I. Introduction

CAR-T cell therapy has emerged as a pivotal modality in the treatment of hematological malignancies; however, traditional *ex vivo* manufacturing processes are characterized by their complexity, high cost, and time-consuming nature. *In vivo* CAR-T engineering—which involves the direct delivery of CAR-encoding sequences within the body—holds the promise of simplifying manufacturing and enhancing accessibility, thereby charting the course for the next generation of cellular immunotherapy.

The team led by Li Chunrui from the Department of Hematology at Tongji Hospital, affiliated with Tongji Medical College of Huazhong University of Science and Technology, recently published a review article in *Biomarker Research* (IF: 9.5) titled "In vivo CAR-T cell engineering: delivery strategies and clinical translation." This article systematically reviews the delivery platforms, translational barriers, and clinical prospects associated with *in vivo* CAR-T technology. Furthermore, the paper offers an in-depth comparison of the advantages and limitations of both viral and non-viral vectors, while also exploring the potential applications of multi-lineage cell engineering.

II. Main Content of the Article

2.1 Comparison of In Vitro and In Vivo CAR-T Generation Strategies

Ex vivo CAR-T therapy entails a multi-step process—including apheresis, genetic modification, expansion, quality control, and reinfusion—that spans several weeks and incurs substantial costs. In vivo strategies bypass ex vivo manipulations by delivering CAR-encoding sequences directly to circulating T cells via systemic administration, effectively transforming the patient's own body into an "autologous bioreactor" and thereby shortening the time from diagnosis to treatment.

Core Advantages and Process Simplification: In vivo engineering eliminates the need for centralized manufacturing, opening up possibilities for regions with limited infrastructure. Furthermore, it avoids the phenotypic and functional heterogeneity often resulting from ex vivo expansion, thereby enhancing batch-to-batch consistency.

Clinical Translation and Cost Reduction: A single administration is sufficient to generate functional CAR-T cells within the body, holding the promise of significantly reducing treatment costs and improving global accessibility.

Figure 1. Strategies for *in vitro* and *in vivo* CAR-T cell generation.

2.2 Molecular Mechanisms of Viral and Non-Viral Delivery Vectors

Lentiviral vectors enter T cells via receptor-mediated endocytosis; through reverse transcription, they integrate the CAR gene into the host genome, thereby achieving long-term, stable expression. In contrast, lipid nanoparticles (LNPs) encapsulate non-integrating nucleic acids; the mRNA is directly translated within the cytoplasm to produce CAR proteins, resulting in a shorter duration of expression.

Advantages and Risks of Viral Vector Integration: Lentiviruses are capable of transducing both quiescent and dividing cells, making them suitable for tumor therapies that require sustained CAR expression. However, they carry the risk of insertional mutagenesis, necessitating the adoption of self-inactivating designs to mitigate safety concerns.

Transient Expression and Controllability of Non-Viral Platforms: The transient expression characteristic of non-viral vectors—such as LNPs—is better suited for indications requiring controllable pharmacodynamics, such as autoimmune diseases. Furthermore, their chemical synthesis processes offer superior scalability and batch-to-batch consistency.。

Figure 2. Molecular Mechanisms of In Vivo CAR Gene Delivery via Viral and Non-viral Vectors

2.3 Design Strategies and Translational Milestones for In Vivo CAR-T Therapy

CAR structural design has evolved through five generations, incorporating enhanced functionalities such as dual co-stimulatory signals, cytokine-inducible modules, and inducible transcriptional control. *In vivo* engineering strategies must strike a balance between expression persistence and safety, often employing scFvs with moderate affinity to minimize background signaling.

Vector Production and Gene Delivery: Viral vectors package CAR transcripts via cellular systems, whereas non-viral platforms encapsulate nucleic acids using chemical or physical methods. Both types of vectors are capable of *in vivo* reprogramming of T cells, NK cells, macrophages, and other immune cells, thereby facilitating synergistic anti-tumor immunity.

Preclinical and Early Clinical Progress: Multiple platforms have demonstrated anti-tumor activity in preclinical models and are advancing through early-stage clinical trials for hematological malignancies, solid tumors, and autoimmune diseases, thereby laying a solid foundation for clinical translation.

Figure 3. Design Strategies and Translational Progress of In Vivo CAR-T Cell Therapy

2.4 Translational Barriers and Mitigation Strategies for In Vivo CAR-T Therapy

Insufficient targeting precision may lead to off-target toxicity; consequently, T-cell specificity must be enhanced through vector engineering and surface functionalization. The integration risks associated with viral vectors, as well as the low transfection efficiency of non-viral vectors, remain critical bottlenecks.

Immunogenicity and Inflammation Control: Vectors and transgene products may elicit immune responses, thereby exacerbating the risks of Cytokine Release Syndrome (CRS) and Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS). These risks must be mitigated through vector de-immunization, optimization of administration routes, and the concomitant use of immunomodulatory agents.

Manufacturing and Regulatory Compliance: Standardized manufacturing and quality control processes face significant challenges, necessitating the establishment of real-time monitoring systems and modular vector platforms. Regulatory frameworks must be adapted to accommodate the unique characteristics of in vivo engineering, placing particular emphasis on process validation and long-term patient follow-up.

