Abstract：
Autosomal dominant osteopetrosis type 2 (ADO2) is a rare inherited bone disorder characterised by dense but brittle bones. It displays striking phenotypic variability, with the most severe symptoms, including blindness and bone marrow failure. Disease management largely relies on symptomatic treatment since there is no safe and effective treatment. Most ADO2 cases are caused by heterozygous loss-of-function mutations in the CLCN7 gene, which encodes an essential Cl-/H+ antiporter for proper bone resorption by osteoclasts. Thus, siRNA-mediated silencing of the mutant allele is a promising therapeutic approach, but targeting bone for first-in-human translation remains challenging. Here, we demonstrate the utility of silicon-stabilised hybrid lipid nanoparticles (sshLNPs) as a next-generation nucleic acid nanocarrier capable of delivering allele-specific siRNA to bone. Using a Clcn7G213R knock-in mouse model recapitulating one of the most common human ADO2 mutations and based on the 129S genetic background (which produces the most severe disease phenotype amongst current models), we show substantial knockdown of the mutant allele in femur when siRNA targeting the pathogenic variant is delivered by sshLNPs. We observed lower areal bone mineral density in femur and reduced trabecular thickness in femur and tibia, when siRNA-loaded sshLNPs were administered subcutaneously (representing the most relevant administration route for clinical adoption and patient adherence). Importantly, sshLNPs have improved stability over conventional LNPs and enable \'post hoc loading\' for point-of-care formulation. The treatment was well tolerated, suggesting that sshLNP-enabled gene therapy might allow successful clinical translation of essential new treatments for ADO2 and potentially other rare genetic bone diseases.
摘要：
常染色体显性骨硬化2型（ADO2）是一种罕见的遗传性骨疾病，其特征是骨骼致密但易碎。它表现出惊人的表型变异性，症状最严重，包括失明和骨髓衰竭。疾病管理很大程度上依赖于对症治疗，因为没有安全有效的治疗方法。大多数ADO2病例是由CLCN7基因的杂合功能缺失突变引起的，它编码必需的Cl-/H反转运蛋白，用于破骨细胞的适当骨吸收。因此，siRNA介导的突变等位基因沉默是一种有前途的治疗方法，但是针对骨骼进行首次人类翻译仍然具有挑战性。这里,我们证明了硅稳定的杂合脂质纳米颗粒（sshLNP）作为能够将等位基因特异性siRNA递送至骨骼的下一代核酸纳米载体的实用性。使用Clcn7G213R敲入小鼠模型，该模型概括了最常见的人类ADO2突变之一，并基于129S遗传背景（在当前模型中产生最严重的疾病表型），当通过sshLNP递送靶向致病变体的siRNA时，我们显示股骨中突变等位基因的大量敲减。我们观察到股骨的骨矿物质密度降低，股骨和胫骨的小梁厚度减少，当皮下施用负载siRNA的sshLNP时(代表临床采用和患者依从性最相关的给药途径).重要的是,与常规LNP相比,sshLNP具有改善的稳定性,并且能够实现即时护理制剂的“事后加载”。治疗耐受性良好，这表明sshLNP基因疗法可能有助于ADO2和潜在的其他罕见遗传性骨病的重要新疗法的成功临床转化.
