Ionis Pharmaceuticals Research Articles

Monoclonal Proteinuria Predicts Progression Risk in Asymptomatic Multiple Myeloma with a Free Light Chain Ratio ≥100

Monoclonal Proteinuria Predicts Progression Risk in Asymptomatic Multiple Myeloma with a Free Light Chain Ratio ≥100

Lack of IL6 Improves Recovery from Anemia of Inflammation Which Gets Hampered in Presence of Excess Iron

Abstract Anemia of inflammation (AI) is the second most common anemia after iron deficiency anemia. The predominant regulators of AI are the cytokine interleukin 6 (IL6) and the hormone hepcidin (HAMP). IL6 is an inflammatory cytokine which also limits iron absorption by inducing HAMP, which promotes the degradation of the iron exporter ferroportin. We hypothesized that knocking down both HAMP and IL6 simultaneously will help us to understand if their mode of action in AI is uniquely limited to iron absorption and erythroid iron intake or if they also have independent roles. Henceforth, we generated IL6/HampKO (DKO) mice and, unexpectedly, observed that IL6KO mice showed the best recovery in bone marrow (BM) erythropoiesis (using flow cytometry analysis and looking at the absolute number of erythroid progenitors) after BA administration when compared to wild type (WT), HampKO and DKO mice. The best differences were observed at 14 days post BA administration. In contrast, the extramedullary erythropoiesis in the spleen was more pronounced in HampKO and DKO mice compared to WT and IL6KO animals, indicating that the mechanism impairing erythropoiesis in the BM did not affect erythroid progenitors in the spleen. These observations suggest that HAMP and IL6 proteins contribute independently to AI, with IL6 having some effect on the erythropoiesis in the BM independent from the IL6-HAMP axis leading to iron restriction. Furthermore, these observations raised the question why both HampKO and DKO mice showed reduced BM erythropoiesis compared to IL6KO animals. We investigated inflammatory cytokines and altered iron parameters as potential mediators of impaired erythropoiesis. We compared several inflammatory cytokines, including IL6, TNFa and INFg following BA administration: cytokine levels were elevated 6 hrs, reduced 48hrs after BA administration and moderately increased again two weeks later. Interestingly, among all the cytokines the levels of IL1b were significantly attenuated in IL6KO mice at day 14 compared to WT and HampKO animals. Moreover, transferrin saturation and NTBI levels were higher in HampKO and DKO animals compared to IL6KO mice. These observations strongly suggested that BM erythropoiesis is more sensitive to inflammatory insult in presence of an excess of iron, while extramedullary erythropoiesis is mildly affected and can eventually thrive under supra-physiological transferrin saturation levels. To test if increased iron affects BM erythropoiesis in presence of inflammation, we treated both WT and IL6KO mice with combination of iron dextran and BA. Both WT and IL6KO mice were treated with a combination of BA and iron at day 0 followed by alternate day of iron injections showed the poorest erythropoiesis in the BM and became rapidly sick, although the effect was significantly more pronounced in WT animals, as suggested by their survival curve. Since mycobacterium infections lead to NLPR3 inflammasome activation and Caspase1 upregulation (Marim et al. Semin Immunopathology 2017), we investigated how erythroid progenitors were affected. By flow cytometry analyses, we observed a significantly higher upregulation of the Caspase1 protein in WT and DKO mice compared to IL6KO animals. This was also reproduced by culturing WT or IL6KO BM progenitor erythroid cells in presence of mouse serum derived from WT or IL6KO mice treated with BA. Most importantly, IL6KO mice treated with BA and iron showed the highest levels of Caspase1 compared to only BA treated IL6KO mice, indicating that excess of iron abrogates the beneficial effect of IL6 deficiency on erythropoiesis under conditions of AI. Furthermore, using flow cytometry, we observed in WT mice treated with BA or BA and iron a significant increase in mitochondrial mass, which is an indicator of mitochondrial stress. The mitochondrial mass was reduced in IL6KO mice treated with BA, but again increased in IL6KO mice treated with BA and iron. We have also observed an increase of mitochondrial superoxide by confocal microscopy in WT mice compared to IL6KO mice treated with BA. Altogether, these data support a model where inflammation in presence of an excess of iron impairs BM erythropoiesis through mechanisms at least in part mediated by Caspase1 and mitochondrial dysfunction, while iron excess itself is sufficient to boost extramedullary erythropoiesis to compensate and sustain RBC production. Disclosures Vinchi: PharmaNutra: Research Funding; Vifor Pharma: Research Funding; Silence Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding. Rivella: Ionis Pharmaceuticals: Consultancy; Meira GTx: Consultancy.

Factors Associated with Renal Impairment at Diagnosis in Multiple Myeloma with Survival Trends over Last Two Decades

