Custom-designed lipid-based nanoparticles efficiently deliver mRNA to reverse the pathogenesis of LAL deficiency in a preclinical model

Lysosomal acid lipase (LAL) is a crucial enzyme responsible for the hydrolysis of triglycerides (TGs) and cholesteryl esters (CEs) in the lysosomes. LAL deficiency results in the extensive storage of CEs and TGs in the liver and the spleen, inducing hepatosplenomegaly, and can be a life-threatening condition with patients rarely surviving beyond 6 months of age in the most aggressive form. Using messenger ribonucleic acid (mRNA) for protein replacement is an innovative approach to treating LAL deficiency. Here, we describe the development of a new lipid-based nanoparticle (LNP) formulation capable of efficiently delivering LAL mRNA and restoring LAL activity in affected organs mediating significant reversal of the pathological progression in a highly predictive preclinical model. A combinatorial library of mRNA-LNPs was generated and screened both in vitro and in vivo to yield a new formulation with a higher potency than an FDA-approved MC3-based nano-formulation. In vivo evaluation revealed that the new formulation could promote a more sustained and superior LAL expression. Multiple injection regimen was able to mitigate hepatosplenomegaly and reduce the lipid accumulation of CEs and TGs by 20-30% in the liver and 50% in the spleen. Furthermore, liver inflammation processes and development of fibrosis were significantly diminished. These findings provide strong evidence that mRNA-LNP is a very promising approach for the chronic treatment of LAL deficiency and support the clinical translation of mRNA therapy to overcome side effects and challenges encountered with traditional enzyme replacement therapies. Overall design: An efficacy study in LAL knockout (LAL-/-) mice was performed to investigate the effect of mRNA therapy for treating LAL deficiency. 3 sex- and age-matched groups with equal number of mice were treated for 8 weeks at a 5-day interval. Wild-type (WT) mice received empty nanoparticles (WT+Blank), untreated LAL-/- mice received also blank nanoparticles (LAL-/-+Blank) and the last group was treated with mRNA-LNP formulation F23 (LAL-/-+F23). 1.0 mg/kg body weight. During the treatment period, 1 LAL-/-+Blank mice died, and one WT mice was excluded from the study yielding at the end point to a total of 28 mice. From each mouse, liver was harvested and RNA extracted for RNA sequencing analysis. PCA analysis showed that one sample (Sample 28, 7121-0629) was wrongly clustered and was therefore withdrawn from subsequent differential gene expression analyses. This mouse was treated at a later age than all other mice, whcih minimized the impact of treatment.

溶酶体酸性脂肪酶（Lysosomal acid lipase, LAL）是负责水解溶酶体内甘油三酯（triglycerides, TGs）与胆固醇酯（cholesteryl esters, CEs）的关键酶。LAL缺乏可导致胆固醇酯与甘油三酯在肝脏及脾脏中大量蓄积，进而引发肝脾肿大，且病情可危及生命；其中最具侵袭性的亚型患者极少能存活超过6个月。
利用信使核糖核酸（messenger ribonucleic acid, mRNA）开展蛋白质替代治疗，是治疗LAL缺乏症的创新策略。本研究阐述了一款新型脂质纳米颗粒（lipid-based nanoparticle, LNP）制剂的开发过程：该制剂可高效递送LAL的mRNA，并在受累器官中恢复LAL活性，能够在高度预测性的临床前模型中显著逆转病理进程。
研究人员构建了mRNA-LNP组合文库，并分别开展体外与体内筛选，最终获得一款效力优于经美国食品药品监督管理局（Food and Drug Administration, FDA）批准的基于MC3的纳米制剂的新型制剂。体内评估结果显示，该新型制剂可促进LAL表达更持久且效果更优异。
多次给药方案可有效缓解肝脾肿大，并使肝脏内胆固醇酯与甘油三酯的脂质蓄积量降低20%~30%，脾脏内则降低50%。此外，肝脏炎症进程与纤维化发展均得到显著抑制。
上述研究结果充分证明，mRNA-LNP疗法用于LAL缺乏症的慢性治疗极具前景，同时为mRNA疗法的临床转化提供了有力支撑，以克服传统酶替代疗法所面临的副作用与挑战。
整体实验设计：本研究在LAL敲除（LAL knockout, LAL-/-）小鼠中开展疗效研究，以探究mRNA疗法治疗LAL缺乏症的效果。设置3个性别、年龄匹配且小鼠数量均等的组别，以5天为间隔连续给药8周；野生型（Wild-type, WT）小鼠给予空白纳米颗粒（WT+Blank组），未接受额外治疗的LAL-/-小鼠同样给予空白纳米颗粒（LAL-/-+Blank组），最后一组接受mRNA-LNP制剂F23治疗（LAL-/-+F23组），给药剂量为1.0 mg/kg体重。
给药期间，1只LAL-/-+Blank组小鼠死亡，另有1只WT小鼠被排除出实验，最终终点时总共有28只小鼠纳入分析。研究人员采集每只小鼠的肝脏组织，提取RNA用于RNA测序分析。主成分分析（Principal Component Analysis, PCA）结果显示，1份样本（样本28，编号7121-0629）出现聚类异常，因此将其从后续差异基因表达分析中剔除。该小鼠的给药年龄晚于其余所有受试小鼠，此举可将该样本对后续分析的干扰影响降至最低。