Figure 4. Translational Barriers and Countermeasures for In Vivo CAR-T Cell Therapy

2.5 Progress in the Commercial Pipeline for In Vivo CAR-T Therapy

Multiple companies are establishing in vivo CAR-T platforms, targeting antigens such as CD19, BCMA, and CD20. Delivery methods include targeted lipid nanoparticles (LNPs) and lentiviral vectors. The scope of indications is expanding from hematological malignancies to encompass autoimmune diseases and solid tumors.

Key Technological Platform Differentiators: Targeted LNP platforms (e.g., CPTX-2309) utilize antibody modifications to enhance T-cell selectivity; meanwhile, lentiviral platforms (e.g., UB-VV111) incorporate RACR regulatory elements to boost CAR expression and function. These distinct platforms each offer unique advantages in terms of durability, safety, and manufacturing processes.

Clinical Stage and Indication Strategy: Multiple candidates have entered Phase I clinical trials, primarily targeting relapsed/refractory hematological malignancies, while some are exploring applications in autoimmune diseases. The pipeline strategy suggests that in vivo CAR-T therapies hold promise for achieving breakthroughs across a wide range of disease areas.

Table 1. Commercial Pipeline for In Vivo CAR-T Cell Therapy

2.6 Comparative Analysis of Viral vs. Non-Viral Delivery Platforms

Viral vectors offer advantages in terms of transduction efficiency and the persistence of gene expression; however, they carry inherent risks associated with immune clearance and genomic integration. Non-viral vectors, conversely, facilitate transient expression and exhibit lower immunogenicity, making them well-suited for indications requiring controllable pharmacodynamics.

Vector Selection and Clinical Suitability: Lentiviral vectors are appropriate for oncology treatments necessitating long-term CAR expression, whereas AAV vectors offer a superior safety profile, albeit with limited packaging capacity. Lipid nanoparticles (LNPs) provide manufacturing flexibility, rendering them ideal for rapid iteration and large-scale production.

Safety and Process Considerations: Viral vectors mandate rigorous testing to rule out the presence of replication-competent viruses, while non-viral vectors must overcome the critical bottleneck of inefficient intracellular escape. The ultimate selection of a vector requires a comprehensive assessment that takes into account the target indication, dosing frequency, and regulatory requirements.

Table 2: Comparison of Viral and Non-viral Delivery Platforms

2.7 Preclinical Evidence for In Vivo CAR-T Therapy

Preclinical studies span various fields, including hematological malignancies, fibrotic diseases, and solid tumors. In models of B-cell lymphoma, fusion nanoparticle-mediated *in vivo* CAR-T therapy demonstrated potent anti-tumor activity without inducing significant cytokine release syndrome (CRS).

Applications in Fibrosis and Myocardial Repair: The delivery of CAR mRNA via CD5-targeted lipid nanoparticles (LNPs) can generate transient anti-fibrotic CAR-T cells, thereby alleviating cardiac fibrosis and restoring cardiac function. Furthermore, uPAR-targeted CAR-T cells can eliminate senescent fibroblasts, leading to improvements in the pathology of liver fibrosis and rheumatoid arthritis.

Expansion into Solid Tumors and Multiple Disease Areas: Studies targeting ROR1 in prostate cancer and CD133 in glioblastoma—among others—have demonstrated the potential of *in vivo* CAR-T therapy in solid tumors, highlighting its capacity to synergize with vaccines and chemotherapy to enhance therapeutic efficacy.

Table 3. Representative Preclinical Evidence for In Vivo CAR-T Therapy

2.8 Multi-Lineage Cell Engineering: Expanding Beyond T Cells

In vivo engineering is not limited to T cells; it can be applied to reprogram NK cells, macrophages, monocytes, and other cell types. CAR-M cells can enhance phagocytic function and remodel the tumor microenvironment, while CAR-NK cells offer potential for allogeneic applications.

Macrophages and Microenvironment Remodeling: CAR-M cells targeting GPC3 or FAP-α can suppress stromal cells and enhance sensitivity to chemotherapy. PD-L1-targeting CAR-M cells can phagocytose immunosuppressive cells and, in synergy with STING agonists, recruit CD8+ T cells.

NK Cells and Allogeneic Applications: CAR-NK cells targeting CD19 or CD20 have demonstrated efficacy in hematological malignancies while avoiding the risk of Graft-versus-Host Disease (GvHD). A multi-lineage synergistic approach can effectively counter immunosuppressive microenvironments and improve control over heterogeneous tumors.

Table 4: Multi-lineage Cell Engineering—Expansion Beyond T Cells

III. Summary and Outlook

This review systematically summarizes the progress of *in vivo* CAR-T technology across various aspects, including delivery strategies, clinical translation, and multi-lineage applications. Both viral and non-viral platforms have demonstrated feasibility in preclinical models, and numerous early-stage clinical trials are currently underway. However, precise targeting, long-term safety, standardized manufacturing, and regulatory frameworks remain key bottlenecks. Future directions include: optimizing vector specificity to mitigate off-target toxicity; developing controllable CAR expression systems to balance efficacy with safety; establishing manufacturing and quality control standards; and refining the regulatory science framework. As the technology matures, *in vivo* CAR-T holds the potential to expand into the fields of autoimmune and infectious diseases, thereby significantly enhancing the accessibility of cell therapies by substantially reducing manufacturing costs.