IntroductionMultiple myeloma (MM) is frequently accompanied by renal impairment (RI) in 30-40% patients at presentation (Dimopoulos et al, IMWG 2010). As per International Myeloma Working Group (IMWG), RI is defined as Creatinine clearance (CrCl) <40 mL/min. Patients with RI at diagnosis have inferior overall survival (OS), however, over the years their outcome has improved due to treatment with novel agents. We examined the baseline clinical characteristics, and outcomes in patients with a new diagnosis of multiple myeloma at our institution to identify factors associated with baseline RI and study prognostic trend over the last 2 decades.MethodsWe retrospectively reviewed records for MM patients with a new diagnosis of MM who were seen at Mayo Clinic, Rochester between 1/2004 and 12/2018. RI was defined as per IMWG criteria. Categorical variables were compared using chi-square test and continuous variables using Wilcoxon test. Overall Survival (OS) was estimated using Kaplan-Meier method and difference between groups was assessed using log-rank test.ResultsIn our cohort of 2702 patients, median age at diagnosis was 65 (range, 22-96) years and 61% were males. Number of patients with CrCl <15, 15-29, 30-59 and ≥60 ml/min at diagnosis was 176 (6.5%), 204 (7.6%), 685 (25.3%) and 1637 (61%) respectively.RI was present in 557 (21%) of patients at the time of presentation. On univariate analysis, factors significantly associated with RI at diagnosis were higher age, ECOG PS ≥2, hemoglobin< 10 g/dL, calcium >11 mg/dL , LDH >222 U/L, β-2-microglobulin >5.5 mg/L, free light chain ratio >100, uric acid >8 mg/dL, higher ISS stage and cytogenetics [gain 1q, t (14;20), absence of any trisomy] as shown in table 1. However, on multivariate analysis, only elevated serum calcium >11 g/dL (OR: 11.5; 95%CI:1.78-74.9; p=0.01) and free light chain ratio>100 (OR: 4.17; 95%CI:1.29-13.4; p=0.016) had significant association with RI.The RI group had inferior OS when compared to patients with no RI at diagnosis (4.5 years vs 7.7 years, p-value <0.001). However, use of novel agents was associated with improvement in OS in patients with RI (4.9 years vs 2.6 years; p=0.0001) (Figure 1).Looking at trends over time, the rate of RI at presentation had not changed. Over the last two decades, OS for the CrCl<30 group had significantly improved in the last 5-year period (3.8 years in 2004-2014 vs not reached for 2014-2018; p= 0.0064) as shown in figure 2.ConclusionIn our cohort of 2702 patient, frequency of RI at baseline was 21%, which corroborates with previous studies. Hypercalcemia and serum free light chain ratio were found to be independent factors associated with RI. RI at diagnosis is associated with inferior survival, however, use of novel agents in this population has led to improvement of patient outcomes. [Display omitted] DisclosuresKapoor: Karyopharm: Consultancy; Cellectar: Consultancy; BeiGene: Consultancy; Pharmacyclics: Consultancy; Sanofi: Consultancy; Amgen: Research Funding; Ichnos Sciences: Research Funding; Regeneron Pharmaceuticals: Research Funding; Glaxo SmithKline: Research Funding; Karyopharm: Research Funding; Sanofi: Research Funding; Takeda: Research Funding; AbbVie: Research Funding. Gertz: Aurora Biopharma: Other: Stock option; Ionis Pharmaceuticals: Other: Advisory Board; Akcea Therapeutics, Ambry Genetics, Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Karyopharm Therapeutics, Pfizer Inc (to Institution), Sanofi Genzyme: Honoraria; AbbVie Inc, Celgene Corporation: Other: Data Safetly & Monitoring; Akcea Therapeutics, Alnylam Pharmaceuticals Inc, Prothena: Consultancy. Dingli: Alexion: Consultancy; Novartis: Research Funding; Apellis: Consultancy; GSK: Consultancy; Sanofi: Consultancy; Janssen: Consultancy. Kumar: Roche-Genentech: Consultancy, Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Beigene: Consultancy; Novartis: Research Funding; Merck: Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding; Bluebird Bio: Consultancy; Oncopeptides: Consultancy; Carsgen: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tenebio: Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Antengene: Consultancy, Honoraria; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding.

Tracking Daratumumab Clearance Using Mass Spectrometric Approaches: Implications on M Protein Monitoring and Reusing Daratumumab

Background:MASS-FIX is a screening method for serum and urine monoclonal proteins in multiple myeloma and related plasma cell disorders, which uses immunoglobulin enrichment coupled with matrix-assisted laser desorption ionization time-of-flight mass-spectrometry (MALDI-TOF). In addition to superior sensitivity over conventional gel-based techniques, MASS-FIX can distinguish therapeutic monoclonal antibodies (MoAb) from patient's M protein. As the utilization of therapeutic MoAbs increases, it is essential to understand the persistence pattern of these therapeutic antibodies in the serum. We designed this study to evaluate the duration of daratumumab detection by MASS-FIX in the serum of treated patients.Methods:We used a prospectively maintained database at Mayo clinic to identify patients with multiple myeloma and related plasma cell disorders who were treated with a daratumumab-containing regimen anytime during their disease course and had serial MASS-FIX data available after discontinuation of daratumumab. A univariate analysis was performed to assess for factors that may impact the clearance of daratumumab.Results:We included 125 patients with plasma cell disorders who received daratumumab as first or subsequent line of treatment between March 15 th, 2016, and March 4 th, 2020. The median age was 60.2 years and 57% were male. The most common diagnoses were multiple myeloma (70%) and light chain amyloidosis (18%). Daratumumab-based treatments were initiated after a median of 28.8 (IQR: 6.4-76.3) months from initial diagnosis. The most common regimen used was daratumumab, bortezomib and dexamethasone (23%); 26% underwent transplant after daratumumab-based induction. The median duration of treatment with a daratumumab-based regimen was 208 (IQR: 99-479) days. The median follow-up from the time of daratumumab discontinuation was 457 (95% CI: 346-NR) days. By last follow up, daratumumab was not detected by MASS-FIX in 93 (74%) patients but remained detectable in 32 (26%) patients. The median time from daratumumab discontinuation to disappearance of daratumumab by MASS-FIX was 160 (IQR: 107-233) days. On univariate analysis, the presence of ≥0.5 grams of urine protein was associated with earlier disappearance of daratumumab on MASS-FIX [risk ratio (RR): 2.0, P=0.02). The median time from daratumumab discontinuation to disappearance of daratumumab on MASS-FIX was 116 (95%CI: 76-160) days in patients with urine protein ≥0.5 grams and 203 (95%CI: 162-216) days in patients with urine protein <0.5 grams (P=0.02). There was no association between the time to disappearance of daratumumab by MASS-FIX and old age ≥70 (RR: 0.9, P=0.81], male gender (RR: 0.9, P=0.60), eGFR <60 (RR: 1.0, P=0.98), daratumumab schedule (every 1/2 weeks vs >2weeks) (RR: 1.0, P=0.97), treatment duration (<200 days vs ≥200 days) ( RR: 1.0, P=0.95), or transplantation status (RR: 1.0, P=0.98).Conclusion:The therapeutic monoclonal antibody daratumumab remains detectable in the serum of treated patients by MASS-FIX for several months after discontinuation and the duration varies between individual patients. This data has implications for diagnostic and monitoring testing and may provide guidance for reuse of daratumumab in clinical trials and practice. Proteinuria is associated with earlier disappearance of daratumumab by MASS-FIX and may have implications in patients with amyloidosis and monoclonal immunoglobulin deposition disease (MIDD). Further studies are needed to identify additional factors associated with the timing of disappearance. DisclosuresMurray: Mayo Clinic: Other: Has received patents for the Mass-Fix technology which has been licensed to the Binding Site with potential royalties.. Dispenzieri: Takeda: Research Funding; Alnylam: Research Funding; Pfizer: Research Funding; Oncopeptides: Consultancy; Sorrento Therapeutics: Consultancy; Janssen: Consultancy, Research Funding. Kapoor: Karyopharm: Consultancy; Cellectar: Consultancy; BeiGene: Consultancy; Pharmacyclics: Consultancy; Sanofi: Consultancy; Amgen: Research Funding; Ichnos Sciences: Research Funding; Regeneron Pharmaceuticals: Research Funding; Glaxo SmithKline: Research Funding; Karyopharm: Research Funding; Sanofi: Research Funding; Takeda: Research Funding; AbbVie: Research Funding. Gertz: Akcea Therapeutics, Ambry Genetics, Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Karyopharm Therapeutics, Pfizer Inc (to Institution), Sanofi Genzyme: Honoraria; Ionis Pharmaceuticals: Other: Advisory Board; Akcea Therapeutics, Alnylam Pharmaceuticals Inc, Prothena: Consultancy; Aurora Biopharma: Other: Stock option; AbbVie Inc, Celgene Corporation: Other: Data Safetly & Monitoring. Dingli: Alexion: Consultancy; Novartis: Research Funding; Apellis: Consultancy; Janssen: Consultancy; Sanofi: Consultancy; GSK: Consultancy. Kumar: Antengene: Consultancy, Honoraria; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bluebird Bio: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Merck: Research Funding; Roche-Genentech: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Beigene: Consultancy; Oncopeptides: Consultancy; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Research Funding; Carsgen: Research Funding; Tenebio: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding.

