Abstract Body (Do not enter title and authors here): Introduction
Cardiometabolic diseases significantly increase the risk of stroke and heart attack, leading causes of death worldwide. Due to the multifactorial nature of cardiometabolic diseases, residual cardiovascular risk remains with current monotherapies, highlighting the need for treatments with pleiotropic effects in at-risk populations. e-therapeutics (ETX) uniquely combines computation and RNAi to discover life-transforming medicines. ETX’s RNAi platform, GalOmic™, enables generation of specific, potent, and long-acting siRNA therapies that effectively silence novel gene targets in hepatocytes. HepNet™, ETX’s computational platform, identified the B4GALT1 gene as a key factor in metabolic disease. This link was supported by genetic evidence that individuals with B4GALT1 missense mutations have pronounced cardiovascular risk reduction. Together, this indicates that a GalOmic™ siRNA targeting B4GALT1 could be a potential treatment for cardiometabolic diseases.

Methods
Potent siRNAs against B4GALT1 were designed in silico. siRNAs were screened in vitro in Huh7 cells, primary mouse and human hepatocytes, and in vivo in C57BL/6 mice for selection of the lead GalOmic™ siRNA, ETX-291. ApoE3*L.CETP mice on high-fat diet and fructose water were subcutaneously injected weekly with ETX-291 for 12 weeks. After 10 weeks, glucose tolerance tests were performed. Plasma fasting insulin, glucose, lipids, and fibrinogen were measured every 4 weeks and at study termination. Terminal cholesterol profiling and measurement of free fatty acids and liver mRNA expression were also performed.

Results
ETX-291 displayed high potency in vitro and in vivo with long duration of action. Treatment of ApoE3*L.CETP mice with ETX-291 significantly reduced the cardiometabolic risk factors LDL and VLDL cholesterol, triglycerides, free fatty acids, and fibrinogen. ETX-291 significantly improved insulin resistance markers and normalized glucose tolerance tests.

Conclusions
ETX-291, a GalOmic™ siRNA targeting B4GALT1, positively affects multiple cardiometabolic risk factors in a humanized mouse model of metabolic disease. The pleiotropic mechanism of action beyond LDL-C lowering, combined with the validated risk reduction observed in individuals with B4GALT1 missense mutations, makes ETX-291 a promising candidate for clinical development. ETX-291 shows potential for therapeutic and preventative treatment in at-risk populations due to its favorable safety profile and prolonged duration of action.