A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing.

The development of clinically viable delivery methods presents one of the greatest challenges in the therapeutic application of CRISPR/Cas9 mediated genome editing. Here, we report the development of a lipid nanoparticle (LNP)-mediated delivery system that, with a single administration, enabled significant editing of the mouse transthyretin (Ttr) gene in the liver, with a >97% reduction in serum protein levels that persisted for at least 12 months. These results were achieved with an LNP delivery system that was biodegradable and well tolerated. The LNP delivery system was combined with a sgRNA having a chemical modification pattern that was important for high levels of in vivo activity. The formulation was similarly effective in a rat model. Our work demonstrates that this LNP system can deliver CRISPR/Cas9 components to achieve clinically relevant levels of in vivo genome editing with a concomitant reduction of TTR serum protein, highlighting the potential of this system as an effective genome editing platform.

Intrathecal delivery of frataxin mRNA encapsulated in lipid nanoparticles to dorsal root ganglia as a potential therapeutic for Friedreich's ataxia.

In Friedreich's ataxia (FRDA) patients, diminished frataxin (FXN) in sensory neurons is thought to yield the predominant pathology associated with disease. In this study, we demonstrate successful usage of RNA transcript therapy (RTT) as an exogenous human FXN supplementation strategy in vitro and in vivo, specifically to dorsal root ganglia (DRG). Initially, 293 T cells were transfected with codon optimized human FXN mRNA, which was translated to yield FXN protein. Importantly, FXN was rapidly processed into the mature functional form of FXN (mFXN). Next, FXN mRNA, in the form of lipid nanoparticles (LNPs), was administered intravenously in adult mice. Examination of liver homogenates demonstrated efficient FXN LNP uptake in hepatocytes and revealed that the mitochondrial maturation machinery had efficiently processed all FXN protein to mFXN in ~24 h in vivo. Remarkably, greater than 50% mFXN protein derived from LNPs was detected seven days after intravenous administration of FXN LNPs, suggesting that the half-life of mFXN in vivo exceeds one week. Moreover, when FXN LNPs were delivered by intrathecal administration, we detected recombinant human FXN protein in DRG. These observations provide the first demonstration that RTT can be used for the delivery of therapeutic mRNA to DRG.

The effect of complexation hydrogels on insulin transport in intestinal epithelial cell models.

A novel class of pH-sensitive complexation hydrogels composed of methacrylic acid and functionalized poly(ethylene glycol) (PEG) tethers, referred to as P(MAA-g-EG) WGA, was investigated as an oral protein delivery system. The PEG tethers were functionalized with wheatgerm agglutinin (WGA), a lectin that can bind to carbohydrates in the intestinal mucosa, to improve residence time of the carrier and absorption of the drug at the delivery site. The ability of P(MAA-g-EG) WGA to improve insulin absorption was observed in two different intestinal epithelial models. In Caco-2 cells P(MAA-g-EG) WGA improved insulin permeability 9-fold as compared with an insulin only solution, which was similar to the improvement by P(MAA-g-EG). P(MAA-g-EG) and P(MAA-g-EG) WGA were also evaluated in a mucus-secreting culture that contained Caco-2 and HT29-MTX cells. Insulin permeability was increased 5-fold in the presence of P(MAA-g-EG) and P(MAA-g-EG) WGA. Overall, it is clear that P(MAA-g-EG) WGA enhances insulin absorption and holds great promise as an oral insulin delivery system.

Wheat germ agglutinin functionalized complexation hydrogels for oral insulin delivery.

Insulin was loaded into hydrogel microparticles after two hours with loading efficiencies greater than 70% for both poly(methacrylic acid-grafted-ethylene glycol) (P(MAA-g-EG)) and poly(methacrylic acid-grafted-ethylene glycol) functionalized with wheat germ agglutinin (P(MAA-g-EG) WGA). The pH-responsive release results demonstrated that the pH shift from the stomach to the small intestine can be used as a physiologic trigger to release insulin from P(MAA-g-EG) and P(MAA-g-EG) WGA microparticles, thus limiting release of insulin into the acidic environment of the stomach. Microplates were successfully treated with PGM to create a surface that allowed for specific binding between mucins and lectins. The 1% PGM treatment followed by a 2 h BSA blocking step gave the most consistent results when incubated with F-WGA. In addition, the PGM-treated microplates were shown to create specific interactions between F-WGA and the PGM by use of a competitive carbohydrate. The 1% PGM treated microplates were also used to show that adhesion was improved in the P(MAA-g-EG) WGA microparticles over the P(MAA-g-EG) microparticles. The interaction between the PGM-treated microplate and P(MAA-g-EG) WGA was again shown to be specific by adding a competitive carbohydrate, while the interaction between P(MAA-g-EG) and the PGM-treated microplate was nonspecific. Cellular monolayers were used as another method for demonstrating that the functionalized microparticles increase adhesion over the nonfunctionalized microparticles. This work has focused on improving the mucoadhesive nature of P(MAA-g-EG) by functionalizing these hydrogel carriers with wheat germ agglutinin (WGA) to create a specific mucosal interaction and then evaluating the potential of these carriers as oral insulin delivery systems by in vitro methods. From these studies, it is concluded that the addition of the WGA on the microparticles produces a specific adhesion to carbohydrate-containing surfaces and that P(MAA-g-EG) WGA shows great promise as an oral insulin delivery system.

Loading and mobility of spin-labeled insulin in physiologically responsive complexation hydrogels intended for oral administration.

Poly(methacrylic acid-g-ethylene glycol) copolymers are pH-responsive complexation hydrogels that have shown promise in in vitro and in vivo results as oral insulin delivery carriers. With the aim of gaining more detailed insight into their performance to further improve the carriers, we spin-labeled insulin and used electron spin resonance (ESR) spectroscopy to follow the loading of the spin-labeled insulin into the copolymer microparticles. A flow through system was developed to monitor continuously and non-invasively the dynamics of the spin-labeled insulin and its surrounding microviscosity during release. Using these methods, the loading efficiency of insulin was determined and was found to match previous HPLC measurements. Additionally, the protein-friendly nature of the hydrogels was demonstrated. The monitoring of the dynamics during flow through provided a rationalization for the unwanted initial burst release in an acidic environment. These studies will aid in the optimization of the system, and will be a basis for subsequent in vivo ESR investigations.

Hydrogels for oral delivery of therapeutic proteins.

In recent years there has been significant new interest in the development of transmucosal (mostly oral) pharmaceutical formulations for the delivery of therapeutic proteins. Emphasis has been given to the molecular design of new carriers for the delivery of insulin, calcitonin and various types of interferons for the treatment of diabetes, osteoporosis, multiple sclerosis and cancer. Most popular carriers include advanced designs of swollen hydrogels prepared from neutral or intelligent polymeric networks. In this review, the most successful of such systems are presented and their promise in the field described.