Henceforth essentially proposed technique is an approach that would help the medical practitioners for precise and superior decision against the lung cancer discovery. The crucial point in the proposed method is that it helps the doctors for taking a firm decision on lung cancer diagnosis. Microscopic lung images are taken for analysis and investigation by using digital image processing techniques which also recovers the quality of images that has been degraded by several reasons including random noise. The statistical parameters are implemented for lung cancer analysis. The statistical and mathematical parameters are implemented like Entropy, Standard Deviation, Mean, Variance and MSE under average information method. The statistical range of each parameter is calculated for number of iterations. The individual statistical parameter analysis with its impact on lung cancer images is carried out and finally the Artificial Neural Network is the final decision maker in lung cancer diagnosis. This paper also rejects the null hypothesis test by implementing one of the standard statistical methods.

In this paper, an Average Information based approach for lung cancer analysis and diagnosis has been proposed. Suggested methodology is established on average information parameters by utilizing image processing tools for lung cancer investigation. The real issue for the lung cancer diagnosis is the time constrictions for physical diagnosis that expands the death possibilities. Henceforth essentially proposed technique is an approach that would help the medical practitioners for precise and superior decision against the lung cancer discovery. Microscopic lung images are taken for analysis and investigation by using digital image processing with MATLAB. The statistical and mathematical parameters under statistical analysis are selected on the basis of the principle working of Average information technique. The input parameters like Entropy, Standard Deviation, Mean, Variance and MSE for average information method are implemented over a large microscopic lung image database. The individual statistical and mathematical parameter analysis with its impact on lung cancer images is successfully carried out and finally the accuracy, selectivity, and sensitivity of the proposed method is calculated by implementing the standard diagnostic test on the proposed method. This method also successfully rejects null hypothesis test by implementing one of the standard statistical methods.

BackgroundInclisiran (ALN-PCSsc) is a long-acting RNA interference (RNAi) therapeutic agent that inhibits the synthesis of proprotein convertase subtilisin–kexin type 9 (PCSK9), a target for the lowering of low-density lipoprotein (LDL) cholesterol. MethodsIn this phase 1 trial, we randomly assigned healthy volunteers with an LDL cholesterol level of at least 100 mg per deciliter in a 3:1 ratio to receive a subcutaneous injection of inclisiran or placebo in either a single-ascending-dose phase (at a dose of 25, 100, 300, 500, or 800 mg) or a multiple-dose phase (125 mg weekly for four doses, 250 mg every other week for two doses, or 300 or 500 mg monthly for two doses, with or without concurrent statin therapy); each dose cohort included four to eight participants. Safety, the side-effect profile, and pharmacodynamic measures (PCSK9 level, LDL cholesterol level, and exploratory lipid variables) were evaluated. ResultsThe most common adverse events were cough, musculoskeletal pain, nasopharyngitis, headach...

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.

SummaryBackgroundHereditary transthyretin-mediated (hATTR) amyloidosis is a rare, progressively debilitating, and fatal disease caused by deposition of aggregated transthyretin amyloid in multiple organs and tissues. Highly variable disease penetrance has made it difficult to predict disease onset and progression. Clinically validated, non-invasive plasma biomarkers may facilitate earlier diagnosis and aid monitoring of disease progression.MethodsPlasma levels of >1000 proteins were measured in patients with hATTR amyloidosis with polyneuropathy who received either placebo or patisiran in the phase 3 APOLLO study (NCT01960348) and in a cohort of healthy individuals. The impact of patisiran treatment on the time profile of each protein was determined by a linear mixed model at 0, 9, and 18 months. Neurofilament light chain (NfL) protein was further assessed using an orthogonal quantitative approach.FindingsA significant change in the levels of 66 proteins was observed with patisiran vs placebo, with change in NfL, a marker of neuronal damage, most significant (p−20). Analysis of the changes in protein levels demonstrated that the proteome of patients treated with patisiran trended towards healthy individuals at 18 months. Plasma NfL levels in healthy controls were four-fold lower than in patients with hATTR amyloidosis with polyneuropathy (16·3 [SD 12·0] pg/mL vs 69·4 [SD 42·1] pg/mL, p−16). Levels of NfL at 18 months increased with placebo (99·5 [SD 60·1] pg/mL) and decreased with patisiran treatment (48·8 [SD 29·9] pg/mL). At 18 months, improvement in modified Neuropathy Impairment Score+7 (mNIS+7) in patisiran-treated patients significantly correlated with a reduction in NfL levels (R=0·43, p−7).InterpretationThe observed correlation of NfL reduction with patisiran treatment and improvement in mNIS+7 suggests it may serve as a biomarker of nerve damage and polyneuropathy in hATTR amyloidosis. This biomarker may enable earlier diagnosis of polyneuropathy in patients with hATTR amyloidosis and facilitate monitoring of disease progression.

