What is Cell-Based Gene Therapy?
Cell-based gene therapies, also known as genetically modified cell therapies, represent an intersection of cell therapy and gene therapy technologies. This approach involves the ex vivo genetic modification of cells, which are then reintroduced into the patient to exert therapeutic effects. The technology combines the regenerative potential of living cells with the precision of gene transfer, to generate cell products with restored or enhanced functions. Biopharmaceutical companies have increasingly adopted genetic modification strategies to confer therapeutic activity of various cell types, resulting in a surge of clinical programs centered on engineered cells. Notably, gene-modified cell therapies now outnumber unmodified cell therapies in clinical development, underscoring the growing importance of genetic engineering as a transformative force in cellular therapeutics.
Among these products, chimeric antigen receptor (CAR) T-cell therapies are the most advanced and widely studied. Other emerging therapies include T-cell receptor (TCR)-modified T cells and CAR-natural killer (CAR-NK) cells. Beyond immunotherapies, genetic modification of hematopoietic stem cells (HSCs) with functional proteins are used to treat inherited conditions. Together, these approaches exemplify how cell-based gene therapies are redefining cancer treatment, converting immune or stem cells into precision therapeutic tools capable of directly eradicating malignant cells or restoring lost cellular function.
Overview of Cell-Based Gene Therapy
Overview of cell-based gene therapy types. This diagram broadly highlights CAR-T, TCR-T, CAR-NK, CAR-M, and HSC-based therapies. Cells are obtained from patients or donors and engineered ex vivo to produce cell-based gene therapy products. Created in BioRender. https://BioRender.com/cp5ssr4
CAR-T Cell Therapy
The defining feature of CAR-T cells is their expression of an engineered receptor that confers antigen specificity independent of the native T cell receptor (TCR). This synthetic receptor consists of three primary components: an extracellular, transmembrane, and intracellular domain. The extracellular portion contains a single-chain variable fragment (scFv) derived from an antibody, which recognizes a tumor-associated antigen (commonly CD19 or BCMA) displayed on malignant cells. Upon antigen binding, signal transduction occurs through the intracellular portion of the receptor, which integrates both co-stimulatory and activation domains. The co-stimulatory motif enhances T cell proliferation, persistence, and cytokine secretion, while the CD3ζ signaling domain (derived from the TCR complex) initiates cytotoxic effector functions. This combination enables CAR-T cells to recognize and kill target cells in an MHC-independent manner.
CAR T cell therapies have demonstrated solid efficacy in cancer cells expressing CD19 and BCMA, leading to the approval of several products by the U.S. FDA. Some notable examples include tisagenlecleucel (Kymriah) for acute lymphoblastic leukemia, axicabtagene ciloleucel (Yescarta) and lisocabtagene maraleucel (Breyanzi) for large B-cell lymphoma, and idecabtagene vicleucel (Abecma) and ciltacabtagene autoleucel (Carvykti) for multiple myeloma.
The manufacturing of chimeric antigen receptor (CAR) T cells generally follows a workflow comprising four major stages: T cell isolation, activation and expansion, genetic modification, and final expansion with cryopreservation. The process begins with the isolation of T cells from the patient’s peripheral blood, collected through leukapheresis. Peripheral blood mononuclear cells (PBMCs) are separated using density gradient centrifugation, followed by magnetic selection based on T cell–specific markers such as CD3. Using anti-CD3 and anti-CD28 antibodies, along with growth factors like IL-2, the T cells are activated, priming them for transgene delivery and expansion. All FDA-approved CAR T cell products to date rely on lentiviral vector-based delivery, which integrate stably into the genome to ensure persistent CAR expression. Alternative strategies such as transposon-based systems and CRISPR/Cas9-mediated genome editing remain under active development. After modification, CAR-expressing T cells undergo further expansion to achieve clinically effective cell numbers. The final product is then subjected to rigorous quality control testing before cryopreservation and infusion into the patient.
