Reducible Branched Ester-Amine Quadpolymers (rBEAQs) Codelivering Plasmid DNA and RNA Oligonucleotides Enable CRISPR/Cas9 Genome Editing

Abstract

Functional codelivery of plasmid DNA and RNA oligonucleotides in the same nanoparticle system is challenging due to differences in their physical properties as well as their intracellular locations of function. In this study, we synthesized a series of reducible branched ester-amine quadpolymers (rBEAQs) and investigated their ability to coencapsulate and deliver DNA plasmids and RNA oligos. The rBEAQs are designed to leverage polymer branching, reducibility, and hydrophobicity to successfully cocomplex DNA and RNA in nanoparticles at low polymer to nucleic acid w/w ratios and enable high delivery efficiency. We validate the synthesis of this new class of biodegradable polymers, characterize the self-assembled nanoparticles that these polymers form with diverse nucleic acids, and demonstrate that the nanoparticles enable safe, effective, and efficient DNA-siRNA codelivery as well as nonviral CRISPR-mediated gene editing utilizing Cas9 DNA and sgRNA codelivery.

Keywords: CRISPR; DNA; RNA oligonucleotides; codelivery; nanoparticle; nonviral; rBEAQs; reducible branched ester-amine quadpolymers.

Figure 1.. R6,8-4-6 polymers enable efficient intracellular siRNA delivery.

(A) Gene knockdown and cytotoxicity of nanoparticles delivering 100 nM siRNA at 180 w/w. Statistical analysis was assessed by one-way ANOVA with Tukey post-hoc tests. N = 4. (B) Nanoparticle uptake measured by flow cytometry after treatment of nanoparticles containing Cy5-labeled siRNA. N = 4. (C) Pre-treatment with 1,000 μM L-buthionine-sulfoximine (BSO) show that intracellular glutathione blockade did not change knockdown levels but significantly increased polymer-mediated cytotoxicity as assessed by Holm-Sidak corrected multiple t-tests; R6,8_20 nanoparticles (180 w/w) were used to deliver 100 nM siRNA. N = 4. (D) Nanoparticle hydrodynamic diameter and (E) zeta potential measured using dynamic light scattering. Bars show average + SEM; N = 3. (F) Representative TEM image of R6,8_40 nanoparticles.

Figure 2.. Polymer branching and reducibility can be modulated to control siRNA binding affinity and release kinetics.

(A) Yo-Pro-1 iodide binding competition assay of R6,8-4-6 polymers to assess siRNA binding affinity. N = 4. (B) % knockdown plotted as a function of polymer EC₅₀ w/w for siRNA binding. N = 4. (C) Gel retardation assays (N = 1) and (D) Yo-Pro binding assay (N = 4) were performed on nanoparticles incubated in 5 mM glutathione to mimic intracellular reducing environments and nucleic acid release slowed as the polymers became more branched with less frequent bio-reducible linkages.

Figure 3.. Hydrophobic R6,7,8-4-6 polymer series enables efficient co-delivery of DNA and siRNA.

Co-delivery efficacy of R6,8-4-6 (0% B7) and R6,7,8-4-6 nanoparticles encapsulating 400 ng total nucleic acid in 293T (A) and Huh7 (B). N = 4. (C) Fluorescence microscopy images of HEK-293T cells treated with R6,7,8_16 nanoparticles co-delivering 200 ng siRNA and 200 ng DNA (10 w/w formulation). Scale bar 100 μm. (D) R6,7,8_64 completely encapsulated plasmid DNA and siRNA at 10 w/w as seen by a gel retardation assay. (E) Confocal microscopy images of 293T cells treated with R6,7,8_64 nanoparticles co-delivering Cy3-siRNA, Cy5-DNA, and unlabeled GFP plasmid DNA (0.5:0.4:0.1 composition by weight) at 3 hr and 24 hr post-uptake. Cy3 and Cy5 signal colocalization could be seen at 3 hours post-uptake (white arrows). At 24 hours post-uptake, diffuse Cy3-siRNA signal could be seen in the cytosol (white asterisk) while some Cy5-DNA signal was detected in the nucleus (yellow arrows) and some cells were visibly expressing GFP. Scale bar 20 μm.

Figure 4.. Co-delivery of anti-GFP sgRNA and Cas9 plasmid enables CRISPR-mediated gene knockout.

(A) HEK-293T cells were transfected with R6,7,8_64 10 w/w nanoparticles encapsulating Cas9 DNA and sgRNA at the indicated nucleic acid molar ratios. N = 4. (B) Flow cytometry histograms of CRISPR- or siRNA-treated cells. CRISPR treatment produced a completely GFP-negative population (null) while siRNA treatment mainly resulted in a general population shift to lower GFP fluorescence (low). (C) Gene suppression kinetics of CRISPR and siRNA treated cells. N = 4.

Scheme 1.. Monomer structures and proposed mechanism for polymer function.

(A) B and S monomers were co-polymerized to form acrylate-terminated base polymers, which were then (B) end-capped with monomer E6. (C) These polymers self-assembled into nanoparticles with anionic nucleic acids and (D) partially degraded at reducible linkages in the reducing cytosolic environment, allowing for intracellular cargo release.