Many of the limited number of patients with blood cancers that have received current chimeric antigen receptor (CAR)-T cell immunotherapies have experienced amazing results, with some still cancer-free more than a decade later. However, eligibility criteria are quite strict, and of the few that do quality, only a small percentage have received these treatments due to lack of accessibility. Variable outcomes and the occurrence of dangerous and sometimes fatal adverse events associated with ex vivo CAR-T cell therapies are additional concerns. In vivo generation of CAR receptors in T (and other immune) cells is an alternative approach that could potentially overcome all these issues. Preclinical and very early clinical results are promising.
Understanding CAR-T Cell Therapy
Chimeric antigen receptor (CAR)-T cell therapy technology was initially developed in the early 1990s as a means for enabling T cells to attack cancer cells that otherwise evade the immune system.1–3 The goal with the first CAR-T cell therapies was to replace some of a patient’s immune cells with genetically modified cells containing CAR receptors targeting specific surface-expressed antigens commonly found on cancerous B cells.
The approved CAR-T cell therapies have been successful in treating leukemia, lymphoma, and myeloma. As the technology has evolved, drug developers have expanded their focus beyond these cancers to include many additional hematologic malignancies and solid tumors, as well as autoimmune disorders, infectious diseases, and other indications.
Ex Vivo Treatments a Huge Advance but Far from Ideal
The six FDA-approved CAR-T cell therapies targeting CD19 on B cells and B cell maturation antigen (BCMA) on plasma cells can be very effective, with some patients with advanced, aggressive cancers experiencing complete responses and remaining cancer-free a decade later.3 For some of these treatments, overall responses rates have reached above 80%.4
Unfortunately, while tens of thousands of patients have benefited from CAR-T cell therapy, they represent a small fraction of those that could. The therapies alone carry a price tag near half a million dollars. When the cost of hospital care and the other treatments required along with administration of the T cell therapy are included, the total price tag can be well over $1,000,000. Some insurance companies do not cover the therapy, and the patient cost can still be significant for those that do provide some coverage. As a result, CAR-T cell therapies are financially inaccessible to many.
The key contributor to the high cost is the fact that current CAR-T cell therapies are autologous, patient-specific treatments in which the genetic modification of the patient’s cells is performed at a central manufacturing facility.2–8 Patients undergo apheresis, and the collected cells are shipped to the production site where they are processed, genetically modified, and expanded. The therapeutic cells are then shipped back to the patient for administration. This one-product-per-batch manufacturing scenario require scale-out rather than scale-up, precluding cost savings generally achieved with scale. In addition, frozen cells only survive for a limited time, and treatment centers must be located near manufacturing facilities.
The overall process is also highly complex and requires appropriately skilled/trained technicians and specialized facilities and equipment. The CAR receptors are introduced to patient T cells using viral vectors, typically γ-retroviral or lentiviral vectors, which themselves must be manufactured, typically via an often-inefficient transient transfection process. The vectors are then introduced to the patient’s T cells after they have been separated from the apheresis material and activated. This complexity has limited the number of centers offering CAR-T cell therapies.
This effectiveness of the transduction process is influenced by the quality of the T cells, which can vary significantly from patient to patient depending on their prior cancer treatments and the state of their disease. The semi-random nature of vector integration into the T cells can also result in variable expression levels from one patient to another. In addition, the in vitro (ex vivo) transduction of viral vectors into T cells can impact their immune function. This can, in some cases, result in limited persistence due to loss of functionality due to chronic antigen stimulation, metabolic dysregulation, and sustained inhibitory receptor signaling. Many patients also experience reduced responses to repeated treatments.
Furthermore, before receiving CAR-T cell therapy, patients must undergo lymphodepletion treatment to eliminate their existing T cells so they do not compete with the genetically modified cells, which can lead to serious infections. Patients must also wait for their treatments once their T cells have been collected and shipped to the production facility. If a manufacturing slot is available, the production process typically takes three to four weeks. In many cases, however, it can be several months before a manufacturing slot can be secured. In the meantime, patients generally require bridging chemotherapy. Unfortunately, because these patients have aggressive cancers that have failed to respond to other treatments, many become too ill to receive the treatment, and 25% or more die while waiting.
Patients that do receive CAR-T cell therapies must deal with potentially severe side effects, including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). CRS is also referred to as cytokine storm and is a systemic inflammatory response to overstimulation of the immune system. ICANS is a form of neurotoxicity that typically occurs after the development of CRS.