CAR-NK Cell Therapy
Like CAR-T cells, CAR-NK cells are engineered natural killer (NK) cells expressing a synthetic receptor designed to confer antigen specificity. This receptor similarly comprises three principal components: an extracellular single-chain variable fragment (scFv) that recognizes a tumor-associated antigen; a transmembrane domain anchoring the receptor to the cell membrane; and intracellular signaling domains that combine co-stimulatory motifs with a CD3ζ activation sequence to initiate cytotoxic responses. By merging the targeted recognition capabilities of CAR technology with the innate antitumor activity of NK cells, CAR-NK therapy leverages both engineered and natural mechanisms of tumor destruction.
NK cells eliminate malignant cells through multiple complementary pathways, including granule-mediated cytotoxicity via perforin and granzymes, death receptor–mediated apoptosis, secretion of pro-inflammatory cytokines and chemokines, and antibody-dependent cell-mediated cytotoxicity (ADCC). Importantly, NK cells secrete a distinct cytokine repertoire compared with T cells—one less likely to provoke the severe systemic inflammation characteristic of cytokine release syndrome. Moreover, NK cells do not induce graft-versus-host disease, opening the possibility of allogeneic, “off-the-shelf” CAR-NK cell products derived from donor cells or established NK cell lines. Although no CAR-NK therapies have yet received regulatory approval, preclinical studies and early-phase clinical trials have demonstrated encouraging results against both hematologic malignancies and solid tumors.
Some key challenges in the development of successful CAR-NK cell therapies include their relatively short persistence, limited tumor infiltration, and the need for CAR designs tailored to the unique microenvironment of solid tumors. A major focus of current research is improving the in vivo persistence and proliferation of NK cells, which is essential for sustaining antitumor activity. Researchers are exploring strategies such as engineering CAR-NK cells with cytokine support (e.g., IL-15 expression), generating memory-like NK cell phenotypes with enhanced longevity and responsiveness, and designing multi-specific CARs capable of recognizing multiple tumor antigens simultaneously.
CAR-Macrophage (CAR-M) Therapy
Chimeric antigen receptor–macrophage (CAR-M) cell therapy is an emerging immunotherapeutic approach that combines the innate cytotoxic and antigen-presenting functions of macrophages with the antigen specificity of engineered CARs. Macrophages can penetrate the tumor microenvironment, where they secrete matrix metalloproteinases that degrade the extracellular matrix and remodel tissue architecture. This inherent capacity for infiltration makes macrophages particularly well suited for targeting solid tumors that are often resistant to T cell-based therapies. They can directly kill tumor cells through multiple mechanisms, including the secretion of tumoricidal mediators such as tumor necrosis factor, nitric oxide, and reactive oxygen species. In addition, CAR-M cells can mediate antibody-dependent cellular cytotoxicity and antibody-dependent phagocytosis via Fc receptor engagement with antibody-coated tumor cells. Indirectly, macrophages secrete cytokines that recruit and activate other immune cells, thereby amplifying the antitumor immune response.
Although CAR-M therapies have not yet reached clinical approval, preclinical studies demonstrate strong antitumor activity, and several clinical trials are underway. Continued research is focused on refining CAR-M biology to enhance therapeutic performance. Key priorities include maintaining a stable, pro-inflammatory M1-like phenotype—supported through IFN-γ gene expression or CD206 modulation—and improving gene delivery efficiency, as macrophages are highly sensitive to foreign nucleic acids. While lentiviral and adenoviral vectors remain standard, emerging nonviral nanoparticle platforms and CRISPR–Cas9 editing offer promising alternatives. Scalable production of clinical-grade macrophages also remains a challenge, with current sources limited to peripheral monocytes or induced pluripotent stem cells (iPSCs). Finally, safety considerations persist, as macrophage-derived interleukin-6 (IL-6) may contribute to cytokine release syndrome, necessitating careful immune monitoring during therapy development.
TCR-T Cell Therapy
T cell receptor–engineered T cell (TCR-T) therapy is an emerging form of adoptive cellular immunotherapy with strong promise in the treatment of solid tumors. Unlike CAR-T cells, which recognize only surface antigens, TCR-T cells are designed to detect peptide antigens presented by major histocompatibility complex (MHC) molecules, enabling them to target both intracellular and surface-derived tumor antigens. This MHC-restricted recognition broadens the range of potential tumor targets and allows for more physiologic antigen engagement.
