(M1230-07-42) Myeloid Targeted Disulfide Lipid Nanoparticle with Enhanced Endosomal Escape Improves HIV-1 Excision

Purpose: HIV-1 remains a global health crisis, with nearly 40 million individuals infected worldwide and a significant number of new infections and deaths occurring each year. Despite the success of antiretroviral therapy (ART) in managing the disease, a permanent cure is elusive due to the persistence of latent HIV-1 proviral DNA in viral reservoirs, particularly in myeloid cells and memory T cells. Current gene delivery methods, such as AAV-based CRISPR delivery, have shown limited efficacy in clinical trials due to poor targeting and complications related to immunogenicity. Recent advancements in lipid nanoparticle (LNP) technology have shown promise in delivering mRNA and CRISPR components to immune cells, including macrophages, which are key reservoirs for HIV. To overcome these limitations, we have developed a compositionally unique disulfide-based ionizable LNPs that 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), offering a promising alternative to existing therapies.

Methods: A library of ionizable lipids featuring cleavable disulfide linkages was synthesized through a four-step chemical process. LNPs were formulated using microfluidic mixing, incorporating the synthesized ionizable lipids, helper lipids (DOPC), cholesterol, PEG-lipids (DMG-PEG), and firefly luciferase (FLuc) mRNA. The resulting LNPs were characterized for size, polydispersity index, zeta potential via DLS, morphology via cryo-electron microscopy, and mRNA encapsulation efficiency using the Ribogreen® assay. Transfection efficiency was assessed in human MDMs using FLuc mRNA, comparing our lead candidate (BA-LNP) against two gold standard FDA-approved ionizable lipids, MC3 and ALC-0315. Biodistribution studies were conducted in NSG humanized mice following intravenous administration of LNPs encapsulating FLuc mRNA, with organ-specific luciferase expression analyzed via an in vivo imaging system (IVIS). Confocal microscopy using lysosomal-associated membrane protein 1 (LAMP-1) staining was employed to evaluate endosomal escape. Protein corona formation was analyzed through SDS-PAGE, Western blotting, and proteomics to understand plasma protein interactions influencing biodistribution. For therapeutic assessment, BA-LNPs were functionalized with a CCR5-targeting D-Ala-peptide T-amide (DAPTA) ligand and loaded with Cas9 mRNA and mosaic guide RNAs targeting conserved regions of the HIV-1 genome (tat1-2, rev1-2, gp41). HIV-1-infected MDMs were treated with these formulations, and proviral DNA excision was evaluated via PCR, gel electrophoresis, and Sanger sequencing.

Results: The BA-LNPs exhibited a spherical morphology with an average hydrodynamic diameter of approximately 90 nm and near-neutral zeta potential. mRNA encapsulation efficiency exceeded 94%, and the particles maintained stability in 10% human plasma over 24 hours, with minimal changes in concentration. Cell viability assays demonstrated that BA-LNPs maintained MDM viability above 80% at therapeutic doses (4 µg/10⁶ cells), comparable to MC3 and ALC-0315 formulations. BA-LNP exhibited 20-fold higher FLuc expression versus controls, indicating superior mRNA translation due to enhanced endosomal escape.  Confocal imaging with lysosomal marker LAMP-1 confirmed BA-LNP's ability to escape lysosomal degradation as compared to MC3-LNP, supporting its higher endosomal escape. Protein corona analysis identified fibrinogen and β2-glycoprotein as key plasma proteins influencing the observed organ tropism. Biodistribution in humanized mice showed mRNA translation in the lungs and spleen, major HIV-1 reservoir, while MC3/ALC-0315 targeted the liver. This finding highlights the HIV reservoir tissue selectivity of BA-LNP over gold-standard MC3-LNP and ALC-LNP. To examine the viral elimination efficacy, MDMs were infected with HIV-1_ADA and subsequently treated with CRISPR-encapsulated CCR5-targeted and non-targeted BA-LNPs. The CCR5-targeted BA-LNP demonstrated an 80% excision of integrated proviral DNA compared to the non-targeted formulation. This invention marks a significant advancement in HIV-1 therapy, providing a safe and efficient CRISPR delivery system for eliminating HIV-1 in infected myeloid cells.

Conclusion: The developed LNP formulation represents a significant advancement in HIV therapeutics, offering improved cytosolic delivery and targeted elimination of latent HIV-1 proviral DNA in myeloid reservoirs. Its superior performance compared to existing FDA-approved lipids, along with its capacity to target HIV reservoirs, positions it as a promising candidate for HIV cure strategies. This work contributes to the expanding field of LNP-based therapeutics for infectious diseases, building on studies that demonstrate LNP efficacy in macrophage-targeted mRNA delivery. Moreover, this platform holds potential for broader applications in gene therapy for other diseases affecting the lungs and spleen, such as chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, and lymphoid malignancies like splenic marginal zone lymphoma. Future studies will aim to optimize the formulation for clinical translation and assess its efficacy in larger animal models, paving the way for a functional cure for HIV and other persistent viral infections.

References: Chatterjee et al., PNAS, 2024
Han et al., Nature Communications, 2024
Dilliard et al., PNAS, 2021
Letchumanan & Theva Das, Epigenetics & Chromatin, 2025

Acknowledgements: The authors declare no conflict of interest


Figure 1 shows the higher mRNA translation efficacy of in-house synthesized BA-LNP, biodistribution, and excision of pro-viral DNA