Development of Poly(β-amino ester)-Based Biodegradable Nanoparticles for Nonviral Delivery of Minicircle DNA

Gene therapy provides a powerful tool for regulating cellular processes and tissue repair. Minicircle (MC) DNA are supercoiled DNA molecules free of bacterial plasmid backbone elements and have been reported to enhance prolonged gene expression compared to conventional plasmids. Despite the great promise of MC DNA for gene therapy, methods for safe and efficient MC DNA delivery remain lacking. To overcome this bottleneck, here we report the development of a poly(β-amino ester) (PBAE)-based, biodegradable nanoparticulate platform for efficient delivery of MC DNA driven by a Ubc promoter <i>in vitro</i> and <i>in vivo.</i> By synthesizing and screening a small library of 18 PBAE polymers with different backbone and end-group chemistry, we identified lead cationic PBAE structures that can complex with minicircle DNA to form nanoparticles, and delivery efficiency can be further modulated by tuning PBAE chemistry. Using human embryonic kidney 293 cells and mouse embryonic fibroblasts as model cell types, we identified a few PBAE polymers that allow efficient MC delivery at levels that are comparable or even surpassing Lipofectamine 2000. The biodegradable nature of PBAE-based nanoparticles facilitates <i>in vivo</i> applications and clinical translation. When injected <i>via</i> intraperitoneal route <i>in vivo</i>, MC alone resulted in high transgene expression, and a lead PBAE/MC nanoparticle formulation achieved a further 2-fold increase in protein expression compared to MC alone. Together, our results highlight the promise of PBAE-based nanoparticles as promising nonviral gene carriers for MC delivery, which may provide a valuable tool for broad applications of MC DNA-based gene therapy

Analysis of five SaCas9 ortholog activities.

(A) Amino acid sequences of the SaCas9 ortholog PI domain are aligned. The residues that are important for PAM recognition are indicated at the top; the conserved residues among newly identified SaCas9 orthologs are shown in red; the names of newly identified Cas9s are shown in green. (B) Design of the GFP activation reporter construct. A target sequence (protospacer) containing a 7-bp random sequence is inserted between ATG and the GFP-coding sequence. The library DNA is stably integrated into HEK293T cells by lentivirus. (C) Transfection of SaCas9 orthologs induced GFP expression. Percentage of GFP-positive cells was shown. The cells without transfection of Cas9 were used as a negative control.</p

Evaluation of Sha2Cas9-HF and SpeCas9-HF on-target activities.

(A) Comparison of activities of high-fidelity Cas9s to the wild-type Cas9s (n = 3). The target sequences are shown on the left. PAM is underlined. If the first nucleotide is C or T, additional “g” is added for U6 promoter transcription. Underlying data for all summary statistics can be found in S1 Data. (B) Quantification of editing efficiency for SaCas9, SmiCas9, Sha2Cas9, and SpeCas9. Underlying data for all summary statistics can be found in S1 Data.</p

Specificity of four SpeCas9 variants.

(A) Schematic of SpeCas9 structure. The amino acid residues important for specificity are shown below. (B) Test of four SpeCas9 variant specificity. Schematic of the GFP activation assay for specificity analysis is shown on the top. A panel of sgRNAs with dinucleotide mutations is shown below. sgRNA activities were measured based on GFP expression. Cells without Cas9 transfection were used as a negative control (NC). Mismatches are shown in red (n = 3). Underlying data for all summary statistics can be found in S1 Data. (TIF)</p

Analysis of the SaCas9 variant PAMs.

(A) Amino acid sequence of the SaCas9 variant PI domains. The residues that are important for PAM recognition are marked at the top; the mutations are highlighted in red. (B) SaCas9 variant PAMs were analyzed by the GFP activation assay. WebLogos generated by analyzing the deep sequencing data. (TIF)</p

Analysis of sgRNAs.

(A) Alignment of sgRNA scaffolds for six SaCas9 orthologs. The GAAA linker are indicated by the black box. (B) Analysis of SaCas9 orthologs’ secondary RNA structures. These structures were generated by an online tool named RNAfold WebServer (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi). (TIF)</p

Analysis of Sha2Cas9-HF and SpeCas9-HF specificity.

(A) Schematic of the GFP activation assay for specificity analysis is shown on the top. A panel of sgRNAs with dinucleotide mutations is shown below. sgRNA activities were measured based on GFP expression. Mismatches are shown in red (n = 3). Underlying data for all summary statistics can be found in S1 Data. (B) Off-targets for EMX1 locus are analyzed by GUIDE-seq. Read numbers for on- and off-targets are shown on the right. Mismatches compared with the on-target site are shown and highlighted in color.</p

Genome editing for endogenous sites.

(A) Schematic of the Cas9 expression constructs. (B) Protein expression level of Cas9s was measured by western blot. Cells without Cas9 transfection was used as a negative control. (C) Comparison of SaCas9, SmiCas9, Sha2Cas9, and SpeCas9 efficiency for genome editing at 13 endogenous loci. Additional “g” is added for U6 promoter transcription (n = 3). Underlying data for all summary statistics can be found in S1 Data. (D) Quantification of editing efficiency for SaCas9, SmiCas9, Sha2Cas9, and SpeCas9. Underlying data for all summary statistics can be found in S1 Data.</p

Genetic locus of CRISPR/Cas9.

(A) The structures of CRISPR loci for six SaCas9 orthologs. (B) Alignment of CRISPR repeat sequences for six SaCas9 orthologs. (C) Alignment of tracrRNA for six SaCas9 orthologs. (TIF)</p

Target sites used in this study.

A list of the endogenous target sites of human and their downstream PAM. PAM, protospacer adjacent motif. (DOCX)</p