Background
A cure for HIV remains elusive as the excision of latent proviral DNA by adenovirus-based therapeutic systems has failed to date due to vector immunogenicity and limited targeting to viral reservoirs. In contrast, lipid nanoparticles (LNPs) are promising for targeting viral reservoirs, offering greater cargo-carrying capacity and reduced immune responses. However, limitations in endosomal escape remain a critical impediment. Therefore, the current system, must show sufficient cytoplasmic release of its cargo to impact clinical outcomes. Herein, disulfide-containing ionizable lipid-based LNPs were developed to leverage the high levels of reactive oxygen species (ROS) in myeloid phagolysosomes. The ROS-sensitive LNPs promote cleavage of disulfide bonds, leading to enhanced endosomal escape and improved cytosolic delivery of CRISPR guide RNAs (gRNAs).
Methods
A four-step synthetic procedure was used to synthesize a library of 20 ionizable lipids with cleavable disulfide linkages. Transfection efficacy was assessed by a FLuc reporter mRNA in human monocyte-derived macrophages (MDMs). FDA-approved ionizable lipids, MC3 and ALC-0315, were compactors against the lead candidate (DS-LNP). IVIS was used to evaluate biodistribution. The toxicity profile of the new ionizable lipid was tested via CTB assay. The lead candidate delivered CRISPR-Cas9 gRNAs targeting five HIV-1 exons (tat1-2/rev1-2/gp41), and CRISPR-Cas9-mediated excision was validated by PCR and gel electrophoresis in HIV-infected MDMs.
Results
Our lead LNP formulation (DS-LNP), consisting of a novel disulfide-based ionizable lipid, DOPC, Cholesterol, and DMG-PEG2K, was nontoxic and demonstrated a 20-fold higher mRNA transfection efficiency in MDMs compared to MC3 and ALC-0315, which are limited by poor endosomal escape in myeloid cells. The DS-LNP biodistribution was specifically observed in the lymphoid and pulmonary viral reservoirs of humanized mice, marking a significant advancement over conventional formulations that inadequately target these reservoirs. CCR5-targeted DS-LNP improved MDMs targeting and endosomal escape, achieving 80% excision of HIV-1 proviral DNA, a clear advancement over conventional formulations.
Conclusions
Disulfide-based LNPs offer a significant advantage in overcoming endosomal escape barriers in MDMs, facilitating effective CRISPR-Cas9 delivery to HIV reservoirs. This approach potentially targets the lymphoreticular and pulmonary system and can improve viral DNA elimination.