Development of Novel mRNA Therapies for Genetic Diseases

Saumya N Heendeniya

doi:10.60867/00000188

Messenger RNA (mRNA) therapeutics offer a revolutionary approach to medicine, enabling the directed synthesis of therapeutic proteins within target cells. mRNA platform offers a distinct advantage over conventional therapeutics through its non-integrating and transient nature, allowing for controlled protein production without permanent genomic alteration. Although mRNA technology achieved global prominence through COVID-19 vaccines, its therapeutic applications extend well beyond vaccination to include the treatment of neurodegenerative diseases, cancer, and inherited metabolic disorders. This research employed a three-pillar strategy to demonstrate the versatility of mRNA therapeutics. The first pillar focused on molecular restoration through development of synthetic Survival Motor Neuron 1 (SMN1) mRNA therapy for Spinal Muscular Atrophy (SMA). Unlike current FDA approved therapies (Nusinersen and Risdiplam) that depend on endogenous SMN2 copy number, this approach delivers functional SMN protein instructions directly, offering potential benefit regardless of patient genotype. Through iterative optimization across three mRNA generations, incorporating CleanCap® technology and N1-methylpseudouridine modifications, robust dose-dependent SMN protein upregulation of up to 2.5-fold was achieved in SMA patient derived fibroblasts. The second pillar explored mRNA as an oncogenic brake for CDKN2A-deficient familial melanoma. Restoration of p16INK4A tumour suppressor function through exogenous mRNA transfection triggered selective cytotoxicity in NZM11 melanoma cells harbouring homozygous CDKN2A deletion, while healthy human fibroblasts tolerated elevated p16 levels without adverse effects. Colony formation assays demonstrated 98% reduction in clonogenic capacity following transient p16 mRNA exposure, outperforming the conventional cytotoxic agent Staurosporine (research-use compound). Transcriptomic profiling revealed substantially greater E2F pathway suppression in melanoma cells compared to healthy cells, with interleukin-24 (IL24) induced 70-fold specifically in melanoma cells undergoing death. The third pillar established proof-of-concept for a dual "see-saw" therapeutic approach for Alpha-1-Antitrypsin (A1AT) deficiency associated serpinopathy, combining antisense oligonucleotide mediated downregulation of toxic mutant protein with simultaneous mRNA mediated restoration of functional A1AT. While ASO displayed decay of the A1AT protein in liver cells, exogenous A1AT mRNA displayed upregulated A1AT protein by ~770 folds in patient cells. Collectively, this work demonstrates that mRNA therapeutics extend beyond prophylactic vaccination to encompass diverse therapeutic applications: replacing deficient proteins in genetic neuromuscular disease, restoring tumour suppressor function to selectively eliminate cancer cells, and coordinating with antisense therapies for metabolic regulation. These findings establish proof of concept for mRNA as a versatile platform for proteomic management, with implications for treating diseases previously considered intractable through conventional pharmacological approaches. This thesis presents novel mRNA therapeutic candidates for SMA and familial melanoma. A provisional patent has been filed for the SMN mRNA therapy, supporting its potential for future clinical translation.

Development of Novel mRNA Therapies for Genetic Diseases

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