Finally, development of CAR-T cell therapies against solid tumors has been challenging due to the immunosuppressive nature of the tumor microenvironment, the inability of CAR T cells to persist within solid tumors, and the lack of single, tumor-specific antigens to target.
Allogeneic and Other Solutions
There are several approaches being pursued to overcome at least some of the challenges posed by current approach to commercial CAR-T cell therapies. A few different point-of-care solutions have been developed. For instance, researchers at the University of Pennsylvania reported development of a CAR-T cell production process that takes just one day.9 Several companies have also developed enclosed manufacturing systems that support production of CAR-T cell therapies at or near hospitals and other healthcare facilities.10
One approach to overcoming the cost, complexity, and time issues associated with autologous CAR-T cell therapies is the development of allogeneic treatments using healthy donor cells.3,4 These off-the-shelf alternatives eliminate the need for patient sample collection, single-batch manufacturing, and long waiting times. However, they present their own set of challenges. Lymphodepletion is still required, and CRS and ICANS remain potential issues. In addition, the donor cells must undergo genetic modification not only with the CAR but also to minimize the likelihood of rejection and the development of graft-versus-host reactions. These modifications can also result in alteration of the T cell phenotype and activity.
It is also worth noting that both autologous and allogeneic CAR cell therapies produced using other immune cells are being developed to overcome some of the limitations of CAR-T celltreatments.4,8 The two of particular interest are therapies using macrophages (CAR-M) and natural killer cells (CAR-NK). Macrophages are of interest because they are better at infiltration solid tumors, while NK cells present a lower risk of graft-versus-host disease (GvHD) and the systemic toxicities.
Many Potential In Vivo Advantages
Generating CAR-modified T cells within the body has become a promising approach to avoiding the issues associated with both autologous and allogeneic ex vivo CAR-T cells.2–4,6–8 The treatment would comprise the genetic material needed to generate the CAR within some type of delivery system that could be produced at large scale and be available off-the-shelf for administration in a manner similar to that for conventional biologics. There would be no need for collection and shipment of patient cells to a central (or even nearby) manufacturing site, thus no waiting time, as well as no concerns about GvHD. Avoiding in vitro cell modification also avoids reduction of cell functionality and variability due to the use of patient cells. Access to the multiple types of T cells present in the body may also lead to greater efficacy.
Lymphodepletion would also not be needed. Leaving the immune system intact would reduce the risk of severe infections and potentially the severity of side reactions, including CRS and ICANS, could be reduced, as well as the likelihood of CAR-target antigen escape, which can lead to poor outcomes. As a result, not only would the time, cost, and complexity of CAR-T cell therapy be dramatically reduced, but a greater number of patients would be eligible for these treatments.
One of the biggest challenges to achieving successful generation of CAR T cells in vivo is the need to selectively deliver the genetic material to only the target cells. This issue does not exist for ex vivo CAR-T cell therapies because the T cells are isolated from patient samples. Off-target delivery not only would reduce the efficacy of the therapy, but could lead to treatment resistance and or exacerbation of disease if tumor cells are transduced.
If viral vectors are used for delivery of the genes encoding the CAR, insertional mutagenesis may also be a concern. Regulatory agencies require that ex vivo CAR-T cell therapies produced using lentiviral vectors (vide infra) carry a warning about potential T cell malignancy, even though quality checks with these products can reduce the risk. No such quality checks would be possible with in vivo treatments.
Viral Vector, Messenger RNA (mRNA), and Single-Stranded DNA (ssDNA) Options
In vivo CAR-T cell therapies in clinical development use one of two delivery systems:3–5,8 lentiviral vectors (LVs) with DNA and lipid nanoparticles (LNPs) encapsulating mRNA.11 Adeno-associated viral (AAV) vectors and other nonviral methods leveraging electroporation of RNA and DNA, polymer nanoparticle delivery of mRNA, and LNP-based delivery of siRNA and ssDNA have also been investigated.4
To overcome the challenge of targeting the appropriate cells, both viral vector and mRNA-based in vivo CAR-T cell therapy developers leverage different ligands designed to bind to specific receptors on the surfaces of the target cells. These ligands may include monoclonal antibodies, antibody fragments (scFv or Fab), nanobodies, or designed ankyrin repeat proteins (DARPins).3,8 Other challenges remain for both approaches, however.
AAV vectors, while generally preferred for in vivo direct gene therapies, do not integrate stably into the genome and have not typically been used for CAR generation. LVs integrate into the cell genome and offer the potential for long-term expression and single-dose treatments but carry the risk of insertional mutagenesis and integration into non-target cells (e.g., germ cells, inhibitory immune cells, malignant B cells). In addition, advances in technology are better enabling efficient processes for large-scale manufacture of LV vectors, truly cost-effective solutions have yet to be implemented for commercial production.
