Jaime Fernández Bimbo

Background Ex vivo chimeric antigen receptor (CAR)-T therapies have revolutionized cancer treatment. However, treatment accessibility is hindered by high costs, long manufacturing times, and the need for specialized centers and inpatient care. Strategies to generate CAR-T cells in vivo have emerged as a promising alternative that could bypass CAR-T manufacturing bottlenecks. Most current in vivo CAR-T approaches, while demonstrating encouraging preclinical efficacy, rely on transient messenger RNA (mRNA) delivery or viral vectors which both have limitations in terms of efficiency, durability, and scalability. To address these challenges, we developed a novel DNA-based targeted lipid nanoparticle (LNP) which we termed NCtx. Methods Minicircle DNA (mcDNA) encoding a CAR construct and SB100x transposase mRNA were encapsulated within a novel lipid formulation which was functionalized with T cell-specific anti-CD7 and anti-CD3 binders. In vitro, we evaluated T cell specificity, mcDNA and mRNA transfection efficiency, transposon-mediated CAR integration and functionality of the resulting CAR-T cells. In vivo efficacy was assessed in peripheral blood mononuclear cell and CD34 + stem cell humanized murine xenograft models of B cell leukemia. Results In vitro, NCtx displayed high specificity and transfection efficiency with both mcDNA and mRNA in primary T cells. Transposase mRNA facilitated genomic integration of the CAR gene, leading to the generation of stable CAR-T cells that exhibited antigen-specific cytotoxicity and cytokine release. In vivo, a single intravenous dose of NCtx induced robust CAR-T cell generation resulting in effective tumor control and significantly improved survival in two distinct xenograft models. Conclusions Our findings demonstrate for the first time that targeted LNPs can be employed for efficient DNA delivery to T cells in vitro and in vivo. We show that when combined with transposase technology, this LNP-based system can efficiently generate stable CAR-T cells directly in vivo, inducing potent and durable antitumor responses. NCtx represents a novel non-viral gene therapy vector for in vivo CAR-T therapy, offering a scalable and potentially more accessible alternative to traditional approaches in CAR-T cell generation.

BACKGROUND/OBJECTIVES: Glioblastoma is the most common and lethal primary brain tumor. Patients often suffer from tumor- and treatment induced vasogenic edema, with devastating neurological consequences. Intracranial edema is effectively treated with dexamethasone. However, systemic dexamethasone requires large doses to surpass the blood brain barrier in therapeutic quantities, which is associated with significant side effects. The aim of this study was to investigate a biodegradable, dextran-hydroxyethyl methacrylate (dex-HEMA) based hydrogel, containing polymeric micelles loaded with dexamethasone and liposomes encapsulating dexamethasone phosphate for localized and prolonged delivery. METHODS: -p(HPMA-Bz)) micelles were loaded with dexamethasone and characterized. The dexamethasone micelles, together with dexamethasone phosphate liposomes, were dispersed in an aqueous dex-HEMA solution followed by radical polymerization using a photoinitiator in combination with light. The kinetics and mechanisms of drug release from this hydrogel were determined. RESULTS: The diameter of the nanoparticles was larger than the mesh size of the hydrogel, rendering them immobilized in the polymer network. The micelles immediately released free dexamethasone from the hydrogel for two weeks. The dexamethasone phosphate loaded in the liposomes was not released until the gel degraded and intact liposomes were released, starting after 15 days. The different modes of release result in a biphasic and sequential release profile of dexamethasone followed by dexamethasone phosphate liposomes. CONCLUSIONS: The results show that this hydrogel system loaded with both dexamethasone polymeric micelles and dexamethasone phosphate loaded liposomes has potential as a local delivery platform for the sequential release of dexamethasone and dexamethasone phosphate, for the intracranial treatment of glioblastoma associated edema.

<h3>Background</h3> <i>Ex-vivo</i> modification of immune cells to express Chimeric Antigen Receptor (CAR) has shown tremendous clinical and commercial success as a cancer treatment. Despite its widespread adoption, <i>ex-vivo</i> CAR-T approaches face challenges such as soaring production costs, extended timelines, inherent toxicity risks and operational complexities. These challenges limit the accessibility of CAR-T treatment and highlight the need for improved methods. Here, we demonstrate a novel non-viral vector capable of permanently modifying T-cells to express CAR by direct intra venous injection or short transfection in whole blood (extra-corporeal <i>transfection)</i>. We will discuss the capabilities of this vector <i>in vitro</i> and <i>in vivo</i>. We believe this cost-effective and efficient vector has the potential to solve the challenges associated with CAR-T generation resulting in an increased accessibility of CAR-T treatment. <h3>Methods</h3> We developed a non-viral vector consisting of a targeted lipid nanoparticle formulation (tLNP) loaded with minicircle DNA encoding for CAR, mRNA encoding for transposase and coated with both T cell targeting and T cell activating proteins. This novel vector was used to engineer CAR-T cells both in whole blood and <i>in vivo</i>. CAR expression was analyzed using flow cytometry and functionality was assessed using target cell killing assays. The vector was then tested <i>in vivo</i> using a huPBMC NSG mouse model of human leukemia. CAR expression in blood and lymphoid tissues was measured using flow cytometry and tumor outgrowth was assessed using bioluminescence imaging of luciferase signal derived from luciferase positive Nalm-6 leukemia cells. Results tLNPs activated resting primary T cells in whole blood, enabling their transfection with minicircle DNA encoding for a CAR construct without exogenous activation. Transposase encoded by mRNA co-loaded into the tLNP facilitated stable integration of this construct, resulting in the generation of fully functional CAR-T cells. <i>In vivo</i>, a single administration of tLNP resulted in the generation of a high number of persistent circulating CAR-T cells. These CAR-T cells were functional as evidenced by a significant reduction in tumor growth and significantly extended survival in treated animals compared to vehicle control mice. <h3>Conclusions</h3> Here we demonstrate successful non-viral CAR-T generation both <i>in vitro</i> and <i>in vivo</i> using a tLNP vector. This approach shows exciting potential and could significantly increase the accessibility of CAR-T treatments in the future.