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In vivo chimeric antigen receptor (CAR)-T cell therapy

Nature Reviews Drug Discovery (2025)Cite this article

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Abstract

Chimeric antigen receptor (CAR)-T cell therapy has transformed the outcomes of patients with haematological malignancies, yet its use is limited by labour-intensive manufacturing, constrained production capacity and variable clinical performance. In vivo CAR-T cell engineering, in which CAR-T cells are generated directly inside the patient’s body, seeks to overcome these challenges by eliminating the need for ex vivo cell processing and complex logistics, as well as improve clinical performance. Recent advances in virology, RNA medicines and nanotechnology have catalysed a radical overhaul of this approach, which uses targeted delivery systems such as lentiviral vectors and lipid nanoparticles to introduce CAR-encoding genetic material into endogenous T cells. Early clinical studies have shown efficient transduction, sustained CAR expression and initial signs of antitumour activity, establishing proof of concept. This Review explores the underlying technologies — including RNA delivered by lipid nanoparticles and engineered viral vectors — and discusses how they are being adapted to develop more broadly applicable, scalable, safe and effective CAR-T cell therapies. By removing the need for ex vivo manipulation and chemotherapeutic conditioning, this strategy could enable the wider application of CAR-T cell therapies not just to blood cancers but to autoimmune diseases for which ex vivo CAR-T cell therapies have shown strong promise, such as systemic lupus erythematosus.

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Fig. 1  : Timeline: major discoveries and milestones leading to clinical translation of in vivo CAR technologies.
Fig. 2: Major in vivo CAR platforms with a focus on the engineering vector and payloads.
Fig. 3: Major in vivo CAR platforms in development, and their mechanism of action.
Fig. 4: Top-line perspective of development of in vivo CAR therapies.
Fig. 5: In vivo engineering platform features inform on target product profile characteristics and therapeutic applicability.
Fig. 6: The rapidly evolving, in vivo CAR therapy ecosystem.

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Acknowledgements

The authors acknowledge the contribution of P. Johnson and D. Fontana to the section ‘Viral vectors’. The authors apologize to those authors whose work was not cited directly owing to space limitations.

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Authors and Affiliations

  1. Capstan Therapeutics, San Diego, CA, USA

    Adrian Bot

  2. Umoja Biopharma, Seattle, WA, USA

    Andrew Scharenberg

  3. Kelonia Therapeutics, Boston, MA, USA

    Kevin Friedman

  4. Moderna Inc., Cambridge, MA, USA

    Lin Guey

  5. Myeloid Therapeutics, Cambridge, MA, USA

    Robert Hofmeister

  6. Interius BioTherapeutics, Philadelphia, PA, USA

    James I. Andorko

  7. Carisma Therapeutics, Philadelphia, PA, USA

    Michael Klichinsky

  8. Orna Therapeutics, Watertown, MA, USA

    Frank Neumann

  9. Sana Biotechnology, Seattle, WA, USA

    Jagesh V. Shah & Kyle Trudeau

  10. Mirai Bio, Cambridge, MA, USA

    Jagesh V. Shah

  11. Sanofi, Cambridge, MA, USA

    Andrew J. Swayer

  12. Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Drew Weissman & Carl H. June

  13. Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, WA, USA

    Matthias T. Stephan

  14. Molecular Biotechnology and Gene Therapy, Paul-Ehrlich-Institute, Langen, Germany

    Christian J. Buchholz

  15. Frankfurt Cancer Institute (FCI), Goethe-University, Frankfurt am Main, Germany

    Christian J. Buchholz

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Contributions

J.I.A., A.B., A.S., K.F., L.G., R.H., M.K., F.N., J.V.S., A.J.S. and K.T. generated the sections ‘Viral vectors’ and ‘RNA-based platforms’. A.B. conceived the article layout. M.T.S., C.J.B., D.W. and C.H.J. contributed to the Abstract, Introduction and Outlook. All authors reviewed and edited the manuscript before submission. C.J.B.’s contribution to the manuscript represents his own perspective and not the official view of the Paul-Erlich Institute.

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Correspondence to Adrian Bot.

