Nucleic acids are important therapeutics yet issues with delivery efficiency continue to hinder widespread advancement in the clinic. Delivery systems are crucial to encapsulate and protect these large and highly sensitive payloads and improve tissue internalization ensuring efficacy. However, we are limited by the vast chemical space and prediction of formulation chemistries indicating discrete selection and optimization of next generation systems. Indeed, new machine learning models aimed at selecting and predicting discrete parameters influencing biological efficacy are needed to advance the next frontier of personalized medicine. We present the facile synthesis of a clickable polymer library with systematic variations in length, binary composition, pK_a, and hydrophobicity (clogP) to optimize intracellular pDNA and CRISPR-Cas9 ribonucleoprotein (RNP) performance. We couple physiochemical characterization and machine learning to interpret quantitative structure-property relationships within the combinatorial design space. For the first time, we reveal unexpected disparate design parameters for nucleic acid carriers; via explainable machine learning on 432 formulations, we discover that lower polymer pK_a and higher percentages of benzimidazole ethanethiol enhance pDNA delivery, yet polymer length and captamine cation identity improve RNP delivery. Closed-loop Bayesian optimization of 552 formulation ratios further enhances in vitro performance. The top three polymers yield higher signal and stable transgene expression over 20-days in vivo, and a 1.7-fold enhancement over controls. Our facile coupling of synthesis, characterization, and machine analysis provide powerful tools to quantitate performance parameters accelerating next generation vehicles for nucleic acid medicines.

Michael Leyden

Appearances