In Vivo CAR-T Cell Therapy: Insights, Delivery Platforms & Clinical Translation

Overview of In Vivo CAR-T Therapy

Conventional ex vivo CAR-T manufacturing involves multiple steps: leukapheresis, T-cell sorting and activation, lentiviral or retroviral transduction, CAR-T cell expansion, release testing, product transportation, and final infusion. This process typically takes 1–3 weeks. Due to its high cost, lengthy and complex procedures, and potential production failures, conventional autologous CAR-T therapy has limited patient accessibility. These limitations have motivated the exploration of alternative strategies.

Allogeneic CAR-T (Universal CAR-T) therapies were developed to address these constraints but introduce immune rejection risks, such as graft-versus-host disease (GVHD). In contrast, in vivo CAR‑T therapy delivers CAR-encoding transgenes directly into endogenous T cells, reprogramming them in situ. This approach greatly simplifies the process, reduces costs, and represents a promising strategy to overcome the limitations of conventional CAR-T therapies.

CAR: chimeric antigen receptor, CRS: cytokine release syndrome, GVHD: graft versus host disease, ICANS: immune effector cell-associated neurotoxicity syndrome, iPSC: induced pluripotent stem cell, SPMs: secondary primary malignancies.

Since 2017, in vivo CAR-T therapy has progressed from proof-of-concept studies using synthetic DNA nanocarriers to optimized viral and non-viral vectors, with preclinical validation in hematological and solid tumor models, and has entered early clinical trials for hematological malignancies and autoimmune diseases. Beyond this rapid development, in vivo CAR-T therapy has demonstrated several practical advantages over conventional adoptive approaches, including streamlined treatment workflows, accelerated therapeutic delivery, and improved patient accessibility (Figure 2).

In adoptive therapy, CAR-T treatment generally takes about 10–25 days. In vivo CAR-T therapy directly generates immune cells using CAR carrier drugs without complex in vitro expansion or individualized operations, significantly simplifying the treatment process. It eliminates the need for lymphodepletion chemotherapy and repeated quality checks. This shortens the time from treatment decision to drug administration, improving overall treatment efficiency.

By eliminating individualized production and long-cycle in vitro operations, in vivo CAR-T enables "off-the-shelf" products, significantly reducing costs and improving patient accessibility. At the same time, it allows for the potential of large-scale production and broader clinical application.

The main procedural difference between adoptive and in vivo CAR-T therapy lies in lymphodepletion pretreatment. While pretreatment in adoptive therapy helps CAR-T cell adaptation and maximizes therapeutic benefits, it carries risks such as infection, leukopenia, and pulmonary vein occlusive disease. In contrast, in vivo CAR-T relies on endogenous T cell generation, preserving the complete immune system, which supports a broad antitumor response and reduces the likelihood of antigen escape. Although in vivo CAR-T may face efficacy limitations, such as immune-mediated CAR-T clearance, it may provide advantages in stemness and proliferation, resulting in superior in vivo activity at lower doses.

Platforms and Strategies for In Vivo CAR-T Cell Production

The development of in vivo CAR-T therapy depends on advances in gene delivery and editing. Unlike adoptive therapy, the therapeutic outcome in this approach is determined during the in vivo generation process, which relies on the precision, efficiency, and accuracy of CAR gene delivery. Consequently, the selection of an appropriate delivery vector and strategy becomes a critical consideration in the manufacturing process. To date, three main technical platforms have been employed for in vivo CAR-T generation, each offering distinct characteristics and advantages.

CAR: chimeric antigen receptor, LNPs: lipid nanoparticles.

Lentiviral vectors and adeno-associated viruses (AAV) are the most commonly used viral vectors. Lentiviral vectors, capable of stable gene integration and widely used in ex vivo CAR-T, are promising for in vivo delivery. However, their in vivo application still faces several challenges, such as host immune responses that may clear the virus, difficulties in specifically targeting T cells, and the potential tumor risk associated with random genomic integration.

To enhance the efficiency and safety of in vivo CAR-T generation, researchers have developed various optimization strategies. For example, some lentiviral platforms display multi-domain fusion proteins (MDFs) on the viral surface, simultaneously providing T cell activation signals and co-stimulatory signals, thereby improving T cell transduction efficiency. Other studies focus on targeting specific T cell subsets (such as CD4+ or CD8+ T cells), with the resulting CAR-T cells demonstrating enhanced persistence and antitumor activity in vivo. AAVs have also been explored, successfully reprogramming T cells and demonstrating antitumor effects in models.

Despite promising preclinical results, genomic integration risks must be carefully monitored to avoid potential malignant transformation. Thus, viral vectors hold great potential, but safety and controllability remain key considerations.

Nanoparticle (NP) platforms, mainly lipid nanoparticles (LNPs), offer non-viral gene delivery for in vivo CAR-T cell generation. Compared to viral vectors, nanoparticles are more readily engineered to target T cells, offer improved safety profiles, and are relatively cost-effective. Moreover, mRNA delivery avoids risks associated with genomic integration. They are also suitable for large-scale production, transport, and storage. LNPs typically contain ionizable lipids, phospholipids, cholesterol, and PEGylated lipids, with surface modifications enhancing T-cell targeting and delivery efficiency.

Early studies explored biodegradable polymeric nanocarriers and transposon systems for in vivo CAR-T cell engineering. Although initial efficiency was limited, these studies demonstrated the feasibility of in situ CAR-T generation. The mRNA-LNP technology advanced by COVID-19 vaccines has opened new opportunities for immune cell therapy. For example, CD5-targeted LNPs delivering CAR mRNA successfully edited T cells and cleared fibroblasts in mouse models, improving cardiac function. Strategies that co-express CARs and cytokines have further enhanced therapeutic efficacy. The latest targeted LNP (tLNP) technologies further reduce off-target delivery to the liver and have shown significant antitumor or B-cell depletion effects in humanized leukemia mouse models and non-human primates, along with favorable safety profiles.