Prognostic Role of IL-6 in POEMS Syndrome

Prognostic Role of IL-6 in POEMS Syndrome

Impact of Achieving an Early Complete Response in Multiple Myeloma and Predictors of Subsequent Outcome

Impact of Achieving an Early Complete Response in Multiple Myeloma and Predictors of Subsequent Outcome

Aberrant CDK7 Activity Drives the Cell Cycle and Transcriptional Dysregulation to Support Multiple Myeloma Growth: An Attractive Molecular Vulnerability

Multiple myeloma (MM) cells are characterized by cell cycle dysregulation, epigenetic heterogeneity, and perturbation of the transcriptional landscape. We have previously shown that chemical and genetic perturbation of transcriptional regulator CDK7 significantly and selectively impacted MM cell growth and viability, supporting it as a pharmacologically relevant target for MM. Indeed, selective CDK7 inhibitor YKL-5-124 was active against a large panel of MM cell lines and primary MM cells, with a significantly lower IC50 compared to PHA-activated normal donor peripheral blood mononuclear cells (PBMCs). The efficacy of YKL-5-124 was confirmed in vivo in several murine models of MM, including disseminated models.Gene expression analysis after CDK7 inhibition in several MM cell lines revealed that transcripts for only a subset of genes were substantially affected by treatment with low dose of YKL-5-124, showing a strong leading-edge enrichment for downregulation of E2F expression program, cell cycle, DNA damage, and MYC targets. We have indeed confirmed a potent reduction in phosphorylation of RB protein, with consequent decrease of E2F activity in MM cells, supporting CDK7 as a central hub in the oncogenic CDK-pRb-E2F pathway in MM cells, with its expression and activity positively correlated with E2F transcriptional output in patient MM cells. Importantly, dual inhibition with low doses of YKL-5-124 and BRD4 inhibitor JQ1, displayed superior activity against a panel of MM cell lines and primary MM cells compared to single perturbation alone by both converging on a subset of key SE-associated dependencies as well as impacting distinct oncogenic expression programs.To identify synthetically lethal targets and mechanisms of resistance to CDK7 inhibition, we performed a genome-wide CRISPR-Cas9 knockout screen in the MM1S cells treated with YKL-5-124 or DMSO. We found that BCL6, NFKBIA and B, TRAF2, TSC1 and CSNK2A1, a subunit of CK2, were top synthetically lethal hits; whereas deletion of RB1, SF3B3 and the DNA-binding transcriptional activator TADA2A that regulates RNA-pol II transcription, led to resistance to YKL-5-124. Molecular mechanisms of intrinsic and acquired resistance to CDK7 inhibition are now under investigation and will be presented.In conclusion, our study demonstrates CDK7 as an attractive molecular vulnerability in MM that can be exploited therapeutically alone or in combination. DisclosuresShirasaki: FIMECS: Consultancy. Chesi: Novartis: Consultancy, Patents & Royalties: human CRBN transgenic mouse; Pfizer: Consultancy; Pi Therapeutics: Patents & Royalties: Genetically engineered mouse model of myeloma; Abcuro: Patents & Royalties: Genetically engineered mouse model of myeloma; Palleon Pharmaceuticals: Patents & Royalties: Genetically engineered mouse model of myeloma. Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Mitsiades: Arch Oncology: Research Funding; Karyopharm: Research Funding; Adicet Bio: Membership on an entity's Board of Directors or advisory committees; Ionis Pharmaceuticals: Consultancy, Honoraria; Abbvie: Research Funding; FIMECS: Consultancy, Honoraria; Nurix: Research Funding; Sanofi: Research Funding; Novartis: Research Funding; H3 Biomedicine: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding; Fate Therapeutics: Consultancy, Honoraria; BMS: Research Funding; TEVA: Research Funding. Munshi: Novartis: Consultancy; Legend: Consultancy; Pfizer: Consultancy; Adaptive Biotechnology: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Abbvie: Consultancy; Karyopharm: Consultancy; Celgene: Consultancy; Bristol-Myers Squibb: Consultancy.

