Combinatorial Polycation Synthesis and Causal Machine Learning Reveal Divergent Polymer Design Rules for Effective pDNA and Ribonucleoprotein Delivery

Ramya Kumar, Ngoc Le, Felipe Oviedo, Mary E. Brown, Theresa M. Reineke

Research output: Contribution to journalArticlepeer-review

17 Scopus citations


The development of polymers that can replace engineered viral vectors in clinical gene therapy has proven elusive despite the vast portfolios of multifunctional polymers generated by advances in polymer synthesis. Functional delivery of payloads such as plasmids (pDNA) and ribonucleoproteins (RNP) to various cellular populations and tissue types requires design precision. Herein, we systematically screen a combinatorially designed library of 43 well-defined polymers, ultimately identifying a lead polycationic vehicle (P38) for efficient pDNA delivery. Further, we demonstrate the versatility of P38 in codelivering spCas9 RNP and pDNA payloads to mediate homology-directed repair as well as in facilitating efficient pDNA delivery in ARPE-19 cells. P38 achieves nuclear import of pDNA and eludes lysosomal processing far more effectively than a structural analogue that does not deliver pDNA as efficiently. To reveal the physicochemical drivers of P38's gene delivery performance, SHapley Additive exPlanations (SHAP) are computed for nine polyplex features, and a causal model is applied to evaluate the average treatment effect of the most important features selected by SHAP. Our machine learning interpretability and causal inference approach derives structure-function relationships underlying delivery efficiency, polyplex uptake, and cellular viability and probes the overlap in polymer design criteria between RNP and pDNA payloads. Together, combinatorial polymer synthesis, parallelized biological screening, and machine learning establish that pDNA delivery demands careful tuning of polycation protonation equilibria while RNP payloads are delivered most efficaciously by polymers that deprotonate cooperatively via hydrophobic interactions. These payload-specific design guidelines will inform further design of bespoke polymers for specific therapeutic contexts.

Original languageEnglish (US)
Pages (from-to)428-442
Number of pages15
JournalJACS Au
Issue number2
StatePublished - Feb 28 2022

Bibliographical note

Funding Information:
We acknowledge Nanite, Inc. and the Defense Advanced Research Projects Agency (DARPA) N660011824041 for funding. We acknowledge Craig Van Bruggen and Zhe Tan, Ph.D. for technical advice. We acknowledge the University Imaging Centers at the University of Minnesota. We gratefully acknowledge Dr. Guillermo Marques from the University Imaging Centers for sharing his expertise. We acknowledge .

Publisher Copyright:
© 2022 American Chemical Society. All rights reserved.


  • combinatorial design
  • machine learning
  • nonviral gene therapy
  • pDNA delivery
  • polymeric vehicles
  • ribonucleoprotein delivery
  • structure-activity relationships

PubMed: MeSH publication types

  • Journal Article


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