Introduction Hereditary transthyretin-mediated (hATTR) amyloidosis is a progressive, life-threatening disease due to deposition of transthyretin (TTR) amyloid in various organs and tissues. Historically the disease has been described by predominant clinical manifestation of cardiomyopathy (CM) or polyneuropathy (PN), however a majority of patients are known to develop a mixed phenotype. Because of this, PN symptoms may not always be assessed in patients with predominant CM and neuropathy progression may continue. The potential of neurofilament light chain (NfL) as a non-invasive biomarker of nerve damage in patients with hATTR amyloidosis with PN has previously been described; however to date no studies have investigated NfL in patients with predominant CM. Hypothesis NfL may serve as a biomarker of co-existing neuropathy in patients with hATTR amyloidosis with predominant CM and could lead to early detection and treatment of nerve damage, thus improving patient care. Methods Plasma NfL levels were measured in baseline samples from patients with hATTR amyloidosis with CM from the Phase 3 ENDEAVOUR study (n = 194; V122I variant = 111) and compared with NfL levels from healthy controls (n = 53) and baseline samples from patients with hATTR amyloidosis with PN from the Phase 3 APOLLO study (n = 193). Polyneuropathy Disability (PND) score was also taken into account for NfL analyses. Results Elevated plasma NfL levels were observed in patients with hATTR amyloidosis with CM relative to healthy controls (54.1 pg/mL vs 16.3 pg/mL, p 0 (n = 101; evidence of neurological impairment) were not significantly different to those in patients with hATTR amyloidosis with PN (p = 0.15), where all patients had a PND>0. Patients with hATTR amyloidosis with CM and the V122I variant, generally considered to cause a predominant CM phenotype, also had elevated plasma NfL levels relative to healthy controls (44.9 pg/mL vs 16.3 pg/mL, p Conclusions NfL may serve as a biomarker of nerve damage in patients with hATTR amyloidosis, regardless of the predominant manifestation. This analysis also suggests PN may be underdiagnosed in patients with predominant CM, such as those with a V122I variant, and NfL may be a useful biomarker to support earlier diagnosis of PN in these patients.

The original article contained incorrect terminology for one of the cardiac measures; throughout the manuscript and supplementary information 'intraventricular septum wall thickness' should have been given as 'interventricular septum wall thickness'. Corrections should also be noted for Tables 1 and 4: in the Table 1 legend 'Low risk - Neither above at baseline' should read 'Low risk - Neither above threshold at baseline'; in Table 4, the rows 'Mild: eGFR > 60 to 30 to 0.20) (Supplementary Fig. 6)' should read 'There were no apparent differences in revusiran Cmax between patients with mild (eGFR: ≥ 60 to 0.20) (Supplementary Fig. 6)'.

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 .).

