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. 2022 Feb 7;2(2):428-442.
doi: 10.1021/jacsau.1c00467. eCollection 2022 Feb 28.

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

Ramya Kumar  1 , Ngoc Le  1 , Felipe Oviedo  2 , Mary E Brown  3 , Theresa M Reineke  1   2
Affiliations

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

Ramya Kumar et al. JACS Au. .

Abstract

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.

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Conflict of interest statement

The authors declare the following competing financial interest(s): Theresa M. Reineke is one of the founders of Nanite, Inc. and has an equity interest. Nanite, Inc. is one of the sponsors of this research. This interest has been reviewed and managed by the University of Minnesota in accordance with its conflict-of-interest policy. Felipe Oviedo consults for Nanite, Inc. and has an equity interest.

Figures

Figure 1
Figure 1
Polymers from a combinatorially designed library are assembled with pDNA payloads and polyplexes characterized thoroughly. Polyplex internalization, pDNA delivery efficiency, and toxicity are evaluated rapidly. Finally, interpretable machine learning approaches are applied to derive structure–function relationships.
Figure 2
Figure 2
Polymer library synthesized via combinatorial RAFT polymerization. (A) Four cationic monomers of varying pKa values: 2-(diethylamino)ethyl methacrylate (DEAEMA), 2-aminoethylmethacrylamide hydrochloride (AEMA), 2-(diisopropylamino)ethyl methacrylate (DIPAEMA), and 2-(dimethylamino)ethyl methacrylate (DMAEMA) were studied. Three neutral monomers of varying hydrophilicities were used as comonomers: 2-methacryloyloxyethyl phosphorylcholine (MPC), poly(ethylene glycol) methyl ether methacrylate (PEGMEMA), and 2-hydroxyethyl methacrylate (HEMA). (B) For each pair of cationic and neutral monomers, we targeted cationic monomer incorporation levels from 0% to 100% in 25% increments, generating 43 polymers. The cationic incorporation was determined by 1H NMR and was used to calculate m and n values.
Figure 3
Figure 3
(A) Polyplexes are formulated at N/P ratios of 5, 10, and 20, and the proportion of cells expressing green fluorescent protein (GFP) evaluated via flow cytometry to identify top polymers. (B) Only N/P 20 formulations of top performers are denoted by white stars although GFP expression is substantial even at lower N/P ratios. Polyplexes formed from p(DIPAEMA52-st-HEMA50) or P38 effect the highest GFP expression.
Figure 4
Figure 4
(A) Polyplexes are formulated with Cy5-labeled pDNA and cellular internalization in HEK293T cells evaluated. (B) The geometric mean Cy5 intensity for each formulation is normalized to the highest value in the library. Unlike with RNP delivery, pDNA delivery is not inhibited by uptake.
Figure 5
Figure 5
HEK293T cells transfected with P38 (hit polymer) and P41 (poor transfection despite high pDNA internalization) at an N/P ratio of 5. Various cellular compartments and intracellular polyplex distribution are visualized as follows: nuclei are stained with DAPI (blue), intracellular GFP expression (green), AlexaFluor 546 stained lysosomal compartments (magenta), Cy5-labeled pDNA payloads (orange), allowing quantification of colocalization. We observe poor transfection efficiencies in the P41 treatment group despite high levels of pDNA internalization. Colocalization analysis yields Pearson’s correlation coefficients (PCC), which reveal the higher propensity of P41 polyplexes to be entrapped within lysosomes compared to P38 polyplexes. Scale bar is 10 μm.
Figure 6
Figure 6
Three-dimensional reconstructions of GFP+ cells from (A) P38 and (B) P41 treatment groups. Cy5-labeled pDNA payloads were classified as cytoplasmic (cyan) or nuclear (gray). Scale bar is 5 μm. (C) From quantile–quantile (Q–Q) plots, we see that P41 nuclei–pDNA distances are shifted further to the right, indicating higher nuclear separation than P38. The Kolmagrov–Smirnov test (p-values shown inset) further confirms that the histograms are unlikely to be drawn from the same distribution. (D) Distribution of pDNA between nuclear and cytoplasmic regions for GFP+ cells. P38 polyplexes display higher nuclear accumulation than P41.
Figure 7
Figure 7
(A) The hydrophobicity (clogP), surface charge (ζ), length (Mn), composition (% cat.), and pKa of polymers were measured while polyplex formulations were described by their size (Rh) and the distance migrated by pDNA during gel electrophoresis (mobility). The contributions of these nine features to delivery efficiency, cellular toxicity, and uptake were computed for pDNA and RNP payloads using SHapley Additive exPlanations (SHAP). SHAP compares structure–function trends across RNP (blue) and pDNA (red) payloads. (B) Direct causal effects (in the form of average treatment effects) of the top five features from SHAP analysis were computed along with 95% confidence intervals. Positive and negative effects indicate protagonistic and antagonistic relationships, respectively.
Figure 8
Figure 8
(A) Schematic of NHEJ and HDR editing pathways. In cells engineered with the traffic light reporter system, the delivery of RNP alone results in imprecise gene editing via the NHEJ pathway (measured via mCherry expression), while codelivery of pDNA donor and RNP leads to gene knock-in via HDR (measured via GFP). (B) Optimization of formulation conditions for codelivering RNP and pDNA donor payloads. The total amount of nucleic acid is kept constant at either 1.5 or 2 μg per well while the weight ratio of single guide RNA (sgRNA) and pDNA donor is varied from 2:1 to 1:5. A formulation of 2 μg nucleic acid loading using a 1:2 w/w mixture of sgRNA and DNA maximizes HDR editing (quantified via GFP expression). (C) Fluorescent micrographs of HDR-edited cells treated with Lipofectamine 2000 or P38. Unpackaged payloads serve as negative controls. Scale bar is 100 μm. (D) Flow cytometry traces highlighting mCherry positive cell populations and GFP positive cells for the optimized formulation.
Figure 9
Figure 9
(A) Summary of transfection and internalization efficiencies in HEK293T (black) and ARPE-19 (gray) cells. In HEK293T, P38 exhibits both high delivery efficiencies (measured by GFP expression) as well as high cellular uptake (measured by Cy5 intensity). In ARPE-19, we observe that delivery performance of P38 is inhibited by low levels of uptake, particularly at an N/P ratio of 10. (B) DLS and turbidity measurements reveal N/P-dependent trends in polyplex aggregation upon the addition of DMEM, with the N/P 10 formulation experiencing severe colloidal instability. We performed turbidimetric titrations in both D-PBS and in DMEM to understand the causes of N/P-dependent polyplex aggregation. Unlike in PBS, where polyplexes recover colloidal stability upon the addition of excess polymer and overcharging, aggregation is irreversible in DMEM because of the poor solubility of P38 in the media. DLS and turbidity measurements indicate that only lower N/P ratios permit colloidally stable polyplexes.
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References

