For more than a decade, ex vivo CAR‑T therapies have defined the frontier of personalised cancer treatment, offering unprecedented remission rates in haematologic malignancies, yet constrained by the logistical, manufacturing, and clinical complexities inherent to cell manipulation outside the body. Today, a new paradigm is emerging. In vivo CAR‑T engineering, which enables a patient’s own T cells to be reprogrammed directly within the body, represents a profound shift in how cell therapies might be delivered, scaled, and ultimately democratised. Autologous CAR-T has demonstrated remarkable clinical benefit in cancer and is now showing promise in autoimmune diseases. However, expanding access to these therapies remains a major challenge. Limited manufacturing capacity and the high cost and complexity of production continue to restrict availability for many patients (1).

Unlike traditional ex vivo CAR‑T workflows, marked by leukapheresis, multi‑day cell processing, vector transduction, expansion, release testing, and reinfusion, in vivo CAR‑T strategies aim to eliminate the entire external manufacturing chain. Instead, targeted gene delivery systems introduce CAR constructs directly into circulating T cells, offering the potential for on-demand, point-of-care immune engineering. Early preclinical investigations and emerging clinical data suggest this approach could overcome critical barriers of current therapies: reducing time-to treatment, improving patient access, and enabling broader indications beyond oncology (1).

Yet the scientific promise comes with equally significant technological demands. In vivo CAR‑T requires a new generation of delivery modalities, analytical methods, and production platforms, from precisely engineered viral and non‑viral vectors to sophisticated formulation, release, and safety strategies that ensure targeted, efficient, and safe in-body cell programming. These innovations must be developed and scaled with the same rigor, reliability, and regulatory robustness expected of any advanced therapy medicinal product (2).

Contract development and manufacturing organisations (CDMOs) play a pivotal role. As innovators push the boundaries of in vivo CAR‑T design, CDMOs are building the specialised capabilities needed to translate these concepts into clinical-grade realities, ensuring the highest quality, purity, and productivity (3).

Emerging evidence: in vivo CAR‑T in preclinical models

OXB has been investigating strategies to enable direct in vivo CAR‑T generation as a complement to established ex vivo approaches. In a recent publication, they describe the development of a retargeted lentiviral vector system designed to transduce T cells in situ, providing a preclinical demonstration of functional CAR‑T production within the body (4).

Vector design and targeting strategy

Central to their approach is a fourth-generation lentiviral platform (the TetraVecta™ system), engineered to optimise both specificity and safety (5). Key features include:

1. Retargeted envelope proteins:

   By modifying the viral envelope, OXB were able to preferentially target T cells, including CD3+ and CD8+ subsets, reducing transduction of non-target cells while enhancing efficiency supporting robust CAR gene delivery.

2. Controlled transgene incorporation:

   They designed the system to minimise incorporation of CAR proteins into vector particles, supporting more predictable and controlled in vivo transduction while mitigating potential off-target transduction (6).

3. Efficient integration and expression:

   Preclinical evaluation demonstrated stable integration of the CAR transgene in T cells, resulting in sustained expression and functional activity (4).

This vector design reflects a focus on precision, efficacy, and translational feasibility, providing a platform that supports both research applications and potential clinical translation.

Functional evaluation in preclinical models

OXB evaluated the performance of the retargeted vectors in preclinical models to assess their ability to generate functional CAR‑T cells in vivo (4). Observations included:

• High levels of CAR-T generation: Circulating T cells were transduced to produce CAR-T cells which expressed the CAR construct and where able to undergo efficient expansion.

• Functional activity: Generated CAR‑T cells demonstrated cytotoxic activity against Bcell targets, resulting in effective depletion comparable to conventional ex vivo CAR‑T therapies.

• Durability of response: CAR‑T populations persisted over time, maintaining functional activity without evidence of early exhaustion, highlighting the potential for sustained therapeutic benefit.

These results provide proof-of-concept that in vivo CAR‑T generation is feasible, and they illustrate how a carefully engineered vector system can produce functional immune cells within the body, laying the groundwork for future translational studies (1).

Translational considerations

Their findings underscore several important considerations for developing in vivo CAR‑T therapies:

• Delivery and specificity: Vector design is critical to maximise transduction efficiency while minimising off-target activity.

• Safety and predictability: Precise cell targeting, controlled incorporation and expression of transgenes enhance safety and support regulatory evaluation.

• Platform adaptability: The underlying vector architecture is adaptable to different CAR constructs and can deliver multiple genes, providing a versatile framework for exploration of diverse disease indications.

• Scalability: By eliminating the need for ex vivo cell processing, in vivo CAR‑T could potentially streamline manufacturing and broaden access to patients (1).