Second Line Treatment Strategies in Multiple Myeloma: A Referral-Center Experience

Second Line Treatment Strategies in Multiple Myeloma: A Referral-Center Experience

A Severe Mouse Model of Alpha-Thalassemia to Study Abnormal Iron Metabolism and Erythropoiesis, Hematopoietic Stem Cell Behavior and Development of a Gene Therapy Approach for Its Treatment

Alpha thalassemia (α-thal) is caused by insufficient production of the α-globin protein because of either deletional or non-deletional inactivation of endogenous α-globin genes. Clinical presentation of deletional α-thal varies from an asymptomatic condition (one inactivated α-globin gene) to a complete knockout (Hb Bart's Hydrops Fetalis). In patients with severe α-thal, a blood transfusion independent state is achievable through allogeneic bone marrow transplantation.The aims of this study are to develop a novel adult mouse model of α-thal and a gene therapy approach for this disease.We generated adult animals that do not produce α-globin chains (α-KO) through transplantation of homozygous B6.129S7-Hbatm1Paz/J fetal liver cells (FLC; isolated at E14.5) into WT recipient mice. These animals demonstrate a worsening phenotype, paradoxically showing elevated hematocrit, high reticulocyte count and a high number of red blood cells (RBC) which expressed only β-globin chains (HbH). RBC show aberrant morphology and aggregation of α- globin tetramers on RBC membranes. Due to severe inability of these RBC to deliver oxygen, the mice eventually succumb to anemia, showing splenomegaly and other organ pathologies, including vaso-occlusive events. These animals show iron deposition in the liver and kidney, in agreement with very low levels of hepcidin expression in the liver, and elevated erythropoietin (EPO) in the kidney.Interestingly, α-KO embryos show lower numbers of FLC compared to WT embryos, lower frequency of engraftable hematopoietic stem cells (HSC; Lin-Sca-1+c-kit+CD48-), decreased clonogenic potential (fewer class 4 CFUs) and elevated erythroferrone. Lethally irradiated mice transplanted with FLC-KO require 5-6x as many cells as those transplanted with FLC-WT for recovery, further suggesting some level of engraftment impairment. Our current hypothesis is that excessive hypoxia in the embryos impairs HSC function and stem cell fitness. Additional assays are in progress to assess the nature of this impairment.To generate a gene therapy tool to rescue these animals and eventually cure severe human α-thal patients, we screened multiple lentiviral vectors to identify the variant capable of producing the highest human α-globin protein per copy. The selection was conducted in murine erythroleukemia cells and human umbilical cord derived erythroid progenitor (HUDEP) cells, modified by knocking out all the human α-globin genes. We identified ALS20α, a vector where α-globin is under control of the β-globin promoter and its locus control region, as the most efficient vector. One copy of ALS20α produces exogenous α-globin at a level comparable to that produced by one endogenous α-globin gene. These results suggest that a relatively low VCN could result in dramatic therapeutic benefits. Transplantation of ALS20α transduced murine BM-KO results in correction of the disease phenotype in a dose-dependent manner. At VCN<1 we observe a delay in death proportional to the VCN value, while at VCN>1 we observe phenotypic normalization, including Hb, hepcidin and EPO levels.We tested ALS20α in CD34 cells isolated from four patients with both deletional and non- deletional HbH disease. We measured the change of β/α-globin mRNA ratio (β/αR) and protein level by HPLC in erythroblasts derived from these cultures. For the specimen with mutational HbH, the initial β/αR matches that of healthy controls, as the mutations do not eliminate the ability for the gene to produce aberrant mRNA transcripts, and decreased with increasing VCN. Erythroblasts with deletional HbH have a β/αR approximately 3x higher than normal cells, decreasing in a dose dependent manner with increasing VCN. HPLC detection of HbH (β4), a hallmark of HbH disease, is observed in hemolysis products from all non-transduced α−thal erythroblasts. A ~50% reduction of HbH is detected in the very same specimens upon integration of ALS20α (VCN between 1 and 2).In conclusion, we generated an adult mouse model of lethal α-thal and, in preliminary experiments, we rescue it with ALS20α. Furthermore, ALS20α successfully improves α-globin levels in patient cells. Further experiments are in progress to establish the consistency of our vector's expression in vivo, as well as to demonstrate its ability to transduce bona fide long-term HSCs. DisclosuresKattamis: Agios Pharmaceuticals: Consultancy; IONIS: Consultancy; VIFOR: Consultancy; CRISPR/Vertex: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria, Research Funding; Chiesi: Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Consultancy. Rivella: Celgene Corporation: Consultancy; Keros Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Disc Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees; MeiraGTx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Forma Theraputics: Consultancy; Incyte: Consultancy; Ionis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Assessing the Prognostic Utility of the Mayo 2018 and IMWG 2020 Smoldering Multiple Myeloma Risk Stratification Scores When Applied Post Diagnosis

Assessing the Prognostic Utility of the Mayo 2018 and IMWG 2020 Smoldering Multiple Myeloma Risk Stratification Scores When Applied Post Diagnosis

Outcomes of Triple Class (Proteasome Inhibitor, IMiDs and Monoclonal Antibody) Refractory Patients with Multiple Myeloma

Outcomes of Triple Class (Proteasome Inhibitor, IMiDs and Monoclonal Antibody) Refractory Patients with Multiple Myeloma

Durability of Hemoglobin Response and Reduction in Transfusion Burden Is Maintained over Time in Patients with Pyruvate Kinase Deficiency Treated with Mitapivat in a Long-Term Extension Study

Durability of Hemoglobin Response and Reduction in Transfusion Burden Is Maintained over Time in Patients with Pyruvate Kinase Deficiency Treated with Mitapivat in a Long-Term Extension Study