Clinical development of TCR-T therapies primarily focuses on two categories of tumor antigens. Tumor-associated antigens (TAAs) are overexpressed in malignant tissues but may also appear at lower levels in some normal tissues (such as HER2, MART-1, mesothelin, and MAGE-A family proteins). Tumor-specific antigens (TSAs), or neoantigens, arise exclusively in tumor cells as a result of genetic mutations or viral oncogenesis. Once a target antigen and corresponding TCR sequence are identified, further engineering optimizes receptor expression, stability, and affinity for the peptide–MHC complex. Transgenic TCRs are typically introduced into patient T cells using viral vector systems or, increasingly, CRISPR–Cas9–based genome editing to enhance precision and control of transgene integration.
TCR-T cell therapy development faces crucial challenges in terms of efficacy and safety. Because TCRs recognize peptide–MHC complexes, one major challenge is “on-target, off-tumor” toxicity, which occurs when the same peptide epitope targeted on tumor cells is also expressed in healthy tissues. In addition, “off-target, off-tumor” toxicities can arise when peptides on normal cells share structural similarity with the intended tumor antigen or when alternative MHC molecules present unrelated peptides that cross-react with the engineered TCR. The ideal antigen for TCR-T therapy would therefore be a peptide-MHC complex that is highly specific, consistently expressed across all tumor cells, and absent from healthy tissues. Comprehensive antigen screening, in-depth preclinical validation, and rigorous safety assessments are crucial to minimize cross-reactivity before advancing TCR-T cell products into clinical trials.
HSC Gene Therapy
Hematopoietic stem cell (HSC) gene therapy is a rapidly advancing field that primarily utilizes autologous ex vivo gene-modification strategies. In this approach, HSCs are obtained from the patient through either direct bone marrow harvest or, more commonly, peripheral blood apheresis following stem cell mobilization. This population is enriched for CD34⁺ hematopoietic stem and progenitor cells, cultured, and then genetically modified using viral vector–mediated transduction or genome-editing technologies to correct the underlying defect—such as by introducing a functional copy of a missing or mutated gene. Conducting genetic modification ex vivo provides a controlled environment for precise cell manipulation, allowing comprehensive evaluation of transduction efficiency, cellular function, and overall product quality before reinfusion into the patient.
Clinically, HSC gene therapies have shown remarkable success in treating a range of inherited and metabolic disorders, particularly those affecting the immune and hematopoietic systems. Several therapies have achieved regulatory approval, including Strimvelis for ADA-SCID, Zynteglo and Casgevy for transfusion-dependent β-thalassemia, Libmeldy for metachromatic leukodystrophy (MLD), Skysona for early cerebral adrenoleukodystrophy (CALD), and Lyfgenia and Casgevy for sickle cell disease (SCD). The restoration of monogenic disorders represents a major clinical application of HSC gene therapy, with more clinical trials currently underway.
MACS GMP Vectofusin-1 is a fully synthetic, non-toxic, cationic amphipathic peptide with viral transduction enhancing capacity, enabling higher transduction levels with low amounts of retroviral vector. MACS GMP Vectofusin-1 promotes the entry of several retroviral pseudo-types into target cells when added to the culture medium. It is applicable for the enhancement of lentiviral and retroviral transduction.
Although HSC gene therapy has made major strides and now rivals allogeneic transplantation for several indications, further innovations are required to enhance efficacy, improve safety, and broaden its therapeutic reach. Current efforts focus on optimizing HSC and progenitor cell mobilization and selection to isolate the most potent, self-renewing stem cell populations, as well as refining ex vivo culture and processing methods to preserve stemness and engraftment potential. Advances in vector and editing technologies such as next-generation self-inactivating lentiviral (SIN-LV) systems, base and prime editing, and more precise gene insertion strategies are needed to improve efficiency and reduce genotoxic risk. Parallel improvements in scalable, high-quality manufacturing platforms will be key to expanding clinical access and enabling the development of new gene therapy products for a wider array of hematologic, infectious, and malignant diseases.