Lung cancer seems to be a very common cause of death among the people all over the world. Hence, accurate detection of lung cancer increases the chance of survival of the people. The major problem with the treatment is the time constraint in several physical diagnoses that increases the death possibilities so basically this method is an approach to help the physicians to take more accurate decision in this regard. This paper comes up with a method which is based on average information statistical parameters using image processing for lung cancer analysis. The basic aim is to help the physicians to take decisions regarding possibilities of lung cancer. Image averaging is a digital image processing technique, which is mostly implemented to improve the quality of images that have been degraded by random noise. The average information parameters are among the statistical parameters that are implemented for lung cancer analysis, and hence, some of the parameters like Entropy, Standard Deviation, Mean, Variance, and MSE are considered in this paper. The selection of average information parameters is thoroughly based on the calculation of number of iterations carried over the lung images through the algorithm. This paper also successfully rejects null hypothesis test by implementing ANOVA. The images are microscopic lung images and the algorithm is implemented in MATLAB.

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.

Lungs play an important role in human respiratory system. There are various diseases that effect the functioning of the lungs. Diagnosis and treatment of the disease is most important for proper functioning of human body. The most difficult part is to detect and diagnose the diseases at primary stage. In this paper, a computerized method for lung disease diagnosis is implemented. The lung diseases like Fibrosis, Emphysema and lung Cancer is detected using MATLAB software as these diseases are having almost similar symptoms and hence difficult to diagnose. The algorithm implemented here is based on Texture analysis, Principle Components Analysis and various image quality measurement statistical parameters such as Entropy, Standard Deviation and Texture index. Fuzzy logic is used to calculate the performance parameters of lung images. The algorithm is implemented and tested on microscopy lung images

RNA interference (RNAi) is a natural mechanism regulating protein expression that is mediated by small interfering RNAs (siRNA). Harnessing RNAi has potential to treat human disease; however, clinical evidence for the effectiveness of this therapeutic approach is lacking. ALN-RSV01 is an siRNA directed against the mRNA of the respiratory syncytial virus (RSV) nucleocapsid (N) protein and has substantial antiviral activity in a murine model of RSV infection. We tested the antiviral activity of ALN-RSV01 in adults experimentally infected with wild-type RSV. Eighty-eight healthy subjects were enrolled into a randomized, double-blind, placebo-controlled trial. A nasal spray of ALN-RSV01 or saline placebo was administered daily for 2 days before and for 3 days after RSV inoculation. RSV was measured serially in nasal washes using several different viral assays. Intranasal ALN-RSV01 was well tolerated, exhibiting a safety profile similar to saline placebo. The proportion of culture-defined RSV infections was 71.4 and 44.2% in placebo and ALN-RSV01 recipients, respectively ( P = 0.009), representing a 38% decrease in the number of infected and a 95% increase in the number of uninfected subjects. The acquisition of infection over time was significantly lower in ALN-RSV01 recipients ( P = 0.007 and P = 0.03, viral culture and PCR, respectively). Multiple logistic regression analysis showed that the ALN-RSV01 antiviral effect was independent of other factors, including preexisting RSV antibody and intranasal proinflammatory cytokine concentrations. ALN-RSV01 has significant antiviral activity against human RSV infection, thus establishing a unique proof-of-concept for an RNAi therapeutic in humans and providing the basis for further evaluation in naturally infected children and adults.

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.

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 Fcγ 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 pro- duced 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-∞ 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.

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

Objective 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. Methods 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). Results 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. Conclusions 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.

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.

Here, we describe the rational design, preclinical, and clinical development of a small interfering RNA (siRNA) directed against human respiratory syncytial virus (RSV). Infection with RSV can cause significant morbidity in young children and in elderly or immunosuppressed adults. In recipients of lung transplants, RSV infection has been associated with early onset of bronchiolitis obliterans syndrome (BOS), which leads to significant morbidity and mortality. Human studies included a Phase II study in adults experimentally infected with RSV. In this study, ALN-RSV01 significantly reduced the rate of RSV infection and resulted in lower overall viral loads and a shorter duration of viral shedding. In two subsequent Phase II studies, ALN-RSV01 was administered as a nebulized aerosol to lung transplant patients naturally infected with RSV. Safety and significant reduction in BOS was first demonstrated in 16 patients. Results from a larger study in 87 patients are currently pending. The approach for using a nebulized siRNA to treat a local lung pathogen is exciting to the field, given the specificity that can be employed by RNA interference technology, the minimal systemic exposure, and the potential to silence endogenous and invading targets that impact lung function and lung diseases.