To address limitations such as mRNA persistence and delivery efficiency, researchers have also investigated circular RNA (circRNA) and DNA platforms. CircRNA, due to its circular structure, is more stable than linear mRNA and can prolong protein expression. DNA carriers, through transposon systems, enable stable integration and sustained expression. Despite optimization, challenges remain in persistence, immunogenicity, and editing efficiency. Overall, nanoparticle platforms offer a flexible, safe, and scalable option for in vivo CAR-T therapy, representing a promising direction for future immune cell therapies.

Beyond direct injection of viruses or nanoparticles, scaffold implants have emerged as a promising strategy for localized in vivo CAR-T delivery. Scaffold-mediated in vivo CAR-T generation is primarily achieved in two approaches.

The first approach pre-isolates T cells from patient blood, co-encapsulates them with a CAR vector in a scaffold, and implants it at the tumor site for in vivo CAR-T generation. Although it requires brief ex vivo isolation, T cell activation and expansion occur entirely in vivo.

The second approach directly implants a scaffold with the CAR vector at the disease site to recruit and edit T cells in vivo. For example, Inamdar et al. used a porous collagen implant to recruit immune cells. The vector assembles CAR molecules, followed by rapid T cell expansion and release into tumors, suppressing growth and prolonging survival in mice^[4]. Compared to direct vector injection, implants enable rapid local CAR-T generation and reduce off-target expression.

In summary, implants provide an in situ platform for CAR integration, T cell expansion, and activation. This approach addresses limitations in CAR-T numbers and enables sustained local release. Compared to systemic approaches, it offers better efficacy and safety and could become a versatile platform for CAR-T engineering.

Clinical Progress and Ongoing Challenges of In Vivo CAR-T Therapy

Leveraging these diverse viral and non-viral platforms, several in vivo CAR-T candidates have progressed into clinical trials (Table 3), representing a critical step toward translating this technology into tangible therapies. The therapeutic applications of in vivo CAR-T platforms vary according to the delivery technology: viral vectors are primarily focused on hematological malignancies and autoimmune disorders, whereas non-viral vectors demonstrate broader applicability, encompassing autoimmune diseases, solid tumors, and fibrotic conditions (Figure 3).

LNP: lipid nanoparticle, LV: lentiviral vector, TROP2: trophoblast cell surface antigen 2, GPC3: glypican-3, BCMA: B-cell maturation antigen, VHH: variable domain of heavy chain of heavy-chain antibody, R/R: relapsed or refractory.

These early clinical studies highlight the translational potential of in vivo CAR-T therapy, demonstrating encouraging progress across multiple disease indications.

While in vivo CAR-T therapy holds great promise, realizing its full clinical potential requires overcoming key manufacturing- and delivery-related challenges (Figure 4).

A key step in in vivo CAR-T therapy is the targeted delivery of the CAR gene to T cells. However, there is a risk of off-target delivery, where the CAR gene may enter non-target cells, causing antigen masking and preventing CAR-T cells from recognizing and killing tumor cells. To mitigate this risk, researchers are developing T-cell-specific promoters and other precise delivery strategies to achieve functional CAR expression within T cells.

The efficiency of CAR gene expression is a critical determinant of therapeutic outcomes. Although viral vectors enable stable genomic integration, their transduction efficiency in primary T cells remains suboptimal. Non-viral vectors, such as nanoparticles, allow for more controllable CAR expression to reduce toxicity; however, delivery efficiency is often low and expression is transient, frequently requiring repeated administration. Emerging technologies, including self-amplifying mRNA (saRNA) and synthetic biology-based tunable promoter systems, offer promising solutions: they enable highly efficient and sustained CAR expression even at low doses, helping to lower production costs, reduce carrier payload, and better balance efficacy with safety.

In vivo CAR-T therapy may provoke immune responses due to non-self components of the CAR protein, residual carrier proteins, mRNA constructs, and nanocarriers, potentially affecting therapeutic efficacy. Strategies to reduce immunogenicity include using humanized CARs, developing low-immunogenicity carriers, chemically modifying RNA, and optimizing the LNP delivery system. These approaches can lower the risk of immune clearance, thereby improving treatment safety and efficacy.

CAR-T cells may attack each other by recognizing target antigens expressed on their own or neighboring T-cell surfaces, a phenomenon particularly observed in therapies targeting T-cell malignancies. Trogocytosis can also lead to antigen transfer, triggering CAR-T cell exhaustion. Strategies to mitigate these effects include dynamic regulation of CAR expression, selecting lower-affinity CARs, and targeting key regulatory molecules (e.g., ATF3/CH25H) to reduce trogocytosis and sustain antitumor activity.

Since the first approval in 2017, CAR-T therapy has revolutionized cancer treatment, particularly for hematological malignancies, and has shown promising potential in non-oncological conditions such as autoimmune disorders. To overcome the manufacturing complexities, supply limitations, and high costs associated with conventional adoptive cell therapy, in vivo CAR-T has emerged as a transformative paradigm. By enabling direct, in situ generation of CAR-equipped immune cells, it offers a more efficient, accessible, and potentially "off-the-shelf" therapeutic approach. As the field continues to evolve, in vivo CAR-T is poised to reshape the landscape of cell-based immunotherapy. Nevertheless, realizing its full potential requires careful management of persistent challenges related to targeted delivery, precise CAR expression control, immunogenicity, and long-term safety.

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