Sapablursen

Cellular uptake of antisense oligonucleotides (ASOs) is one of the main determinants of in vivo activity and potency. A significant advancement in improving uptake into cells has come through the conjugation of ASOs to triantenarry N-acetyl-galactosamine (GalNAc₃), a ligand for the asialoglycoprotein receptor on hepatocytes. The impact for antisense oligonucleotides, which are already taken up into hepatocytes, is a 10-fold improvement in potency in mice and up to a 30-fold potency improvement in humans, resulting in overall lower effective dose and exposure levels. 2'-Methoxyethyl-modified antisense oligonucleotide conjugated to GalNAc₃ (ISIS 702843) is specific for human transmembrane protease serine 6 and is currently in clinical trials for the treatment of β-thalassemia. This report summarizes a chronic toxicity study of ISIS 702843 in nonhuman primates (NHPs), including pharmacokinetic and pharmacology assessments. Suprapharmacologic doses of ISIS 702843 were well tolerated in NHPs after chronic dosing, and the data indicate that the overall safety profile is very similar to that of the unconjugated 2'-(2-methoxyethyl)-D-ribose (2'-MOE) ASOs. Notably, the GalNAc₃ moiety did not cause any new toxicities nor exacerbate the known nonspecific class effects of the 2'-MOE ASOs. This observation was confirmed with multiple GalNAc₃-MOE conjugates by querying a data base of monkey studies containing both GalNAc₃-conjugated and unconjugated 2'-MOE ASOs. SIGNIFICANCE STATEMENT: This report documents the potency, pharmacology, and overall tolerability profile of a triantenarry N-acetyl-galactosamine (GalNAc₃)-conjugated 2'-(2-methoxyethyl)-D-ribose (2'-MOE) antisense oligonucleotide (ASO) specific to transmembrane protease serine 6 after chronic treatment in the cynomolgus monkey. Collective analysis of 15 independent GalNAc₃-conjugated and unconjugated 2'-MOE ASOs shows the consistency in the dose response and character of hepatic and platelet tolerability across sequences that will result in much larger safety margins for the GalNAc₃-conjugated 2'-MOE ASOs when compared with the unconjugated 2'-MOE ASOs given the increased potency.

Management of patients with β-thalassemia is undergoing a swift evolution, considering the number of novel agents recently receiving marketing approval or entering clinical development. Although (thankfully) the availability of new treatment options will address several persisting unmet needs in this patient population, several questions remain on how such advances should be optimally integrated into standard of care. The only currently approved therapy for patients with non-transfusion-dependent β-thalassemia (NTDT) is iron chelation (for patients ≥ 10 years).1 This only followed recent evidence of clinically significant iron overload and subsequent multiorgan morbidity, even in NTDT patients who never received transfusion therapy.2 Patients with NTDT accumulate iron from increased intestinal iron absorption and release from the reticuloendothelial system, signaled by low hepcidin levels attributed to ineffective erythropoiesis.2 Although this milestone in disease management addressed a key morbidity risk factor, there are currently no approved therapies specifically targeting the underlying ineffective erythropoiesis and anemia, which are not only the drivers for primary iron overload, but are also linked with a variety of clinical complications stemming from chronic tissue hypoxia, hemolysis, and hypercoagulability.1 There is evidence that transfusion therapy may be associated with lower morbidity rates in NTDT,1 but physicians are understandably hesitant to start these patients on regular transfusion programs which would open the door to transfusional siderosis and progressive organ dysfunction. In this context, several agents are currently being evaluated in clinical trials with the aim of targeting ineffective erythropoiesis or iron dysregulation in NTDT, with the expectation that addressing one pathophysiologic mechanism will ameliorate the other; since a bidirectional relationship between both anomalies has been confirmed.3 Clinically, this would translate to improvement in anemia and prevention of iron overload. Luspatercept (ACE-536) is a recombinant fusion protein that binds to select transforming growth factor β superfamily ligands and enhances late-stage erythropoiesis.4, 5 Luspatercept showed encouraging data in a single-arm, open-label, phase two study including improvement in hemoglobin level in NTDT and reduction in transfusion burden in transfusion-dependent β-thalassemia (TDT).6 A phase three trial (BELIEVE) confirmed these findings in TDT patients and led to product approval in this subset of patients.7 The ongoing randomized (2:1), double-blind, placebo-controlled, phase two BEYOND trial (NCT03342404) is evaluating the efficacy of subcutaneous luspatercept (every 3 weeks) in increasing hemoglobin level (by ≥ 1.0 g/dL) in adult patients with NTDT and a baseline hemoglobin ≤ 10 g/dL. Assessment of effects on tiredness, weakness, and shortness of breath is also being undertaken to confirm clinical benefit. The impact of therapy on iron indices and use of iron chelation will also be reported. Mitapivat (AG-348) is an oral, small-molecule, allosteric activator of the red blood cell (RBC)-specific form of pyruvate kinase (PK-R). Adenosine triphosphate (ATP) supply appears to be insufficient in thalassemic RBCs to maintain RBC membrane fitness and clearance of globin precipitates. In β-thalassemia mouse models, mitapivat increased ATP levels, reduced markers of ineffective erythropoiesis, and improved anemia, RBC survival, and indices of iron overload.8 An open-label, phase two trial (NCT03692052) in adults with NTDT and a baseline hemoglobin of ≤ 10 g/dL is evaluating the efficacy of mitapivat in improving hemoglobin level (by ≥ 1.0 g/dL) as well as markers of hemolysis and ineffective erythropoiesis. Interim data showed response in eight of nine patients following 12 weeks of therapy.9 Anti-sense oligonucleotides (ASO) downregulating TMPRSS6, a metalloprotease which plays a key role in hepcidin expression, stimulated hepcidin, reduced iron burden, and improved ineffective erythropoiesis