Lower respiratory tract infections due to respiratory syncytial virus (RSV) are associated with development of bronchiolitis obliterans syndrome in lung transplant (LTX) recipients. ALN-RSV01 is a small interfering RNA targeting RSV replication.To determine the safety and explore the efficacy of ALN-RSV01 in RSV infection.We performed a randomized, double-blind, placebo-controlled trial in LTX recipients with RSV respiratory tract infection. Patients were permitted to receive standard of care for RSV. Aerosolized ALN-RSV01 (0.6 mg/kg) or placebo was administered daily for 3 days. Viral load was determined by quantitative reverse transcriptase-polymerase chain reaction on serial nasal swabs. Patients completed symptom score cards twice daily. Lung function, including the incidence of new-onset or progressive bronchiolitis obliterans syndrome, was recorded at Day 90.We enrolled 24 patients (ALN-RSV01, n = 16; placebo, n = 8); randomization was stratified by ribavirin use. ALN-RSV01 was well tolerated, with no drug-related serious adverse events or post-inhalation perturbations in lung function. Interpretation of viral measures was confounded by baseline differences between the two groups in viral load and time from symptom onset to first dose. Mean daily symptom scores were lower in subjects receiving ALN-RSV01, and the mean cumulative daily total symptom score was significantly lower with ALN-RSV01 (114.7 ± 63.13 vs. 189.3 ± 99.59, P = 0.035). At Day 90, incidence of new or progressive bronchiolitis obliterans syndrome was significantly reduced in ALN-RSV01 recipients compared with placebo (6.3% vs. 50%, P = 0.027).ALN-RSV01 was safe and may have beneficial effects on long-term allograft function in LTX patients infected with RSV. Clinical trial registered with www.clinicaltrials.gov (NCT 00658086).

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.

Excessive complement activation plays a pivotal role in a variety of disorders. Complement component C5 is a clinically validated therapeutic target for treatment of both paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic-uremic syndrome. We developed a robust RNAi therapeutics platform for the delivery of siRNAs to the liver using trivalent GalNAc conjugates, enabling specific silencing of hepatocyte-expressed genes following subcutaneous (SC) injection. The liver produces essentially the entirety of C5 and other complement pathway proteins. We are developing ALN-CC5, an investigational RNAi therapeutic targeting human, primate and rodent C5. C5 silencing and complement activity inhibition were examined in rodents and primates. Multi-dose SC ALN-CC5 treatment resulted in sustained lowering of cyno serum C5 with ≤3% residual protein remaining. C5 reduction was associated with >90% and >95% inhibition of classical and alternative complement pathways as measured by ELISA-based assays. Additionally, >80% lowering of complement serum hemolytic activity was observed. ALN-CC5 was safe and well tolerated in both rat and non-human primate toxicology studies. In addition to wild type animals, ALN-CC5 was tested in several animal models of disease in which complement activation plays a prominent role. Silencing of murine C5 was highly efficacious in a model of anti-collagen antibody-induced arthritis with a disease modifying activity equivalent to that of an anti-C5 antibody. Furthermore, C5 silencing was effective at reducing proteinuria in a rat model of membranous nephropathy. Up-regulation of C5 expression, observed in both models, had no effect on the extent of C5 silencing, suggesting that ALN-CC5 could be efficacious in the context of inflammation. These data demonstrate a prominent role for circulating, liver-derived C5 in mediating pathology at extrahepatic sites and the potential utility of an RNAi therapeutic targeting C5. In summary, RNAi-mediated silencing of liver-derived C5 is a promising novel therapeutic approach for inhibiting systemic complement activity, with the potential to enable, low volume, subcutaneous treatment for patients with PNH and other disorders where complement activation plays a role in disease progression. Disclosures Borodovsky: Alnylam: Employment. Yucius:Alnylam: Employment. Sprague:Alnylam: Employment. Banda:Alnylam: Research Funding. Holers:Alnylam: Research Funding. Vaishnaw:Alnylam Pharmaceuticals: Employment, Equity Ownership. Maier:Alnylam: Employment. Kallanthottathil:Alnylam: Employment. Charisse:Alnylam: Employment. Kuchimanchi:Alnylam: Employment. Manoharan:Alnylam: Employment. Salant:Alnylam: Honoraria. Fitzgerald:Alnylam: Employment. Meyers:Alnylam: Employment. Sorensen:Alnylam Pharmaceuticals: Employment, Equity Ownership.

