Poly(Beta-Amino Ester) Nanoparticles Enable Nonviral Delivery of CRISPR-Cas9 Plasmids for Gene Knockout and Gene Deletion.

The CRISPR-Cas9 system is a powerful gene-editing tool with wide-ranging applications, but the safe and efficient intracellular delivery of CRISPR components remains a challenge. In this study, we utilized biodegradable poly(beta-amino ester) nanoparticles to codeliver plasmid DNA encoding Cas9 and short guide RNA (sgRNA), respectively, to enable gene knockout following a CRISPR-mediated cleavage at one genomic site (1-cut edit), as well as gene deletion following DNA cleavage at two sites flanking a region of interest (2-cut edits). We designed a reporter system that allows for easy evaluation of both types of edits: gene knockout can be assessed by a decrease in near-infrared fluorescent protein (iRFP) fluorescence, whereas deletion of an expression stop cassette turns on a red-enhanced nanolantern fluorescence/luminescence dual reporter. Nanoparticles enabled up to 70% gene knockout due to small indels, as well as 45% gain-of-function expression after a 600-bp deletion edit. The efficiency of 2-cut edits is more sensitive than 1-cut edits to Cas9 and the sgRNA expression level. We demonstrate promising biodegradable nanoparticle formulations for gene editing. Our findings also provide new insights into the screening and transfection requirements for different types of gene edits, which are applicable for designing nonviral delivery systems for the CRISPR-Cas9 platform.

Efficiency of Cytosolic Delivery with Poly(ß-amino ester) Nanoparticles is Dependent on the Effective pKa of the Polymer.

The mechanism by which cationic polymers containing titratable amines mediate effective endosomal escape and cytosolic delivery of nucleic acids is not well understood despite the decades of research devoted to these materials. Here, we utilize multiple assays investigating the endosomal escape step associated with plasmid delivery by polyethylenimine (PEI) and poly(ß-amino esters) (PBAEs) to improve the understanding of how these cationic polymers enable gene delivery. To probe the role of these materials in facilitating endosomal escape, we utilized vesicle membrane leakage and extracellular pH modulation assays to demonstrate the influence of polymer buffering capacity and effective pKa on the delivery of the plasmid DNA. Our results demonstrate that transfection with PBAEs is highly sensitive to the effective pKa of the overall polymer, which has broad implications for transfection. In more acidic environments, PBAE-mediated transfection was inhibited, while PEI was relatively unaffected. In neutral to basic environments, PBAEs have high buffering capacities that led to dramatically improved transfection efficacy. The cellular uptake of polymeric nanoparticles overall was unchanged as a function of pH, indicating that microenvironmental acidity was important for downstream intracellular delivery efficiency. Overall, this study motivates the use of polymer chemical characteristics, such as effective pKa values, to more efficiently evaluate new polymeric materials for enhanced intracellular delivery characteristics.

Carboxylated branched poly(ß-amino ester) nanoparticles enable robust cytosolic protein delivery and CRISPR-Cas9 gene editing.

Efficient cytosolic protein delivery is necessary to fully realize the potential of protein therapeutics. Current methods of protein delivery often suffer from low serum tolerance and limited in vivo efficacy. Here, we report the synthesis and validation of a previously unreported class of carboxylated branched poly(ß-amino ester)s that can self-assemble into nanoparticles for efficient intracellular delivery of a variety of different proteins. In vitro, nanoparticles enabled rapid cellular uptake, efficient endosomal escape, and functional cytosolic protein release into cells in media containing 10% serum. Moreover, nanoparticles encapsulating CRISPR-Cas9 ribonucleoproteins (RNPs) induced robust levels of gene knock-in (4%) and gene knockout (>75%) in several cell types. A single intracranial administration of nanoparticles delivering a low RNP dose (3.5 pmol) induced robust gene editing in mice bearing engineered orthotopic murine glioma tumors. This self-assembled polymeric nanocarrier system enables a versatile protein delivery and gene editing platform for biological research and therapeutic applications.