and RBC survival in β-thalassemia mouse models.10 TMPRSS6-LRx is a generation 2+ ligand-conjugated ASO subcutaneous drug (given every 4 weeks) that is now being evaluated in a randomized, open-label, phase two trial (NCT04059406) in adults with NTDT and baseline hemoglobin ≤ 10 g/dL. The main endpoints include increasing hemoglobin level (by ≥ 1.0 g/dL) and decreasing LIC. Finally, VIT-2763 is an oral ferroportin inhibitor which restricted iron availability, ameliorated anemia, and reversed the dysregulated iron homeostasis in β-thalassemia mouse models.11 VITHAL (NCT04364269) is a randomized, double-blind, placebo-controlled, phase two trial evaluating the efficacy of VIT-2763 in improving hemoglobin and iron indices in NTDT patients aged ≥ 12 years with a baseline hemoglobin ≤ 11 g/dL. All novel agents primarily aim to ameliorate anemia and iron overload in patients with NTDT, and final data from ongoing and subsequent registration trials are awaited. Considering multiple agents may become available not long from today; it is imperative to think ahead and try to visualize the outlook for overall disease management (Figure 1), so gaps in knowledge can be promptly identified and addressed alongside clinical development. Realizing the impact of improvement in anemia in the context of clinical trials may be challenging, since the aim of such improvement is to prevent the development of serious, long-term morbidity. Observational studies and disease registries would be key to evaluate such association, once these agents become available for clinical use. In the short term, clinical benefit from raising hemoglobin level may be evaluated through observation of changes in patient reported outcomes, although this should not exclude asymptomatic patients who may still benefit from drug-induced long-term changes in the underlying pathophysiology. The main effect of such agents on iron overload will be prevention of "new" and ongoing iron accumulation. Decreases in existing iron burden may also be observed in view of mobilization of iron to support production of RBCs, but this latter effect is expected to be minimal. Considering most ongoing clinical trials are including adult participants only (or adolescents in few cases), it is expected that patients would already have some degree of iron overload requiring iron chelation therapy. Combination of these novel agents with iron chelation may improve chelator efficiency since the latter is dependent on both pre-existing and ongoing iron intake. This may result in lower iron chelator dosing and/or reaching target (safe) iron levels faster. This synergistic effect has been observed in mouse models receiving a combination of TMPRSS6-ASO and iron chelation.12 If ongoing iron accumulation is completely reversed, this may in fact lead to long-term or permanent discontinuation of iron chelation therapy following reductions in pre-existing iron overload. This also brings up the interesting question of whether future studies in children who are not yet on iron chelation therapy, will avoid the need for the latter altogether. The more important question that we will probably be faced with is which agent should we use, especially that the design of current clinical trials and eligibility criteria are largely similar. Data from individual studies can be compared, but in an era of evidence-based medicine clinicians would likely want to see data from head-to-head comparative trials before making any management decisions. We have witnessed this challenge for over two decades with oral iron chelation therapy in TDT, and choices regarding the type of iron chelation therapy remain largely based on physician preference rather than comparative studies. Combinatorial trials with multiple novel agents (or with novel agents and stimulants such as erythropoietin) may also be warranted, since the mechanisms of action are different and may prove complimentary. For example, in β-thalassemic mice, administration of TMPRSS6-ASO and erythropoietin produced significantly higher hemoglobin levels and reduction in splenomegaly compared with each agent alone. This suggests that combinatorial approaches may in fact be superior to single treatments and could provide guidance to translate some of these approaches into treatment strategies.13 Lastly, progress has already been made with gene therapy and genome editing for patients with TDT. It was once argued that bone marrow transplantation should not be considered in NTDT, considering the disease is less severe. However, we continue to see strong evidence that an NTDT diagnosis is associated with serious, irreversible morbidities. Moreover, safer conditioning approaches during transplant are evolving and data from gene therapy studies are pointing towards better response in patients with less severe mutations (non-β0/β0). Together, these observations suggest that NTDT patients may be considered in future studies with interventions targeting the underlying genetic abnormality, especially when the risk-benefit ratio is low. K.M.M. has been or is a consultant for Novartis, Celgene Corp (Bristol Myers Squibb), Agios Pharmaceuticals, CRISPR Therapeutics and Vifor Pharma. S.R. is a member of scientific advisory board of Ionis Pharmaceuticals, Meira GTx, Incyte and Disc Medicine and owns stock options from Disc Medicine and Meira GTx. He has been or is consultant for Cambridge Healthcare Res, Celgene Corp (Bristol Myers Squibb), Catenion, First Manhattan Co., FORMA Therapeutics, Ghost Tree Capital, Keros Therapeutics, Noble insight, Protagonist Therapeutics, Sanofi Aventis U.S., Slingshot Insight, Techspert.io and BVF Partners L.P., Rallybio LLC and venBio Select LLC. A.T.T. has been or is a consultant for Novartis, Celgene Corp (Bristol Myers Squibb), Vifor Pharma, Silence Therapeutics and Ionis Pharmaceuticals; and received research funding from Novartis, Celgene Corp (Bristol Myers Squibb), La Jolla Pharmaceutical Company, Roche, Protagonist Therapeutics and Agios Pharmaceuticals. All authors contributed to manuscript drafting or critical review and final approval for submission. Data sharing is not applicable to this article as no new data were created or analyzed in this study.