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Dual-Targeted LNPs Enable Nonviral DNA Delivery and In Vivo CAR-T Generation

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2026-09-24 10:51

In a 2025 study published in the *Journal for ImmunoTherapy of Cancer*, Bimbo and colleagues reported a dual-targeted lipid nanoparticle platform that generated persistent, functional CAR-T cells directly in vivo. Following a single intravenous administration, the platform produced sustained CAR expression, controlled tumor growth, and improved survival in humanized mouse models of B-cell leukemia.

How did the researchers achieve stable CAR expression without using a viral vector? And why were both CD3 and CD7 targeting required to deliver the genetic payload to T cells?

 

Experimental Design and Key Findings

1）Dual CD3/CD7 Targeting Supports T-Cell Delivery

The researchers first developed an LNP formulation capable of carrying both reporter mRNA and minicircle DNA. The particles were left untargeted or modified with an anti-CD7 nanobody, an anti-CD3 single-chain variable fragment, or both ligands.

The two ligands served different functions. CD7 targeting improved particle uptake and supported selective mRNA expression in T cells, while CD3 targeting activated resting T cells and enabled functional DNA delivery without separate CD3/CD28 stimulation. The dual-targeted formulation produced the highest minicircle DNA expression in both resting and preactivated T cells.

In mixed peripheral blood mononuclear cell cultures, adding CD7 targeting also improved the specificity of functional mRNA delivery. Some uptake and expression occurred in natural killer cells, consistent with their expression of CD7.

These findings help explain the value of the dual-targeting design. The anti-CD3 binder acts as both a targeting and activating component, creating a cellular state that supports DNA expression. The anti-CD7 binder improves cell recognition and internalization. However, the observed selectivity should be interpreted as preferential rather than exclusive T-cell delivery.

2）Minicircle DNA and SB100x mRNA Enable Stable CAR Expression

NCtx was designed to codeliver minicircle DNA encoding the CAR and SB100x transposase mRNA. Transient expression of the transposase allows the CAR cassette to integrate into the T-cell genome, supporting more durable expression than an mRNA-only payload.

NCtx-CD19 generated CAR expression in primary T cells that persisted for at least 20 days. The resulting cells demonstrated antigen-specific activity against CD19-positive target cells, and stable CAR expression was also achieved in resting T cells without external activation.

These findings show that targeted LNPs can support persistent, nonviral CAR-T cell engineering. However, genomic integration also requires careful evaluation of insertion sites, long-term expression, and potential insertional risks.

3）Single-Dose Treatment Generates CAR-T Cells in Humanized Mice

The researchers next evaluated NCtx-CD19 in a human peripheral blood mononuclear cell-engrafted mouse model of Nalm6 leukemia. A single intravenous dose of 0.5 mg/kg generated increasing numbers of circulating CAR-T cells, reaching an average of 122 CAR-positive T cells per microliter of blood on day 22.

At the end of the study, approximately 24% of T cells in the spleen and bone marrow expressed the CAR. Mice treated with NCtx-CD19 showed tumor regression and improved survival compared with animals receiving the vehicle control.

Dose-de-escalation experiments produced complete tumor control at 40 μg/kg and partial control at 4 μg/kg. CAR-positive T cells were detected in the blood, spleen, and bone marrow at both doses, indicating that the platform remained active across a broad dose range in this model.

These findings suggest that one administration can initiate sustained in vivo CAR-T cell expansion and antitumor activity. However, the magnitude of expansion may depend on the number and condition of the T cells available for transfection.

4）A Dual CD19/CD22 CAR Was Evaluated in a Second Model

To test whether NCtx could accommodate a more complex payload, the researchers replaced the CD19 CAR with a dual CD19/CD22 CAR.

In CD34+ hematopoietic stem cell-engrafted mice, a single 50 μg/kg dose generated circulating CAR-T cells that continued to increase through day 40. Seven of 12 treated mice showed complete tumor elimination on day 42, and 75% survived to the end of the study. By comparison, all vehicle-treated control animals reached the humane endpoint by day 28.

The CAR-positive cells showed no clear increase in PD-1 expression relative to CAR-negative T cells and shifted toward a central memory phenotype over time. Treatment response also appeared to correlate with the number of circulating T cells before administration, suggesting that the availability of target cells may influence in vivo engineering efficiency.

This second model strengthens the proof of concept and demonstrates that the platform can deliver a more complex CAR construct. Nevertheless, the findings remain preclinical. The study did not evaluate NCtx in non-human primates or patients and was not designed to establish systemic safety.

 

Implications for In Vivo Cell Engineering

By combining minicircle DNA with transposase mRNA, NCtx enabled stable CAR expression without relying on a viral vector. The results also suggest that the platform can accommodate more complex payloads, including a dual CD19/CD22 CAR, providing another strategy for applications that may require sustained CAR-T cell activity.

However, stable genomic integration introduces different development considerations from transient mRNA delivery. Further studies are needed to evaluate integration sites, off-target delivery, clonal expansion, long-term safety, and manufacturing consistency. The study therefore provides a promising mouse proof of concept, but not yet evidence of clinical efficacy or safety.

 

Supporting In Vivo CAR-T Research

As CD3 and CD7 play central roles in the targeting strategy described in this study, Levostar offers off-the-shelf CD3- and CD7-related products to support early-stage target validation and in vivo CAR-T delivery research.

For projects requiring other targets or formulation designs, Levostar also provides custom antibody-conjugated LNP development, including antibody selection, LNP formulation, conjugation, particle characterization, and functional evaluation.

Reference

Read the original article: T cell-specific non-viral DNA delivery and in vivo CAR-T generation using targeted lipid nanoparticles

 

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