Therapeutic applications of cell engineering using mRNA technology.

Engineering and reprogramming cells has significant therapeutic potential to treat a wide range of diseases, by replacing missing or defective proteins, to provide transcription factors (TFs) to reprogram cell phenotypes, or to provide enzymes such as RNA-guided Cas9 derivatives for CRISPR-based cell engineering. While viral vector-mediated gene transfer has played an important role in this field, the use of mRNA avoids safety concerns associated with the integration of DNA into the host cell genome, making mRNA particularly attractive for in vivo applications. Widespread application of mRNA for cell engineering is limited by its instability in the biological environment and challenges involved in the delivery of mRNA to its target site. In this review, we examine challenges that must be overcome to develop effective therapeutics.

Tailored Monoacyl Poly(2-oxazoline)- and Poly(2-oxazine)-Lipids as PEG-Lipid Alternatives for Stabilization and Delivery of mRNA-Lipid Nanoparticles.

The successful use of lipid nanoparticles (LNPs) for clinical development of the COVID-19 mRNA vaccines marked a breakthrough in mRNA-LNP therapeutics. As one of the vital components of LNPs, poly(ethylene glycol)-lipid conjugates (PEG-lipids) influence particle biophysical properties and stability, as well as interactions within biological environments. Reports suggesting that anti-PEG antibodies can be detected quite commonly within the human population raise concerns that PEG content in commercial LNP products could further stimulate immune responses to PEG. The presence of anti-PEG antibodies has been linked to accelerated clearance of LNPs, potentially a source of variability in the biological response to mRNA-LNP products. This motivated us to explore potential PEG alternatives. Herein, we report physicochemical and biological properties of mRNA-LNPs assembled using poly(2-oxazoline) (POx)- and poly(2-oxazine) (POz)-based polymer-lipid conjugates. Notably, we investigated monoacyl lipids as alternatives to diacyl lipids. mRNA-LNPs produced using monoacyl POx/POz-lipids displayed comparable biophysical characteristics and cytocompatibility. Delivery of reporter mRNA resulted in similar transfection efficiencies, in both adherent and suspension cells, and in mice, compared to PEG-lipid equivalents. Our results suggest that monoacyl POx/POz-lipid-containing LNPs are promising candidates for the development of PEG-free LNP-based therapeutic products.

CRISPR-Cas13b-mediated suppression of HBV replication and protein expression.

BACKGROUND & AIMS: New antiviral approaches that target multiple aspects of the HBV replication cycle to improve rates of functional cure are urgently required. HBV RNA represents a novel therapeutic target. Here, we programmed CRISPR-Cas13b endonuclease to specifically target the HBV pregenomic RNA and viral mRNAs in a novel approach to reduce HBV replication and protein expression. METHODS: Cas13b CRISPR RNAs (crRNAs) were designed to target multiple regions of HBV pregenomic RNA. Mammalian cells transfected with replication competent wild-type HBV DNA of different genotypes, a HBV-expressing stable cell line, a HBV infection model and a hepatitis B surface antigen (HBsAg)-expressing stable cell line were transfected with PspCas13b-BFP (blue fluorescent protein) and crRNA plasmids, and the impact on HBV replication and protein expression was measured. Wild-type HBV DNA, PspCas13b-BFP and crRNA plasmids were simultaneously hydrodynamically injected into mice, and serum HBsAg was measured. PspCas13b mRNA and crRNA were also delivered to a HBsAg-expressing stable cell line via lipid nanoparticles and the impact on secreted HBsAg determined. RESULTS: Our HBV-targeting crRNAs strongly suppressed HBV replication and protein expression in mammalian cells by up to 96% (p <0.0001). HBV protein expression was also reduced in a HBV-expressing stable cell line and in the HBV infection model. CRISPR-Cas13b crRNAs reduced HBsAg expression by 50% (p <0.0001) in vivo. Lipid nanoparticle-encapsulated PspCas13b mRNA reduced secreted HBsAg by 87% (p = 0.0168) in a HBsAg-expressing stable cell line. CONCLUSIONS: Together, these results show that CRISPR-Cas13b can be programmed to specifically target and degrade HBV RNAs to reduce HBV replication and protein expression, demonstrating its potential as a novel therapeutic option for chronic HBV infection. IMPACT AND IMPLICATIONS: Owing to the limitations of current antiviral therapies for hepatitis B, there is an urgent need for new treatments that target multiple aspects of the HBV replication cycle to improve rates of functional cure. Here, we present CRISPR-Cas13b as a novel strategy to target HBV replication and protein expression, paving the way for its development as a potential new treatment option for patients living with chronic hepatitis B.