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Competing interests

J.I.A. is a paid employee of Interius BioTherapeutics, Inc., holds equity in the company, and is an inventor on patents issued and/or pending related to this work. A.B. is a shareholder and employee of Capstan Therapeutics, a company developing in vivo chimeric antigen receptor (CAR) therapies, and inventor or co-inventor on multiple relevant patents in the CAR and related fields. C.J.B. is a co-inventor on patents covering T cell-targeted lentiviral vectors. K.F. is a shareholder and member of the Board of Directors of Kelonia Therapeutics, and is also an inventor on patent applications assigned to Kelonia. L.G. is a shareholder and employee of Moderna, and a shareholder of Tessera. A.S. is a shareholder and employee of Umoja Therapeutics, a company developing in vivo CAR therapies. R.H. is a shareholder and employee of Myeloid Therapeutics, a company developing in vivo CAR therapies. C.H.J. is an inventor of multiple CAR patents and shareholder of several companies developing CAR products. M.K. is a founder, employee and shareholder of Carisma Therapeutics; a founder, shareholder and board director of Chymal Therapeutics; and an inventor of multiple patents related to CAR macrophages and monocytes (CAR-M) that have been licensed by Carisma Therapeutics. F.N. is a shareholder and employee of Orna Therapeutics, a company developing in vivo CAR therapies. A.J.S. is a shareholder and current employee of Sanofi. J.V.S. is an employee and shareholder in Mirai Bio, and a consultant and shareholder in Sana Biotechnology. M.T.S. is co-founder of and has received stock options from Persistence Therapeutics (Jupiter Bioventures); and has IP Licensing with Sanofi, Juno Therapeutics (now Bristol Myers Squibb) and Jupiter Bioventures. K.T. is a shareholder and employee of Sana Therapeutics, a company developing in vivo CAR and cell gene therapies. D.W. has filed patent applications based on some aspects of this work; those interests were fully disclosed to the University of Pennsylvania, with an approved plan in place for managing any potential conflicts arising from licensing of these patents.

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Glossary

Circular RNA

A single-stranded RNA molecule that forms a continuous loop. Although naturally a type of non-coding RNA that is found in many species, circular RNA can be adapted for cell engineering.

Designed ankyrin repeat protein

(DARPin). A genetically engineered antibody mimetic protein typically exhibiting highly specific and high-affinity target protein binding, derived from natural ankyrin repeat proteins.

Fusogen

A protein on the surface of a virus that helps the virus enter a host cell by fusing the viral membrane with the host cell membrane. This process releases the genetic material of the virus into the host cell’s cytoplasm.

Hypogammaglobulinaemia

A condition characterized by abnormally low levels of immunoglobulins in the blood, making individuals more susceptible to infections.

Internal ribosomal entry site

A specific RNA sequence that allows ribosomes to bind to mRNA and initiate protein synthesis at an internal location, bypassing the typical cap-dependent initiation process.

Ionizable lipids

Lipid molecules that can change their charge depending on the pH level of their environment. They are a key component of lipid nanoparticles (LNPs), which are used to deliver RNA therapeutics.

Lymphodepletion conditioning

A treatment that prepares the body for chimeric antigen receptor (CAR)-T cell therapy or adoptive cell transfer in general. It involves using chemotherapy drugs (most frequently cyclophosphamide and fludarabine) to deplete endogenous immune cells.

Minimal residual disease

A term used for a small number of cancer cells that remain in the body after treatment. Minimal residual disease can occur in blood cancers such as leukaemia and lymphoma, as well as solid tumours.

N 1-Methylpseudouridine

A chemical compound found in tRNA and mRNA vaccines. Although a natural component of archaea, it is presently utilized in biochemistry and molecular biology.

Nipah virus (NiV) glycoproteins

NiV uses two key glycoproteins on its surface: the attachment glycoprotein (G) and the fusion glycoprotein (F), which are crucial for viral entry into host cells. The G protein facilitates attachment to the host cell, whereas the F protein triggers membrane fusion, allowing the virus to enter the cell.

PiggyBac transposon system

A genetic engineering tool used to introduce and integrate DNA sequences into a genome, often in a stable and reproducible manner. It utilizes a transposase enzyme to ‘cut and paste’ a transposon (a DNA sequence) into a new location in the genome.

Pseudotype

A virus particle that has been engineered to display a foreign viral envelope protein on its surface.

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Bot, A., Scharenberg, A., Friedman, K. et al. In vivo chimeric antigen receptor (CAR)-T cell therapy. Nat Rev Drug Discov (2025). https://doi.org/10.1038/s41573-025-01291-5

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