Taken together, these observations support the continued investigation of in vivo CAR‑T as a complementary approach to conventional ex vivo therapies, offering the potential for more rapid, flexible, and accessible cellular immunotherapy.

Comparisons with other in vivo CAR‑T strategies

OXB’s lentiviral platform is one of several approaches under exploration. Comparative strategies include:

• Lipid nanoparticle (LNP) systems: LNPs delivering CAR-encoding mRNA can transiently reprogram circulating T cells. These rely on efficient cellular uptake and intracellular expression, which can vary with T-cell subsets, circulation dynamics, and tissue distribution (7).

• Alternative viral platforms: Diverse viral vector platforms like adenoviral vectors, AAV, lentiviral vectors and retroviral vectors have been employed towards in vivo gene therapy development. While each platform has its own advantages and limitations, lentiviral vectors and AAV are currently the most widely used systems for in vivo gene engineering (8).

• Combinatorial strategies: Emerging research explores combining in vivo CAR-T with checkpoint modulation, cytokine support, or co-stimulatory payloads to overcome immunosuppressive microenvironments, particularly relevant for solid tumours (9).

In this context, the OXB study contributes rigorous preclinical validation of a fully retargeted lentiviral system with demonstrable functional outcomes, positioning it as a foundational reference for ongoing comparative research.

Technological demands and the role of CDMOs

Translating in vivo CAR-T from preclinical promise to clinical reality requires integrated platform capabilities. The technological demands are extensive:

• Vector manufacturing: High-titer, GMP-compliant vector production with consistent quality is essential to support clinical trials.

• Vector engineering and delivery: Ensuring stability, bioavailability, and targeted biodistribution in vivo requires sophisticated vector engineering strategies.

• Analytical and release testing: Accurate quantification of vector potency, transduction efficiency, and purity is essential to meet regulatory expectations.

• Safety and pharmacokinetics: In vivo therapies must be evaluated for off-target activity, immunogenicity, and persistence, necessitating robust monitoring strategies (2).

Contract development and manufacturing organizations (CDMOs) are pivotal in bridging the gap between innovation and clinical application, providing the infrastructure, expertise, and regulatory compliance frameworks that allow experimental vectors like OXB’s to reach human studies.

Challenges and considerations

Despite promising preclinical and early clinical results, significant hurdles remain to realise the full potential of in vivo CAR-T:

• Safety and immunogenicity: Host immune responses against viral vectors or CAR constructs could impact efficacy and patient safety.

• Targeted CAR expression: Ensuring the right balance between therapeutic activity and avoidance of toxicity requires precise vector engineering to ensure targeted integration and expression in the desired cells.

• Translational relevance: Preclinical models do not fully recapitulate human immune dynamics; careful design of early clinical studies will be essential.

• Regulatory oversight: In vivo genetic modification introduces unique regulatory considerations, particularly regarding long-term follow-up and monitoring for insertional mutagenesis.

Addressing these challenges will require iterative development, rigorous preclinical evaluation, and robust manufacturing and analytical support, highlighting the interdisciplinary nature of this next-generation therapy (1,2).

Future directions

The OXB work provides a basis for iterative advancement in in vivo CAR-T development:

• Expansion to additional CAR constructs targeting haematologic and solid tumour antigens.

• Integration with immune-modulatory strategies to enhance T-cell persistence and overcome suppressive tumour microenvironments.

• Optimisation of vector design and dosing strategies to maximize efficacy and minimize off-target risks.

• Development of scalable GMP production to support first-in-human trials.

As these elements converge, in vivo CAR-T holds the potential to redefine accessibility, manufacturing efficiency, and therapeutic reach, making cellular immunotherapy feasible for a broader patient population (10).

Conclusion

For over a decade, ex vivo CAR-T therapies have transformed haematologic oncology. Yet, as the field seeks to overcome operational and logistical barriers, in vivo CAR-T emerges as a paradigm shift, enabling point-of-care immune engineering that is faster, more scalable, and potentially more accessible.

The OXB publication on lentiviral vector-mediated in vivo CAR-T generation illustrates this potential in action, demonstrating precise, functional, and sustained CAR-T production in preclinical models. By combining careful vector engineering, safety-conscious design, and translational foresight, this work offers a tangible example of how scientific innovation and manufacturing infrastructure can converge to bring next-generation cellular therapies closer to patients (4).

As the field progresses, continued collaboration among vector developers, translational scientists, and CDMOs will be crucial. Only by integrating mechanistic understanding, rigorous safety evaluation, and scalable production capabilities can in vivo CAR-T move from experimental promise to clinical reality, potentially transforming the landscape of cellular immunotherapy for the next generation of patients (11).

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