WARF TECHNOLOGIES

With an increasing interest in using the CRISPR/Cas9 system for gene editing, there comes a strong need to develop tools for delivering the CRISPR machinery safely and efficiently into cells. Existing non-viral strategies may suffer from one or more disadvantages including high cytotoxicity, poor in vivo stability, large particle size, lack of cell/tissue targeting delivery and variable loading of gene editing machinery. These hurdles continue to limit the ability to fully explore new clinical applications for CRISPR/Cas9 gene editing, especially in living organisms.

UW-Madison researchers have developed an improved biocompatible polyplex that can successfully deliver various biomolecular payloads (e.g., nucleic acids, RNP, RNP together with a single-stranded oligonucleotide DNA donor template). This polyplex contains two polymeric chains that can interact with negatively charged nucleic acid and/or protein payloads – a cationic polymer (poly-β-amino acid, PBAA) containing glutathione (GSH)-responsive disulfide bonds in the backbone and a second polymer that includes modifiable ends (polyethylene glycol, PEG) . Randomly incorporated imidazole groups in the cationic polymer enhance endosomal escape of the polyplex. To further enhance the stability of the polyplexes, adamantane (AD) and β-cyclodextrin (β-CD) are conjugated to the polymers. The crosslinked polyplexes formed by host–guest interaction between β-CD and AD are more stable than non-crosslinked polyplexes in physiological conditions. Once in the cytosol, the disulfide bonds are cleaved by GSH, thereby releasing the protein/nucleic acid from the polyplex. Importantly, at the PEG terminal ends, simple chemistry can be used to add various cellular or tissue-specific targeting ligands and imaging probes.

When compared to a commercially available transfection reagent (Lipofectamine 2000), the polyplex significantly decreased toxicity and showed equal or better nucleic acid transfection efficiency in HEK 293 and RAW 264.7 cells. Testing both sgRNA/Cas9 complex (i.e., RNP) and RNP together with single-stranded DNA complex, the inventors showed that using the polyplex delivery system yielded superior gene knockout and gene editing results.

• Delivery of large biomolecular payloads into cells (e.g., DNA, mRNA, sgRNA/Cas9 RNP, and RNP together with ssODN)

• Versatile nanodelivery system capable of safely and efficiently delivering a wide range of biomolecules (e.g., DNA, mRNA, CRISPR RNP, RNP together with a DNA repair template)
• Significantly reduced toxicity compared to existing transfection reagents
• Tunable platform for application-specific modifications (via PEG moieties)

For More Information About the Inventors

• Shaoqin Gong
• Krishanu Saha

Publications

• Wang et al. Versatile Redox-Responsive Polyplexes for the Delivery of Plasmid DNA, Messenger RNA, and Crispr-Cas9 Genome-Editing Machinery. 2018. ACS Applied Material Interfaces 10(38), 31915-31927.
• Wang et al. Enhancing the In Vitro and In Vivo Stabilities of Polymeric Nucleic Acid Delivery Nanosytems. 2019. Bioconjugate Chemistry 30(2), 325-337.

Tech Fields

• Drug Delivery : Other drug delivery technologies