Highly efficient CD4+ T cell targeting and genetic recombination using engineered CD4+ cell-homing mRNA-LNPs

Purpose: T cells are notoriously resistant to exogenous mRNA transfection, limiting in vivo T cell engineering for immunotherapy, CAR T generation, and HIV cure. A safe, specific, and efficient in vivo mRNA delivery platform for CD4+ T cells was needed.

Hypothesis: Conjugating anti-CD4 antibody to nucleoside-modified mRNA-LNPs will enable specific targeting, functional mRNA translation, and genetic recombination in CD4+ T cells in vitro and in vivo.

Aims: Develop anti-CD4 antibody-conjugated mRNA-LNPs. - Evaluate specific binding and functional mRNA delivery to human CD4+ T cells in vitro. - Assess in vivo biodistribution and targeting to CD4+ T cells in spleen and lymph nodes. - Demonstrate Cre mRNA-mediated genetic recombination in CD4+ T cells using Ai6 reporter mice. - Test T cell subtype uniformity, durability, repeated dosing, and compatibility with an alternative LNP formulation (ALC-0315).

Delivery system: Platform: Nucleoside-modified mRNA lipid nanoparticles (mRNA-LNPs). - Ionizable lipid: ALC-0307 (primary); ALC-0315 also tested. - Other components: Phosphatidylcholine, cholesterol, PEG-lipid; ~80 nm; ~3.5 antibodies per LNP. - Targeting ligand: Anti-CD4 monoclonal antibody conjugated via SATA-maleimide chemistry. - Payloads: Cre recombinase mRNA, firefly luciferase mRNA, Poly(C) RNA. - Controls: Unconjugated LNPs and control IgG-conjugated LNPs. - Route: Retro-orbital intravenous injection in mice.

Approach: In vitro: Human CD4+ T cells from healthy donors; binding and luciferase transfection assays; Ai6 splenocytes for Cre recombination. - In vivo: C57BL/6J and Ai6 Cre-reporter mice. - Doses: 8 µg Luc mRNA; Cre mRNA doses 1–9 µg in vitro; 3, 10, 30, 90 µg in vivo (90 µg toxic); repeated daily injections of 10 µg for 3 or 5 days. - Group sizes: Typically n = 3–11 animals depending on experiment. - Disease context: Healthy mice; no therapeutic disease model.

Key methods: Radiolabeled ¹²⁵I-LNP biodistribution and tissue uptake (%ID/g). - Flow cytometry for ZsGreen1, CD3, CD8, CD4, CD25, CD44, CD62L. - Luciferase activity assay in tissues and isolated CD3+ T cells. - IVIS bioluminescence imaging. - Dynamic light scattering for LNP size and PDI. - Antibody conjugation efficiency measurements.

Key results: In vitro: anti-CD4/mRNA-LNPs bound specifically to human CD4+ T cells; ~80% of CD3+CD8− splenocytes became ZsGreen1+ with anti-CD4/Cre mRNA-LNPs, even at the lowest dose. - In vivo biodistribution: spleen uptake was 131.59 %ID/g for anti-CD4 LNPs vs 37.6 %ID/g for control IgG; ~6-fold higher spleen accumulation; localization ratio ~61 at 24 h. - Luciferase activity: ~7-fold higher in spleen and 33-fold higher in isolated CD3+ T cells with anti-CD4 LNPs vs control IgG. - Cre recombination: ~60% of CD4+ T cells in spleen and ~40% in lymph nodes at 10 µg; unconjugated LNPs reached only ~20% at 30 µg. - T cell subtypes: uniform transfection across naive, central memory, and effector memory cells; ~57% of resting CD25− vs ~40% of activated CD25+ CD4+ T cells. - Durability: splenic ZsGreen1+ cells dropped from ~50% at day 1 to ~32% at day 4 and ~26% at day 7; lymph node signal remained stable. - Repeated dosing: five injections gave higher recombination than three, showing additive effect. - ALC-0315 LNPs showed comparable CD4 targeting.

Interpretation: The CD4-targeted mRNA-LNP platform enables specific and efficient in vivo mRNA delivery and genetic recombination in CD4+ T cells. This provides a promising platform for in vivo T cell manipulation, including CAR T generation, HIV cure strategies, and immunotherapy of cancer and infectious diseases.

Limitations: Only mouse in vivo models; no human in vivo or large-animal validation. - High 90 µg dose was toxic. - Transgene expression/recombination declined in spleen over 7 days. - Substantial liver delivery remained, indicating incomplete CD4 specificity. - No therapeutic efficacy in a disease model; only reporter genes. - Long-term safety, immunogenicity, and repeated-dose toxicity were not fully evaluated. - Anti-CD4 antibody binding transiently reduced CD4 detection, complicating direct CD4 staining.