Regulatory Considerations
Regulatory evaluation of hematopoietic stem cell (HSC) gene therapies requires a comprehensive assessment of product specificity, safety, and long-term biological behavior. A critical component of this process is evaluating antigen recognition specificity and affinity to identify potential on-target/off-tumor and off-target toxicities. Safety assessments commonly include tissue cross-reactivity studies using monoclonal antibodies or fusion proteins sharing the engineered antigen-recognition domain, protein array screening, and cytotoxicity or cytokine-release assays conducted on panels of human primary cells, established cell lines, or induced pluripotent stem cell–derived models. In addition, relevant animal studies may be employed to assess in vivo adverse effects and biodistribution. Equally important is the comprehensive characterization of the target antigen, as prior clinical data and known tissue-expression profiles provide critical context for predicting and mitigating off-tumor reactivity.
Tumorigenicity is a critical consideration in the development of cell-based gene therapies, particularly those involving stem or progenitor cells. The U.S. FDA recommends thorough evaluation of factors that may influence malignant transformation risk, including the engineered product’s differentiation status, the extent of ex vivo manipulation, and the genomic integration potential of the vector or gene-editing platform. Additional parameters to assess can include the nature of the expressed transgene, as well as the product’s in vivo biodistribution, persistence, and proliferative capacity. Collectively, these evaluations form the foundation of a comprehensive preclinical safety framework designed to ensure that HSC gene therapy products deliver precise, durable, and safe therapeutic benefit and clinical success.
References
Brudno JN, Maus MV, Hinrichs CS. CAR T Cells and T-Cell Therapies for Cancer: A Translational Science Review. JAMA. 2024;332(22):1924-1935. doi:10.1001/jama.2024.19462
Zugasti I, Espinosa-Aroca L, Fidyt K, et al. CAR-T cell therapy for cancer: current challenges and future directions. Signal Transduct Target Ther. 2025;10(1):210. Published 2025 Jul 4. doi:10.1038/s41392-025-02269-w
Balkhi S, Zuccolotto G, Di Spirito A, Rosato A, Mortara L. CAR-NK cell therapy: promise and challenges in solid tumors. Front Immunol. 2025;16:1574742. Published 2025 Apr 7. doi:10.3389/fimmu.2025.1574742
Zhong Y, Liu J. Emerging roles of CAR-NK cell therapies in tumor immunotherapy: current status and future directions. Cell Death Discov. 2024;10(1):318. Published 2024 Jul 10. doi:10.1038/s41420-024-02077-1
Lu, J., Ma, Y., Li, Q. et al. CAR Macrophages: a promising novel immunotherapy for solid tumors and beyond. Biomark Res 12, 86 (2024). doi:/10.1186/s40364-024-00637-2
Chen K, Liu ML, Wang JC, Fang S. CAR-macrophage versus CAR-T for solid tumors: The race between a rising star and a superstar. Biomol Biomed. 2024;24(3):465-476. Published 2024 May 2. doi:10.17305/bb.2023.9675
Baulu E, Gardet C, Chuvin N, Depil S. TCR-engineered T cell therapy in solid tumors: State of the art and perspectives. Sci Adv. 2023;9(7):eadf3700. doi:10.1126/sciadv.adf3700
Golikova, E.A., Alshevskaya, A.A., Alrhmoun, S. et al. TCR-T cell therapy: current development approaches, preclinical evaluation, and perspectives on regulatory challenges. J Transl Med 22, 897 (2024). doi:10.1186/s12967-024-05703-9
John T, Czechowicz A. Clinical hematopoietic stem cell-based gene therapy. Mol Ther. 2025;33(6):2663-2678. doi:10.1016/j.ymthe.2025.04.029
U.S. Food and Drug Administration, Center for Biologics Evaluation and Research. Frequently Asked Questions — Developing Potential Cellular and Gene Therapy Products: Draft Guidance for Industry. Silver Spring, MD: US Food and Drug Administration; November 2024. Accessed October 2025.
U.S. Food and Drug Administration. Considerations for the Development of Chimeric Antigen Receptor (CAR) T Cell Products: Guidance for Industry. U.S. Department of Health and Human Services; January 2024.