Cholesterol Analogues Incorporated Into LNPs For Enhanced mRNA Delivery

Hydroxycholesterols are used to formulate lipid nanoparticles (LNPs) for enhanced mRNA delivery into T cells.
Problem:
In recent times, the delivery of mRNA has become a major focus for therapeutics. Intracellular delivery is the major challenge. Intracellular delivery requires ionizable lipids for mRNA delivery. In addition to this, these constituents of LNPs can affect the potency of the treatment.
Solution:
This invention demonstrates that cholesterol analogues such as Hydroxycholesterol incorporated into LNPs enhance mRNA delivery into T cells. In addition, an example of incorporating dexamethasone into the LNP formulation is shown to reduce inflammatory responses.
Technology:
Cholesterol analogues such as Hydroxycholesterol and Dexamethasone which has a structure similar to cholesterol, when incorporated into LNPs enhance the mRNA delivery into cells. The addition of hydroxyl groups to the cholesterol molecule can alter the cholesterol recycling mechanism exhibited by the endosome; and Dexamethasone having anti-inflammatory properties, reduces inflammation in cells which leads to higher expression of the payload mRNA, leading to greater potency of the treatment. A library of 24 LNPs was formulated using 6 X-hydroxycholesterol modifications (7𝛼, 7𝛽, 19, 20, 24, 25). Each of the 6 hydroxycholesterols were substituted in various ratios relative to unmodified cholesterol (12.5%, 25%, 50%, and 100% substitutions).
Advantages:

• Hydroxycholesterol substitutions, particularly in the 7𝛼 position, at substitution ratios of 25% and 50%, demonstrate a statistically significant increase in delivery of mRNA to T cells
• The incorporation of hydroxycholesterols has an impact on the shape of LNPs when observed with TEM, suggesting that morphological changes may contribute to improved mRNA delivery
• Size, PDI, zeta potential, and encapsulation efficiency did not differ significantly between the particle formulations and S2, indicating that the incorporation of hydroxycholesterols allowed for LNP formation
• Reduced inflammation and potentially fewer side effects with Dexamethasone than conventional LNPs
• 1.5X expression of payload mRNA with Dexamethasone than conventional LNPs 

Stage of Development:

• Preclinical Discovery

Engineering lipid nanoparticles (LNP) with hydroxycholesterol substitution: motivation, design, and synthesis. (A) Schematic of LNP components, formulation, and expected structure. An ethanol phase containing lipid-anchored PEG (polyethylene glycol), cholesterol, X-hydroxycholesterol, DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine), and C14–4 ionizable lipid and an aqueous phase containing mRNA are mixed in a microfluidic device to produce LNPs. (B) Diagram of LNP delivery into a T cell and the endosomal trafficking mechanisms involving the Rab family of proteins. Rab5, Rab7, and Rab11 associate with the early, late, and recycling endosomes, respectively. (C) Design of an LNP library incorporating the substitution of various hydroxycholesterols for unmodified cholesterol.
Intellectual Property:

• PCT Application Filed

Reference Media:

• Patel et. al., J. Control Release 2022 Jul; 347: 521
• Zhang et. al., J. Biomed Mater Res A. 2022 May; 110(5): 1101

Desired Partnerships:

• License
• Co-development 

Docket #22-9845