Abstract
Despite these impressive clinical outcomes, chimeric antigen receptor (CAR) T-cell therapy is hindered by multiple intrinsic limitations that go beyond logistics and are truly biological in nature, which limit its use to treat hematologic malignancies. The ex vivo manufacturing paradigm is time-intensive, involving leukapheresis, genetic modification, expansion and quality control over 2-4 weeks, adding to the manufacturing costs and complexity, and also introducing heterogeneity in the product and a risk of T cell exhaustion or differentiation before infusion. These challenges have driven the development of the concept of in vivo CRISPR-based reprogramming of hematopoietic stem cells (HSCs) to produce endogenous CAR immune cells, which are a paradigm shift towards therapeutic development. This approach aims to provide a single intravenous injection of the CRISPR components that would specifically and stably edit LTR HSCs within their native niche to generate persistent, multilineage output of CAR-expressing T, NK, and macrophages. The aim of the current review is thus to critically consolidate and articulate the emerging but growing knowledge base that includes molecular tools for HSC-directed CRISPR delivery, preclinical data that document the antitumor activity of CRISPR in both hematologic and solid tumor models, the significant delivery hurdles of the bone marrow microenvironment, the range of immunological and genotoxic safety concerns, and the evolving translational landscape. A compilation of major results shows that antibody-lipid nanoparticles selectively target HSCs, and adeno-associated virus serotype 6 (AAV6) and virus-like particles (VLPs) mediate efficient knock-in of CAR cassettes by homology-directed repair under lineage-restricted endogenous promoters. In preclinical studies, long term multifunctional antitumor activity has been shown, including immunological memory upon re-challenge. Nonetheless, the potential for off-target editing in LTHSCs, variability in CAR expression in non-effector cells (e.g., erythroid or megakaryocytic progenitors) and pre-existing or acquired immunity to Cas9 requires careful mitigation strategies. In the future, we envision a roadmap from bench to bedside, with regulatory harmonization of in vivo gene editing products, GMP innovations to allow production and scaling of lipid nanoparticles, and the development of next-generation platforms such as prime editing, base editing and miRNA-based lineage restriction switches that could all potentially be used to speed up first-in-human trials in the next three to five years