For in vivo applications, LVs have been pseudo typed with a range of virus enveloped beyond vesicular stomatitis virus glycoprotein (VSV G), which has typically been used for ex vivo applications. Examples include measles virus, Nipah virus, Sindbis virus, and cocal fusion glycoprotein.3 The vectors are also engineered to express targeting ligands and improve T cell function.
LNP-based delivery of mRNA offers several advantages over production of CAR-T cells. Because there is no integration into the T cell genome, expression is of short duration and insertional mutagenesis concerns are avoided. In addition, LNP compositions can be customized to support targeted delivery and can accommodate multiple mRNA sequences simultaneously for multiantigen targeting, which would be highly beneficial when targeting solid tumors.5 Careful design of mRNA sequences can also affect regulation and expression in specific cell types. LNPs can also act as adjuvants, and their tailored design can help boost the innate immune activity of in vivo CAR-T therapies.
Transient expression, meanwhile, leads to the need for repeat dosing, which allows for real-time dose adjustment in response to occurrences of CRS and ICANs and improvement of efficacy if needed and helps limit the induction of exhaustion in CAR-bearing T cells. Us of other RNA species, such as self-amplifying or circular RNAs, may prove effective at increasing performance. Furthermore, cost-effective large-scale production of mRNA–LNPs has already been demonstrated, although improving targeting through ligand conjugation does add manufacturing complexity. Other concerns with mRNA–LNP approaches include the potential toxicity of the cationic/ionizable lipids used in LNP formulations, the lower transfection rates compared with viral vector delivery, and transient CAR expression.
With regard to other nonviral approaches, electroporation is not expected to be a viable clinical method due to tissue damage. While several polymer nanoparticle systems have been investigated for delivery of mRNA cargos, no therapy using this approach has yet received regulatory approval, and no current in vivo CAR therapies in early clinical studies use this technology. Transposon systems, like viral vectors, present a risk of insertional mutagenesis and require a delivery system such as LNPs, increasing complexity and cost.
One approach at an early development stage but showing significant promise is the in vivo modification of T cells using ssDNA, which can be delivered using LNPs or unique ssDNA-based nanocarriers.12,13 There are a few advantages to using ssDNA, including superior cellular uptake, its immunosilence, and the ability to achieve durable expression without concern of insertional mutagenesis. Structural stability elements and chemical modifications can also be incorporated to increase resistance to nuclease degradation and support intracellular transport. In vitro studies have demonstrated expression in both proliferating and arrested cells and the suitability of ssDNA for repeat dosing if required (such as for treatment of chronic conditions).
Targeted delivery can be achieved through encapsulation in appropriately formulated LNPs and through conjugation through with biodegradable polymers. Another approach is to encapsulate the ssDNA transgene into nanoscale programmable DNA frameworks that are formed with precise spatial control and functionality to support highly targeted and controlled delivery. These self-folding nanocarriers have enhanced structural stability and reduced immunogenicity, allow fine-tuning of gene expression, and enable selective binding to cellular receptors through ligand functionalization. Furthermore, cost-effective, scalable manufacturing of the customized short and long ssDNA strands require for the payload (CAR transgene) and nanocarrier framework has been demonstrated.
Promising Preclinical Results
Many preclinical studies of various types of in vivo CAR-T cell therapies have provided encouraging results. In many cases, efficacy similar to that observed with ex vivo therapies has been observed.1,3–5,8,12–15 Researchers have explored in vivo CAR cells targeting not only various blood cancers but also solid tumors and non-oncology diseases, such as autoimmune disorders and cardiac-related fibrosis. CARs have been investigated that leverage LV and AAV vectors and various DNA and RNA species delivered using both LNPs and polymeric nanoparticles, as well as programmable DNA nanocarriers.