Inhibition of Fibroblast Growth Factor-23 (FGF-23) Rescues Bone and Hematopoietic Stem Cell Niche Defects in Beta-Thalassemia, Uncovering the Missing Link between Hematopoiesis and Bone

The bone marrow (BM) niche regulation and interactions with hematopoietic stem cells (HSC) have been extensively studied in steady state conditions and malignancies, but are still underexplored in hematological inherited disorders. We provided the first demonstration of impaired HSC function caused by an altered BM niche in a non-malignant disease, beta-thalassemia (BT) (Aprile et al., Blood 2020). BT is a globally widespread congenital hemoglobin disorder, resulting in severe anemia, ineffective erythropoiesis and multi-organ secondary complications, including bone alterations. Correction of the genetic defect is achieved by transplantation of HSC from healthy donors or autologous HSC from patients upon gene therapy. Since the quality and the engraftment of HSC depend on the BM microenvironment, niche-HSC crosstalk plays a crucial role for transplantation outcome. During the analysis of different components of the niche, we found reduced bone density in BT th3 mice, along with a defective HSC supporting activity by the BM stromal niche. Interestingly, osteoporosis is a constant hallmark in BT patients.We investigated the mechanisms underlying bone and HSC niche defects focusing on the role of fibroblast growth factor-23 (FGF-23), a hormone mainly secreted by osteocytes, but also by erythroid cells, which negatively modulates bone metabolism. Since FGF-23 is stimulated by the anemia-related factor erythropoietin (EPO), we hypothesized that the high EPO levels in BT might contribute to increase FGF-23, potentially affecting bone and BM niche homeostasis.We found high levels of circulating FGF-23 in th3 mice (wt vs. th3: 290.5±27.3 vs. 1823±136.1 pg/ml, p<0.0001) and in BT patients (HD vs. THAL: 94.7±1.8 vs. 117.2±5.3 RU/ml, p<0.01), associated to its increased expression by bone and BM erythroid cells. In vivo neutralization of EPO axis was sufficient to normalize FGF-23 levels (th3 vs. th3+anti-EPO: 1591±162.2 vs. 496.1±33.3 pg/ml, p<0.001), thus demonstrating the causative role of EPO. EPO stimulation and signaling inhibition strategies highlighted the involvement of Erk1/2 and Stat5 pathways in activating Fgf-23 transcription in bone and BM erythroid cells, respectively.To provide proof of concept data on the contribution of FGF-23 to BT bone and stromal niche alterations, we inhibited FGF-23 signaling. In vivo administration of FGF-23 blocking peptide rescued the trabecular bone density in th3 mice (th3 vs. th3+FGF23inh: 138.2±4.9 vs. 166.9±5.2 mg/cm 3, p<0.01). Short-term inhibition treatment (38 hours) was sufficient to enhance bone mineralization by acting on Alkaline Phosphatase and on the expression of the main regulators of mineralization Dmp1 and Mepe by osteocyte, whereas long-term administration (12 days) restored also osteoblast number and bone deposition. Importantly, FGF-23 inhibition normalized the expression of key niche molecules, such as Jagged-1 and osteopontin, involved in the functional crosstalk between HSC and the stromal niche. Consistently, the treatment restored the frequency of th3 HSC by expanding the pool of quiescent cells (th3 vs. th3+FGF23inh: 0.026 vs. 0.045% on Lin neg BM cells, p<0.01). FGF-23 inhibition had also a positive anti-apoptotic effect on the expanded BM erythroid compartment (th3 vs. th3+FGF23inh: 61.6±1.3 vs. 51.1±2.1% of BM Ter119 + cells, p<0.001), promoting the maturation of early erythroid precursors (th3 vs. th3+FGF23inh: 8.5±1 vs. 16.4±1.1% of BM Pro-Erythroblasts, p<0.0001), as already shown in models of secondary anemias.Evidence in BT patients showed negative correlations between FGF-23 levels and markers of bone homeostasis (e.g. osteocalcin R 2=0.88, p<0.05) and positive correlations with makers of ineffective erythropoiesis (e.g. circulating reticulocytes R 2=0.83, p<0.05), thus positioning FGF-23 as the molecule at the crossroads of erythropoiesis and bone metabolism in BT.Our findings uncover an underexplored role of FGF-23 in bone and BM niche defects in a primary anemia, as a condition of chronic EPO stimulation, and propose FGF-23 as the missing link between hematopoiesis and bone regulation. The inhibition of FGF-23 signaling might provide a novel strategy to ameliorate bone compartment and restore HSC-BM niche interactions in BT by a ‘two birds with one stone’ approach, with a potential translational relevance in improving HSC transplantation and gene therapy. DisclosuresCappellini: Celgene: Consultancy, Research Funding; Vifor: Consultancy; La Jolla: Research Funding; Protagonist Therapeutics: Research Funding; IONIS Pharmaceuticals: Consultancy; CRISPR Therapeutics: Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Biogen’s ALS drug falters in Phase 3 trials

Biogen’s experimental treatment for amyotrophic lateral sclerosis (ALS) failed to meet its goal of reducing symptoms in a Phase 3 clinical trial—a disappointing result for the highly anticipated treatment. Despite the setback, the company calls some of its secondary findings encouraging and is asking the US Food and Drug Administration to broaden availability of the drug, which is already being administered under a compassionate use program. The treatment, tofersen, was part of a deal struck by Biogen and Ionis Pharmaceuticals in 2018 that cost Biogen $1 billion. It would have been the first antisense drug for ALS, a neurodegenerative disease in which people lose motor function and become progressively weaker. Small-molecule treatments exist, but experts say they don’t offer enough benefits. “We were pretty disappointed,” says Nicholas Maragakis, a neurologist at Johns Hopkins Medicine who was one of the investigators in Biogen’s trials. But he notes that other tofersen trials

Clinical characteristics of patients with hereditary transthyretin mutations primarily associated with cardiomyopathy and other rare transthyretin mutations: insights from a genetic testing programme