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.

treatment group began to show evidence of statistically significant lower left ventricular ejection fraction (LVEF) levels at 6 to 12 month followup, the trial was halted. Conclusions: Despite comparable patient population characteristcs, the addition of pentoxifylline treatment to standard heart failure treatment of PPCM patients showed no benefits and failed to match the LVEF gains that were experienced by the non-pentoxifylline treatment group.

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.

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.

Abstract 1045 The liver hormone Hepcidin (encoded by Hamp1) regulates serum iron levels by controlling the efflux of iron from intestinal enterocytes and macrophages. Maintaining sufficient iron levels to support erythropoiesis while preventing iron overload requires tight control of Hepcidin expression. Transcription of Hamp1 in hepatocytes is stimulated by high serum iron levels, via Transferrin Receptor signaling, as well as by activation of the BMP/SMAD pathway. The membrane serine protease Matriptase-2 (encoded by Tmprss6) inhibits BMP induced Hamp1 induction through the regulation of the BMP co-receptor, Hemojuvelin. In humans, loss of function mutations in TMPRSS6 lead to elevated Hepcidin levels resulting in iron-resistant iron-deficiency anemia (IRIDA). In diseases associated with iron overload, such as Thalassemia intermedia (TI) and Familial Hemochromatosis (FH), Hepcidin levels are low despite elevated serum iron concentrations. Studies in murine models of TI and FH have shown that elevating Hepcidin levels by genetic inactivation of Tmprss6 can prevent iron overload and correct aspects of the disease phenotype. Therefore, therapeutic strategies aimed at specifically inhibiting Tmprss6 expression could prove efficacious in these, and other, iron overloading diseases. Here we show that systemic administration of a potent lipid nanoparticle (LNP) formulated siRNA directed against Tmprss6 leads to durable inhibition of Tmprss6 mRNA in the mouse liver, with concomitant elevation of Hamp1 expression. This leads to significant decreases in serum iron concentration and Transferrin saturation, along with changes in hematologic parameters consistent with iron restriction. Further testing in mouse genetic models of TI and FH will support the rationale for developing LNP formulated Tmprss6 siRNA as a novel therapeutic modality. Disclosures: Toudjarska: Alnylam Pharmaceuticals, Inc.: Employment. Cai:Alnylam Pharmaceuticals, Inc.: Employment. Racie:Alnylam Pharmaceuticals, Inc.: Employment. Milstein:Alnylam Pharmaceuticals, Inc.: Employment. Bettencourt:Alnylam Pharmaceuticals, Inc.: Employment. Hettinger:Alnylam Pharmaceuticals, Inc.: Employment. Sah:Alnylam Pharmaceuticals, Inc.: Employment. Vaishnaw:Alnylam Pharmaceuticals, Inc.: Employment. Bumcrot:Alnylam Pharmaceuticals, Inc.: Employment.