Engineering the blood: Lipid nanoparticle platforms for ex vivo immune and hematopoietic cell therapies.

Targeted delivery of mRNA to immune cells for in vivo cell therapy

Abstract Messenger RNA (mRNA) therapeutics have revolutionized biomedicine by enabling direct in vivo programming of immune cells. This strategy bypasses the complex manufacturing and high costs associated with ex vivo cell therapies. However, efficient and specific systemic delivery of mRNA to target immune cell subsets remains a major translational hurdle. This review systematically examines engineering strategies that address this challenge. We first outline the key biological barriers to mRNA delivery, such as serum instability, nonspecific biodistribution, cellular uptake heterogeneity, and inefficient endosomal escape. Next, we comprehensively review advances in lipid nanoparticle (LNP) engineering, including discovering novel lipids, modulating compositions, conjugating targeting ligands, and incorporating stimuli-responsive elements, to enable enhanced tropism toward specific immune cells. Representative applications in oncology, protein replacement, autoimmune disease, and tissue regeneration are highlighted. Finally, we address translational challenges in safety, scalable manufacturing, and regulatory issues. The integration of rational material design, high-throughput screening, artificial intelligence, and interdisciplinary collaboration will be essential to advance next-generation targeted in vivo mRNA cell therapies toward clinical translation.

In vivo engineering of CAR immune cells by nonviral nanoparticles.

Chimeric antigen receptor (CAR) immune cell therapy has revolutionized the treatment of hematologic malignancies, yet conventional ex vivo manufacturing remains costly, complex, and logistically demanding. Emerging in vivo engineering strategies, especially those based on nonviral nanoparticles (NPs), offer a transformative alternative by enabling direct programming of immune cells within the body. Recent advances in NPs-mediated delivery of CAR constructs, including mRNA, circular RNA (circRNA), self-amplifying RNA (saRNA), DNA and gene editing tools, have demonstrated promising potential for achieving transient, tunable and repeatable expression with reduced genotoxicity. Immune cell targeting has been achieved by multiple targeting strategies for T cells, NK cells and macrophages, allowing precise control of CAR expression and functional activation in disease-relevant contexts. For T cells specifically, antibody-functionalized NPs categorized by antibody types including CD3, CD4, CD5, CD7 and CD8, demonstrate differential engagement of T cell subsets and influence their activation profiles across various therapeutic contexts. This review highlights key advances in the development of immune cell targeting NPs, discusses evolving CAR architectures and quality control considerations, and outlines future directions for integrating in vivo CAR platforms into broader immunotherapeutic landscapes. Nonviral NPs are poised to redefine CAR therapy by enabling scalable, off-the-shelf immune interventions for cancer, autoimmune, and fibrotic diseases.

Nanomaterials Drive In Vivo CAR Immune Cells Engineering.

Chimeric antigen receptor (CAR) immune cell therapy has emerged as a cornerstone of modern cell-based medicine, demonstrating potent clinical efficacy against a range of malignant tumors and autoimmune diseases. Nevertheless, conventional ex vivo CAR immune cell manufacturing is hindered by complexity, high costs, and significant inter-individual variability, which have limited its broad clinical application. These bottlenecks have prompted a paradigm shift toward in vivo CAR engineering, wherein nanocarriers directly deliver genetic material to circulating or tissue-resident immune cells, substantially simplifying the production process. This review first outlines the evolution of CAR immune cell therapy and key limitations of ex vivo approaches and then examines major delivery platforms for in vivo approaches. Comparative analyses are presented across delivery efficiency, cellular tropism, and expression kinetics, with particular emphasis on delineating the mechanistic distinctions and application boundaries between transient and durable expression strategies. We further dissect the critical challenges, including receptor-mediated non-specific immune activation, nanocarrier-associated immunogenicity constraining repeated administration, and delivery barriers imposed by the solid tumor microenvironment. Finally, the intrinsic trade-off between therapeutic durability and biosafety across distinct delivery platforms is discussed, along with the necessity that their clinical translation hinges on systematic optimization of delivery precision, immune compatibility, and expression controllability.

In vivo transduction of CAR T cells: Pharmacological principles, delivery platforms, and the road to clinical translation.

A novel silicon-based intracellular delivery platform enables multiplexed delivery across cell types and cell states 2310229

Immune cell engineering is a critical element of basic immunological research and translational applications. The ability to deliver modifying materials to cells without substantial phenotypic or survival impacts is a limiting factor for many immune cell types. To enable engineering in sensitive cell types or cell states, we have developed a novel intracellular delivery platform that works through mechanoporation. Cells are pushed through pores on a thin silicon membrane which creates temporary openings in the cell membrane that allow the payload of interest to diffuse into the cytoplasm before the membranes repair themselves. This method has been used to introduce CRISPR RNPs, mRNA, circRNA, siRNA and peptides and is compatible across cell types including naive and activated T cells, B cells, NK cells, monocytes, neutrophils, iPSC and HSC. Using our device, we simultaneously delivered a B2M targeting CRISPR RNP, GFP mRNA, and fluorescent dextran to primary unstimulated T cells and observed that 85% of the live population was B2M negative, GFP and dextran positive. The temporary membrane disruption is gentle enough to use with sensitive cells like neutrophils which allowed us to deliver dextran and GFP mRNA to neutrophils in whole blood. We measured 83% GFP positivity in the live, dextran positive population the next day. Similarly, we delivered a GFP-mRNA to PBMCs and observed expression in T cells, B cells, NK cells, and monocytes. This technology can also enable the use of viral vectors in challenging cell types. Using our method, we engineered B cells to express a circRNA ‘transduction enhancer’ (TE) and then infected them with a GFP lentivirus. After 3 days, the cells that received the TE had the highest GFP expression. Our technology unlocks scalable, gentle, and versatile intracellular delivery for sensitive immune cells, expanding the feasibility of complex genetic and molecular engineering for research and clinical cell therapies. n/a Technological Innovations in Immunology (TECH)

In vivo CAR-T therapy: The shift from ex vivo culturing to direct in situ immune reprogramming.

CAR T-cell therapy using chimeric antigen receptors (CARs) has provided a radical shift in the treatment of several hematological malignancies, producing high response rates and durable remissions. However, conventional ex vivo manufacturing is limited by complex processing steps, high costs, variability in product quality, and clinically relevant delays that restrict patient eligibility. In vivo manufacturing has emerged as a next-generation approach in which immune cells are reprogrammed directly within the patient, eliminating the need for exogenous handling and culture. This strategy uses viral and non-viral delivery platforms, including lentiviral vectors, adeno-associated viruses, lipid nanoparticles, and targeted polymer systems, together with DNA, mRNA, and genome editing tools such as CRISPR-based technologies. Early feasibility data are supported mainly by preclinical models and translational studies, while safety remains a central concern due to potential immunotoxicity, off-target transduction, and regulatory challenges. This review highlights key engineering strategies enabling in vivo CAR T-cell generation, summarizes emerging clinical research and development, and discusses future opportunities for expanding in vivo CAR T-cell therapies as scalable immunotherapy platforms.