Abstract Background/Introduction Hereditary transthyretin amyloidosis (hATTR; ATTRv) is a progressive, fatal disease caused by mutations in the transthyretin gene (TTR) that results in deposition of amyloid throughout the body, including in the heart. The p.V142I and p.T80A mutations typically manifest with a cardiomyopathy (CM) phenotype. Early diagnosis, which can be facilitated by genetic testing, is key to achieving optimal patient outcomes. Purpose To characterise the clinical profile and symptom burden of patients with hereditary transthyretin mutations associated primarily with a CM phenotype and rare hereditary transthyretin mutations. Methods This analysis used data from hATTR Compass, a genetic testing programme in the United States and Canada for patients suspected of having hATTR with polyneuropathy (PN) and patients with a family history of hATTR. Sequencing was performed using a TTR single-gene test, a gene panel of inherited cardiovascular disorders (CardioNext), or a gene panel of inherited neuromuscular disorders (NeuropathySelect). Akcea is aware of isolated data quality issues. Importantly, these do not affect the conclusions of this analysis. Results Cardiology specialists referred 466 patients with p.V142I, 15 with p.T80A, and 28 with rare TTR mutations to this programme. Of patients who reported sex, 57%, 53%, and 52% with p.V142I, p.T80A, and rare mutations, respectively, were male. Of patients who reported ethnicity, most with p.V142I were African American (94%), whereas the majority of patients with p.T80A and rare TTR mutations were Caucasian (100% and 69%, respectively). 24%, 60%, and 50%, of patients with p.V142I, p.T80A, and rare TTR mutations, respectively, had a family history of hATTR. The majority of patients with p.V142I (74%), p.T80A (53%), and rare TTR (54%) mutations were 65 years of age or older. Although most patients with p.V142I, p.T80A, and rare TTR mutations experienced symptoms/manifestations of heart disease (94% vs 100% vs 85%), many also presented with bilateral carpal tunnel syndrome (23% vs 44% vs 30%) and with sensory (27% vs 44% vs 65%), motor (15% vs 11% vs 25%), and autonomic (19% vs 11% vs 30%) dysfunction. Conclusion Most patients with the p.V142I mutation were African American, whereas many with p.T80A and other rare TTR mutations were Caucasian. Family history of hATTR was more common among patients with p.T80A and other rare TTR mutations than among patients with p.V142I. Regardless of the underlying mutation variant, many hATTR patients can present with various symptoms/manifestations aside from CM, such as PN and bilateral carpal tunnel syndrome. Recognising the neurological symptoms that can occur alongside CM and performing subsequent genetic testing facilitates diagnosis of hATTR. Early diagnosis is critical in hATTR because it is progressive and fatal, and early initiation of disease-modifying therapy is essential to optimising patient outcomes. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): This study was sponsored by Akcea Therapeutics, an affiliate of Ionis Pharmaceuticals, Inc.

Long-term efficacy and safety of inotersen in patients with hereditary transthyretin amyloid polyneuropathy with or without cardiomyopathy: post hoc analysis of NEURO-TTR open-label extension

Abstract Background/Introduction Cardiomyopathy (CM) with associated heart failure and polyneuropathy (PN) are common manifestations of hereditary transthyretin amyloidosis (ATTRv), a progressive, debilitating, and fatal disease that results from the deposition of misfolded transthyretin (TTR) protein throughout the body. NEURO-TTR (NCT01737398) showed that inotersen, an antisense oligonucleotide inhibitor of TTR protein production, slowed the progression of PN and maintained quality of life in patients with ATTRv. Purpose To report efficacy and safety from the open-label extension (OLE) of the NEURO-TTR study in patients with ATTRv PN overall and in CM subgroups. Methods Patients who completed NEURO-TTR, enrolled in the OLE (NCT02175004), and either switched from placebo in NEURO-TTR to inotersen in the OLE (placebo-inotersen) or received inotersen in NEURO-TTR and remained on inotersen in the OLE (inotersen-inotersen) were included. Assessments included the modified Neuropathy Impairment Score +7 composite score (mNIS+7 [range –22.3 to 346.3], a measure of neuropathy with higher scores indicative of poorer function), TTR levels, and safety monitoring. Utilizing patients from Europe and North America (EU+NA) as of 28 July 2020, this post hoc analysis examined two subgroups: CM ECHO and severe CM ECHO. CM was defined as a diagnosis of ATTRv CM at study entry or all of the following criteria: a left ventricular wall thickness of ≥1.3 cm on transthoracic echocardiography at baseline, no known history of persistent hypertension (systolic blood pressure ≥150 mm Hg) within 12 months before study screening, and evaluable baseline ECHO obtained by central assessment. Severe CM was defined as an interventricular septum thickness ≥1.5 cm at baseline. Descriptive statistics are reported. Results In the overall population and both CM subgroups, the placebo-inotersen group demonstrated slowing of neurological disease progression compared with natural history based on NEURO-TTR placebo projection (estimated natural history will be presented). Furthermore, in the overall population and both CM subgroups, the inotersen-inotersen group demonstrated sustained benefit compared with the placebo-inotersen group (Table). Change in serum TTR levels will be presented. There have been no reports of grade 4 thrombocytopenia or acute glomerulonephritis under enhanced monitoring in patients in the EU+NA despite the increased duration of exposure. No new safety concerns were identified. Conclusions Inotersen treatment for &amp;gt;3 years slowed the progression of PN associated with ATTRv in patients with CM, including severe CM. In both subgroups, greater neurological preservation was observed in those who initiated inotersen earlier (inotersen-inotersen group), underscoring the benefits of early treatment. No new safety signals were detected in this OLE analysis; enhanced monitoring is successful in managing the risk for thrombocytopenia and acute glomerulonephritis. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): This study was sponsored by Akcea Therapeutics, an affiliate of Ionis Pharmaceuticals, Inc.