    1. Newby G. A.; et al. Base Editing of Haematopoietic Stem Cells Rescues Sickle Cell Disease in Mice. Nature 2021, 595, 295–302. 10.1038/s41586-021-03609-w. - DOI - PMC - PubMed
    1. Foust K. D.; Wang X.; McGovern V. L.; Braun L.; Bevan A. K.; Haidet A. M.; Le T. T.; Morales P. R.; Rich M. M.; Burghes A. H. M.; Kaspar B. K. Rescue of the Spinal Muscular Atrophy Phenotype in a Mouse Model by Early Postnatal Delivery of SMN. Nat. Biotechnol. 2010, 28, 271–274. 10.1038/nbt.1610. - DOI - PMC - PubMed
    1. Mendell J. R.; et al. Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy. N. Engl. J. Med. 2017, 377, 1713–1722. 10.1056/NEJMoa1706198. - DOI - PubMed
    1. Lee K.; et al. Nanoparticle Delivery of Cas9 Ribonucleoprotein and Donor DNA in Vivo Induces Homology-Directed DNA Repair. Nat. Biomed. Eng. 2017, 1, 889–901. 10.1038/s41551-017-0137-2. - DOI - PMC - PubMed
    1. Anguela X. M.; High K. A. Entering the Modern Era of Gene Therapy. Annu. Rev. Med. 2019, 70, 273–288. 10.1146/annurev-med-012017-043332. - DOI - PubMed