(M1230-05-27) CCR5 Receptor Targeted Lipid Nanoparticles for HIV-1 Proviral DNA Excision

Purpose: The research is aimed at developing CCR5 receptor targeted lipid nanoparticles to achieve enhanced proviral DNA elimination from central HIV-1 cellular and tissue reservoirs. Therapeutic interruption of ART in HIV-1 results in viral rebound, the reasons underlying this rebound viremia is the existence of latent viral reservoirs. Latency development in HIV-1 infection is associated with an exclusive increase in CCR5 receptor expression in the latently infected cells; Hence, we formulated CCR5-targeted Lipid Nanoparticles containing an FDA approved ionizable lipid MC3. Encapsulating CRISPR-Cas9 mRNA in LNPs to excise the latent proviral DNA resulted in selective targeting and elimination of HIV-1 from both T-lymphocyte and myeloid cellular compartments.

Methods: CCR5-targeted lipid nanoparticles (LNPs) constructs encapsulating CRISPR-Cas9 and guide mRNA (gRNA) were formulated using microfluidics. Ribogreen assay was performed to determine the mRNA encapsulation efficiency. Nanoparticle size and zeta potential were analyzed by Dynamic Light Scattering (DLS). Luciferase and cell viability assay was conducted to analyze the nanoparticle’s toxicity profile and differential uptake efficiency in human macrophages. CCR5-targeted CRISPR-Cas9 encapsulated LNPs were tested for their HIV-1 excision efficiency in primary human monocyte derived macrophages (MDMS), T-lymphocytic, and monocytic cell lines. Protein corona assessment studies were conducted to obtain mechanistic insights into the endogenous targeting abilities of the LNP.

Results: The formulated CCR5 receptor targeted LNPs (M5-LNPs) had mRNA encapsulation efficiency of more than 90% and particle size of less than 100nm. The developed LNPs were non-toxic at dose ranging from 16µg to 1µg per million cells in primary macrophages. The protein translation efficacy was higher in M5-LNPs compared to the control LNPs in a dose dependent manner as determined by the expression of reporter luciferase mRNA. Furthermore, M5-LNPs encapsulating CRISPR-Cas9 mRNA targeted towards the latent proviral HIV-1 DNA achieved higher HIV-1 excision efficiency in both monocytic and lymphocytic cell lines relative to the control LNPs. Conclusively, M5-LNPs also exhibited superior HIV-1 excision efficiency in HIV-1 infected MDMs relative to the control. In addition, SDS page analysis revealed the spectrum of proteins associated with the unique protein corona of MC3 containing LNPs with and without the presence of helper lipid in the LNP composition.

Conclusion: CCR5-targeted M5 LNPs demonstrated more than 90% mRNA encapsulation efficiency. M5 LNPs resulted in higher luciferase mRNA translation efficacy than untargeted LNPs in MDMs. Conclusively, CRISPR-Cas9 encapsulating M5 LNPs exhibited greater HIV-1 excision in both monocytic and lymphocytic cells as well as in HIV-1 infected MDMs relative to control LNPs.

Acknowledgements: The authors declare that Dr. Howard Gendelman is a co-founder of Exavir Therapeutics, Inc. The biotechnology company is developing ultra-long-acting drugs. The drugs in development are not linked to those created in the current report. All other authors declare no competing interests. NIH Funding sources: R01MH121402, R01MH115860

(A) Formulation scheme for the CCR5 peptide conjugated Lipid Nanoparticles using microfluidics. (B,C,D) Schematic illustrating determination of mRNA encapsulation efficiency by Ribogreen Assay, zeta potential & particle size of LNPs.

(E,G) Assessment of cell viability via the CTB assay in Human Macrophages at different mRNA dose concentrations. (F, H) Luciferase mRNA translation efficacy of CCR5-targeted and control LNPs in Human Macrophages; data were recorded as RLU in a dose-dependent manner.

(I) Schematic representing the LNP treatment to HIV-1 infected Primary cells and latently infected Monocytic and T cell lines. (J) DNA isolation and PCR analysis post-LNP treatment of the cells. (K)Ex vivo evaluation of HIV-1 excision efficacy of CCR5-targeted and control LNPs in MDMs infected with HIV-1ADA . (L) PCR gel electrophoresis blots showing excision of HIV-1 DNA in monocytic cells (U1) treated with CRISPR-Cas9 encapsulating M5 and MC3-LNPs. (M) PCR gel electrophoresis blots showing excision of HIV-1 DNA in latent HIV-1 infected JE6 cells treated with CRISPR-Cas9 encapsulating M5 and MC3-LNPs. Quantification of HIV-1 DNA excision efficacy in MDMs of LNPs using image J and densitometric analysis.