(M1530-05-28) Piperazine Based Lipid Nanoparticles Facilitate Enhanced Endosomal Escape and CRISPR-Cas9 Mediated HIV-1 Proviral DNA Excision

Purpose: The study aims to develop and evaluate piperazine-based LNPs for targeted CRISPR/Cas9 delivery to latent HIV-1 reservoirs to facilitate excision of integrated proviral HIV-1 DNA. Despite the effectiveness of antiretroviral therapy (ART), the persistence of latent HIV-1 proviral DNA in CD4⁺ T cells remains a major barrier to achieving a functional cure. Therefore, CRISPR-Cas9-mediated excision of integrated viral DNA offers a promising therapeutic strategy for eliminating these latent reservoirs. To this end, our laboratory has developed compositionally distinct lipid nanoparticles (LNPs) designed to deliver CRISPR-Cas9 and guide RNA (gRNA) specifically to HIV-1 reservoirs. The gRNAs target structurally conserved regions (tat, rev, and gp41) of the HIV-1 genome to mediate precise excision of viral sequences. However, the success of LNP-based CRISPR delivery is often hindered by poor endosomal escape and suboptimal mRNA translation efficiency. To overcome these limitations, we synthesized a library of biodegradable, piperazine-based ionizable lipids and formulated them into LNPs. These novel LNPs are engineered to enhance endosomal release and improve cytosolic delivery of CRISPR-Cas9 components, offering a promising strategy for effective HIV-1 proviral DNA excision.

Methods: A library of ionizable lipids characterized by piperazine-based amine head groups and unsaturated lipid tails was synthesized and used to formulate mRNA‐loaded LNPs via microfluidic mixing. The physicochemical properties of the LNPs, including particle size and zeta potential, were characterized using dynamic light scattering (DLS), while mRNA encapsulation efficiency was quantified with the RiboGreen assay. In vitro transfection efficiency was evaluated using firefly luciferase (FLuc) reporter mRNA in T-lymphocytic cells and monocytic cell lines. In vivo biodistribution and mRNA translation were assessed in BALB/c mice using in vivo imaging system (IVIS imaging). Cytotoxicity was determined using the CellTiter-Blue (CTB) assay. Additionally, the efficiency of CRISPR/Cas9-mediated excision of HIV-1 proviral DNA was examined in latently infected CD4⁺ T cells.

Results: Our lead ionizable lipid-based LNPs (Bpip-LNPs) have an average particle size of approximately 56 nm and a near-neutral surface charge. It demonstrates over 90% mRNA encapsulation efficiency while maintaining greater than 80% cell viability at therapeutic doses. In latent HIV-1 infected T cells, Bpip-LNPs achieved a 5-fold increase in mRNA transfection efficiency compared to FDA-approved ionizable lipids MC3 and ALC-0315, highlighting their enhanced endosomal escape and cytosolic delivery capabilities. In vivo, Bpip-LNPs resulted in a 2-fold improvement in mRNA translation efficiency relative to MC3. Furthermore, when formulated with CRISPR/Cas9 mRNA, Bpip-LNPs enabled approximately 60% excision of HIV-1 proviral DNA in HIV-1 infected T lymphocytes.

Conclusion: Bpip-LNPs demonstrated a fivefold increase in mRNA translation efficiency in CD4⁺ T cells compared to both MC3 and ALC-0315 formulations. In vivo bioimaging studies further confirmed a twofold enhancement in mRNA translation relative to MC3. At the optimal gRNA/Cas9 molar ratio, Bpip-LNPs achieved approximately 60% excision of HIV-1 proviral DNA in latently infected T lymphocytes. Overall, these results highlight the crucial role of the piperazine-based ionizable lipid backbone in facilitating efficient endosomal escape in CD4⁺ T cells and enhancing CRISPR-Cas9 delivery to HIV-1 reservoirs.

Acknowledgements: Conflict of Interest: 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. Biodistribution Studies: The animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center (UNMC), and all procedures followed the Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals by the National Institutes of Health.

Figure 1. Schematic representation of LNP formulation using a microfluidic device. The illustration also depicts the preliminary screening of Fluc-mRNA-loaded LNPs and the gene delivery of CRISPR-Cas9-encapsulated LNPs into T cells.

Figure 2. Preparation and preliminary screening of LNPs in vitro. (A) Donut charts showing the molar composition of the lipids used to formulate LNPs. (B) Size, PDI, and zeta potential of LNP measured by dynamic light scattering (DLS) and mRNA encapsulation efficiency measured by RiboGreen assay. (C) Schematic representation of latent HIV-1-infected cells (T-lymphocytic JLat, JE6, and promonocytic U1). (D) Luciferase mRNA translation efficacy of LNPs in the JE6, JLat, and U1 cells. The mRNA translation efficiency was assessed 48 hours post-LNP treatment (at 1µg/ million cells mRNA dose) using the luciferase assay and presented as relative luminescence units (RLU). (E, F) Dose-dependent mRNA translation efficiency of Bpip and MC3 LNPs in JE6 and JLat cells. (G, H) The mRNA dose-dependent cell vitality of Bpip and MC3 LNPs in U1, JLat, and JE6 cells. LNPs show more than 80% cell vitality at a dose of 1µg/106 cells.

Figure 3. In vivo biodistribution of Bpip and MC3 LNPs. (A) A representative biodistribution image of MC3 and Bpip LNPs in BALB/c mice captured under IVIS. LNPs with a Fluc-mRNA dose of 0.5mg/kg were injected via the tail vein, and biodistribution was observed at 6h post-injection. (B) The luminescence intensity in the form of total flux in the MC3 and Bpip-treated mice was quantified. Bpip shows a significantly higher mRNA translation efficiency than MC3.