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.