Abstract MP47: Assessment Of Atherosclerosis In A Novel Transgenic Mouse Expressing Pathogenic Levels Of Human Lipoprotein(a)

Studying the role of elevated plasma levels of lipoprotein(a) (Lp(a)) in atherogenesis is challenging given that the LPA gene encoding apo(a) is absent in typical animal models. As such we created a novel transgenic mouse line expressing both human apolipoprotein(a) (apo(a)) and human apoB100 ((Tg(LPA +/0 ;APOB +/0 )) . The LPA transgene, assembled in the pLIV vector, produces a physiologically relevant 12-kringle apo(a) isoform. The APOB transgene contains the entire human APOB gene with a mutation preventing APOB editing. In our atherosclerosis model, male and female mice were injected weekly with an antisense oligonucleotide targeting the mouse LDL receptor (IONIS Pharmaceuticals), and fed a high-fat, high-cholesterol diet for 12 weeks. High plasma Lp(a) levels were observed in both male and female Tg(LPA +/0 ;APOB +/0 ) mice (219.0±11.9 and 133.7±12.1 mg/dL, respectively; n=12/group). Importantly, high levels of Lp(a) in plasma did not result in metabolic alterations, including differences in weight gain, energy expenditure, activity, RER, VO 2 /VCO 2 consumption/output, and glucose tolerance compared to Tg(APOB +/0 ) control mice. Both male and female Tg(LPA +/0 ;APOB +/0 ) and Tg(APOB +/0 ) mice exhibited enhanced proatherogenic lipoprotein profiles with the majority of the cholesterol and TG present in the VLDL and LDL fractions. Complex lesions developed in all transgenic mice, including large, lipid-rich necrotic cores, with overlying fibrous caps and intimal calcium deposition. Immunohistochemistry with a monoclonal apo(a) antibody revealed the presence of Lp(a) in the plaques of Tg(LPA +/0 ;APOB +/0 ) mice. Analysis of aortic sinus lesions revealed a 23% increase in total plaque area in female Tg(LPA +/0 ;APOB +/0 ) mice compared to female Tg(APOB +/0 ) mice (p=0.0836). Moreover, atherosclerotic plaques in female Tg(LPA +/0 ;APOB +/0 ) mice contained significantly more calcium deposition than female Tg( APOB +/0 ) mice (1.79±0.4% vs. 2.96 ±0.3% of total plaque area (p&lt;0.05)). No differences in plaque area or calcium were observed in male mice. In this ongoing study, detailed analyses of atherosclerotic plaque components in Tg(LPA +/0 ;APOB +/0 ) mice will be required to uncover the unique contribution of Lp(a) to atherogenesis.

Ionis, Bicycle sign oligonucleotide pact

Ionis Pharmaceuticals will pay Bicycle Therapeutics $45 million to license the firm’s bicyclic peptide technology. Bicycle is developing the synthetic short peptides as oncology treatments. But Ionis will use them to deliver oligonucleotide therapeutics to tissue, taking advantage of the high affinity for the transferrin receptor of some of Bicycle’s peptides. Bicycle will retain rights to use the peptides for all nonoligonucleotide delivery applications.

Pharmacokinetics and Pharmacodynamics of Ionis-FXIRx, an Antisense Inhibitor of Factor XI, in Patients with End-Stage Renal Disease on Hemodialysis

BACKGROUNDPatients with end-stage renal disease (ESRD) on hemodialysis are at high risk of both thrombotic and bleeding events. Experimental and clinical data indicate that reducing factor XI (FXI), a key component of the intrinsic pathway, prevents thrombosis without causing bleeding. FXI activity can be lowered with IONIS-FXIRx, a 2nd generation antisense oligonucleotide that specifically reduces human FXI mRNA expression in the liver. This study sought to determine the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of IONIS-FXIRx in participants with ESRD. METHODSIn this Phase 2 multicenter study, we enrolled a total of 49 ESRD patients: (A) 6 participants receiving chronic in-center hemodialysis (HD) to receive an open-label single-dose of 300 mg IONIS-FXIRx either 5 minutes before or 10 minutes after HD and assessed the effects of HD on peak (Cmax) and the extent of exposure (AUC0-24hr) parameters of IONIS-FXIRx and (B) another 43 participants in a double-blind design to one of two multiple-dose regimens (200 mg or 300 mg) IONIS-FXIRx or placebo for 12 weeks with 12 weeks of additional follow up. In the double-blind portion, IONIS-FXIRx was administered subcutaneously ~10 minutes after HD. For PK, plasma trough concentration and apparent elimination half-life (t1/2λz) were evaluated. The PD parameters including FXI activity and antigen, aPTT, prothrombin time (PT), and INR were evaluated in the per-protocol population (N= 34) that was defined as all participants who received the complete protocol-specified administration of IONIS-FXIRx or placebo through week 8 and who did not have any major protocol violations. The rate and frequency of clotting on the HD filters and circuit were qualitatively measured with a clotting scale as an exploratory PD analysis. RESULTSThe PK cohort included 6 males (5 white) with a mean age of 61 years. The plasma PK profile of IONIS-FXIRx was not significantly altered whether injected before or after HD. After multiple dosing, plasma Cmax values were dose-dependent and similar between Day 1 (first dose) and Day 78 (last dose), suggesting no accumulation of IONIS-FXIRx in ESRD participants after 12-weeks of dosing. After treatment cessation, IONIS-FXIRx had an apparent elimination half-life of approximately 2 weeks. In the double-blind portion of the study, 22 (51.2%) of the participants were male, 24 (55.8%) were white and the mean age was 59 years. Attenuation of FXI activity was associated with a reduction in the incidence of severe clotting events on both the air filter and dialyzer (Table). The reduction in severe clotting events was observed when FXI activity was < 0.4 U/mL. Consistent with prior observations in healthy subjects, IONIS-FXIRx prolonged aPTT in a time and dose-dependent manner with maximum mean percent changes from baseline of 53.3% (p < 0.05) for the 200 mg dose group and 75.6% (p < 0.05) for the 300 mg dose group at Week 14, but had no effect on PT. Treatment emergent adverse events (TEAEs) were mostly mild; participants reporting serious TEAEs were 1 (16.7%) for the PK cohort, 4 (30.8%) for placebo and 3 (20.0%) each for the 200 mg and 300 mg IONIS-FXIRx dose groups. There were no drug-related SAEs and no drug-related major or clinically-relevant non-major bleeding events. Minor bleeds were primarily observed in the 300 mg multiple-dose group at AV fistula/graft sites but did not correlate with reduction of FXI activity. No clinically significant reductions in platelet counts were reported during the study. CONCLUSIONSThis study demonstrated that (i) HD had no effect on IONIS-FXIRx PK, (ii) IONIS-FXIRx was well tolerated and produced sustained, and dose-dependent reductions in FXI antigen and activity, and (iii) IONIS-FXIRx reduced severe dialysis circuit clotting events beyond standard heparin use. Collectively, these data support further evaluation of IONIS-FXIRx as a potentially safe, effective antithrombotic therapy in ESRD patients on HD. [Display omitted] DisclosuresBethune:Ionis Pharmaceuticals: Employment. Walsh:Population Health Research Institute: Employment; Ionis Pharmaceuticals: Consultancy. Jung:Ionis Pharmaceuticals: Employment. Yu:Ionis Pharmaceuticals: Employment. Geary:Ionis Pharmaceuticals: Employment. Bhanot:Ionis Pharmaceuticals: Employment.

