CD8纳米抗体靶向的脂质纳米颗粒包裹CD13 nanoCAR环状RNA用于原位CAR-T细胞根除急性髓系白血病

Acute myeloid leukemia (AML) is an aggressive and recurrent cancer with a high mortality rate. Chimeric antigen receptor (CAR)-T cell therapy is an FDA approved therapy for acute lymphoblastic leukemia (ALL) but not for AML mainly due to hematopoietic stem and progenitor cell side effects. This is driven by persistent CD13, CD33, CD123 targeting from lentivirus-transduced CAR-T cells. Yet, CD13, a prognostic marker for AML, identifies 95% of the AML blasts and most leukemic stem cells (LSCs). AML is also a heterogenous cancer with diverse antigen profiles, posing treatment challenges. Finally, current CAR-T cell therapies are immensely costly due to cumbersome ex vivo T cell and viral procedures, restricting patient access. We aim to overcome these technical challenges by leveraging the low tonic signaling of VHH-based nanobodies, transient expression of circular RNAs (circRNAs), and non-viral properties of lipid nanoparticles for effective eradication of AML while minimizing negative hematopoietic effects. Specifically, we attached CD8 nanobodies to lipid nanoparticles, constructed CD13 nanoCARs on circRNA backbones, and assembled the targeted circRNA lipid nanoparticle complexes. These complexes enable in situ, cell-specific engineering of splenic human CD8+ T cells and generate cytotoxic CD13 nanoCAR-T cells that target the prevalent AML population. We demonstrated the feasibility of this approach by generating a CD8 nanobody with 86% reactivity toward human CD8+ T cells. We then customized the CD8 nanobody with a negatively charged polyglutamic acid tail to form strong electrostatic associations with our positively charged, proprietary lipid nanoparticles. We produced CD13 nanoCAR circRNAs with undetectable levels of double-stranded RNA impurities through cellulose purification and encapsulated them in the lipid nanoparticles by microfluidic mixing. The entire targeted circRNA lipid nanoparticle complex selectively transfected 24% of the human CD3+ lymphoid T cells in the spleen of NPG mice previously reconstituted with human PBMCs. We then assayed and selected the THP-1 human AML cell line showing high CD13 expression and low nonspecific T cell killing in vitro. In an immunodeficient NPG CDX mouse model, CD8 nanobody-targeted lipid nanoparticles encapsulating CD13 nanoCAR circRNAs exhibit specific and effective in vivo killing of a subcutaneously engrafted THP-1 tumor.