Several Early Clinical Trials Now Underway
Interius BioTherapeutics, Umoja BioPharma, EsoBioTech (acquired by AstraZeneca for $1 billion in 2024), Kelonia Therapeutics (collaborating with Astellas Pharma), and Vyriad (partnering with Novartis) all have elected to use LV vectors as their delivery approach. Companies developing mRNA-based products include Capstan Therapeutics, Myeloid Therapeutics, Orbital Therapeutics, Orna Therapeutics, Tessera Therapeutics, and Moderna. Several of these firms have or are planning to launch early-phase clinical trials, and according to GlobalData there are 12 in vivo CAR-T assets in preclinical trials and 14 more at the discovery stage targeting blood cancers and solid tumors.7
Other companies of note include Azalea Therapeutics, which was co-founded by Jennifer Doudna, one of the scientists who developed CRISPR-based gene editing, and is exploring CRISPR-based in vivo CAR-T cell therapies,2 and CPTx, which is developing ssDNA-based in vivo CAR-T cell therapies leveraging both LNP- and DNA-based nanocarrier delivery systems.12,13
Interius BioTherapeutics dosed the first patient in its Australian phase I trial of INT2104 for the treatment of B cell malignancies in 2024. The lentiviral-based therapy delivers a CAR transgene that target CD20+ B cells to CD7+ T and NK cells.6 The technology avoids off-target integration and insertional mutagenesis by using an HIV integrase. Results in small and large animal preclinical cancer models demonstrated the effectiveness of the therapy. A second phase I trial was approved in Europe in early 2025.16 Interius believes its in vivo CAR-T cell therapies could be delivered to patients at a cost comparable to that of conventional biologics.
Umoja Biopharma, which signed a deal with AbbVie in January 2024,17 received approval of its IND for lead candidate UB-VV111 from FDA in 2024 for a phase I dose-escalation study in hematologic malignancies. Produced using Umoja’s VivoVecTM gene delivery platform leveraging third generation lentiviral vectors and a T cell targeting and activation surface complex, this in vivo therapy generates CD19-directed CAR-T cells.18 In non-human primates, Umoja showed that UB-VV111 resulted in a longer, more sustained responses than benchmarked models prepared using conventional ex vivo processes.11 Notably, the company has its own manufacturing facility in Louisville, Colorado, which it says supports faster commercialization at lower cost and risk.
In January 2025, EsoBioTech dosed its first patient with ESO-T01 for treatment of relapsed/refractory multiple myeloma participating in its early phase I China-based trial.18 This treatment uses EsoBioTec’s ENaBL, a third-generation immune-shielded cell specific lentiviral vector platform, and a BCMA CAR-T transgene developed by Pregene Biopharma. The treatment is administered in a single intravenous dose that takes less than 10 minutes. In July 2025, the company reported that all four of the initial patients responded to treatment as reported by the company in July 2025, with two having their cancer completely resolved.19 All of the patients developed some level of CRS, and the patient with the most cancerous tissue also developed ICANS.
The first patient in Capstan Therapeutics’ Australian phase I trial to evaluate the safety, tolerability, and pharmacodynamic activity of CPTX2309, Capstan’s lead mRNA-based anti-CD19 in vivo CAR-T candidate for the treatment of B cell–mediated autoimmune disorders in healthy volunteers took place in June 2025.20 Preclinical studies in non-human primates showed the therapy generates CD8+ CAR T cells, achieves profound B cell depletion in blood and tissues, and supports repopulation with predominantly naïve B cells. Capstan was founded by CAR-T pioneers Bruce Levine and Carl June, along with Nobel Prize awardee for mRNA vaccines Drew Weissman.2
Myeloid Therapeutics has two intravenously delivered mRNA–LNP-based in vivo CAR-T cell therapy candidates in the clinic. MT-302 is designed to generate a TROP2-targeted CAR-myeloid cells that trigger an adaptive immune response to metastatic epithelial tumors.21,22 The first patients were dosed in September 2023. MT-303 targets GPC3, which is highly expressed in the majority of liver cancers (e.g., hepatocellular carcinoma, HCC).23 The ATAK™ Receptor CARs comprise novel combinations of myeloid signaling domains coded within a simple mRNA and presentation of tumor neoantigen to stimulate T cells, leading to a more comprehensive immune response.
Time will Tell, but Hopes are High
The field of in vivo CAR therapies is just emerging, and it is too soon to tell which technology may prove to be most effective. It may turn out that the use of LV vectors is bests suited for certain applications and mRNA and ssDNA for others. Developers will also need to be prepared to compete with newer ex vivo solutions, most notably those manufactured in small, closed systems designed for use at the point of care and to eliminate many of the logistics and manufacturing complexity issues associated with currently approved treatments.
It can be hoped, though, that if in vivo CAR therapies are successful, they will help bring the price of CAR therapies down to a level that supports much broader patient access from many perspectives, treatment at earlier stages of disease, and effective solutions for many more indications affecting large numbers of people, such as rheumatoid arthritis, systemic lupus, multiple sclerosis, and various forms of fibrosis.
References
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