Differential Role of Plasma and Platelet-Derived Factor V In Vivo

Coagulation factor V (FV) has a profound effect on coagulation and deficiency states lead to bleeding. In blood, there are two pools of FV with 80% found in plasma and 20% contained in the α-granules of platelets. The biological basis for this separation is not clear; however, structural distinctions between the pools have been identified suggesting they may have unique effects or play different roles following injury. Broadly however, in vivo evidence for this is lacking. Transgenic mice with synthesis limited to liver or megakaryocytes have been generated to study the role of plasma and platelet FV (Sun et al, Blood 102:2856, 2003). Either pool is sufficient for basal hemostasis and tail-bleeding times were only slightly prolonged compared to wild-type (wt) mice. The impact of the two pools following different hemostatic challenges in different vessels is not known. To expand our in vivo understanding of the FV pools on clot formation, we further studied mice with only platelet FV (platelet-FV+) or plasma FV (plasma-FV+). As a control, we also generated mice with essentially little to no total FV (FV-low). To generate platelet-FV+ mice, wt mice were treated with a single dose of a liver targeted antisense oligonucleotide (ASO) to knockdown plasma FV (ASO-FV; 40 mg/kg). After 3 weeks of ASO-FV treatment, the plasma FV levels assessed with a murine FV-specific ELISA were at the lower limits of detection while platelet FV levels were normal. This confirms prior work showing platelet FV originates from megakaryocyte synthesis in mice, which is different from humans. To generate plasma-FV+ mice, we used previously established transgenic mice only expressing FV (~30% of wt) from the liver with no platelet FV. To create FV-low mice, plasma-FV+ mice were treated with ASO-FV; this resulted in mice with markedly reduced (<0.5%) plasma FV and no platelet FV. Using whole blood from these mice, ROTEM assessment showed that compared to wt mice (n = 5; 5.9 ± 0.3 min), the clot time (CT) for plasma-FV+ mice (n = 3; 5.9 ± 0.4 min) was normal while the CT was prolonged 2-fold for platelet-FV+ mice (n = 10; 13 ± 1.2 min). As expected, the almost complete lack of both pools (FV-low) substantially increased the CT (n = 3; 24 ± 2.2 min). Evaluation of these mice in several injury models probing different vessels was next pursued to examine the hemostatic potential of either pool. Using either a partial (mild) or total (severe) tail injury model, total blood loss for both wt (n = 5) and platelet-FV+ mice (n=10) were similar. In contrast, plasma-FV+ mice (n=5, p<0.001) exhibited a 2- (mild injury) or 5-fold (severe injury) increase in blood loss compared to wt mice. As expected, FV-low mice (n=5; p<0.001) had the highest levels of blood loss. Further, we used a microcirculatory model of hemostasis to image the distribution of plasma vs. platelet FV following laser injury and assess fibrin and platelet deposition. In plasma-FV+ mice, a rapid and robust fluorescence signal for murine FV/Va was detected on the vessel wall surrounding the microscopic injury and on a subset of platelets adherent at the injury site. For platelet-FV+ mice, murine FV/Va was detected only on core subset of platelets, which we speculate are fully activated. Further, analysis of the median integrated florescence as a function of time in multiple mice (n=3-5/group; 15-25 injuries) revealed that the absence of either platelet or plasma FV did not considerably alter the kinetics of platelet accumulation (~2-fold less than wt). However, in contrast to plasma-FV+ mice, platelet-FV+ mice had significantly reduced and delayed fibrin deposition following laser injury suggesting the FV released from platelets is not sufficient to produce normal amounts of fibrin in this model. Our data indicate that the overall impact and contribution of the platelet and plasma FV pools is different depending on the type of injury and/or vessel type. In the microcirculation, plasma FV appears to play an important role in binding to damaged endothelium and contributing to robust thrombin formation, while platelet FV may play a reduced role. However, in more severe injury models, platelet FV plays a prominent role. Collectively these data highlight potentially different roles for the FV pools and/or indicate that the injury models employed impact FV activation or release from platelet α-granules to different extents. DisclosuresIvanciu:Bayer: Research Funding; Novo Nordisk: Research Funding. Crosby:Ionis Pharmaceuticals: Employment, Research Funding. Revenko:Ionis Pharmaceuticals: Employment, Research Funding. MacLeod:Ionis Pharmaceuticals: Employment. Monia:Ionis Pharmaceuticals: Employment. Davidson:Spark Therapeutics: Consultancy. Camire:Bayer: Consultancy, Research Funding; Pfizer: Consultancy, Patents & Royalties, Research Funding; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Research Funding.