Molecular Therapy最新文献

Direct cardiac reprogramming has emerged as a promising therapeutic strategy to remuscularize injured myocardium. This approach converts non-contractile fibroblasts to induced cardiomyocytes (iCMs) that spontaneously contract, yet the intrinsic metabolic requirements driving cardiac reprogramming are not fully understood. Using single-cell metabolic flux estimation and flux balance analysis, we characterized the metabolic heterogeneity of iCMs and identified fatty acid oxidation (FAO) as a critical factor in iCM conversion. Both pharmacological and genetic inhibition of FAO impairs iCM generation. We further identified stearoyl-coenzyme A desaturase 1 (SCD1) as a metabolic switch that suppresses iCM reprogramming. Mechanistically, Scd1 knockdown activates PGC1α and PPARβ signaling, enhancing FAO-related gene expression and mitochondrial biogenesis, thereby improving reprogramming efficacy. Pharmacological manipulations targeting SCD1, PGC1α, and the PPARβ signaling axis further improved iCM generation and mitochondrial function. Our findings collectively highlight FAO as a key determinant of iCM fate and offer new therapeutic avenues for advancing reprogramming strategies.

Enhancer of zeste homolog 2 (EZH2) catalyzes trimethylation of histone H3 at lysine 27 (H3K27me3), which promotes heterochromatin formation and gene silencing. Expression of EZH2 is frequently elevated in various malignancies, including hepatocellular carcinoma (HCC). Silencing of EZH2 has been pursued as a promising strategy to halt cancer progression. Here, we identified antisense oligonucleotides (ASOs) that efficiently silence EZH2 through promoting skipping of its exon 14, an exon encoding part of the essential CXC domain, increasing production of an internally shortened isoform that exerts dominant negative effect on the full-length EZH2. A lead ASO, hybridizing to an exonic splicing enhancer element bound by SRSF3, robustly promoted exon 14 skipping not only in cultured human HCC cell lines but also in mouse peripheral tissues after systemic administration, leading to dramatic reduction of EZH2 and H3K27me3 levels. The lead ASO potently inhibited HCC cell proliferation through multiple mechanisms including enhanced apoptosis, cell-cycle arrest, and reversed epithelial-mesenchymal transition, which is likely attributable to the suppression of diverse cancer-related pathways. In an orthotopic xenograft HCC mouse model, ASO treatment repressed tumor growth, improved tissue phenotype, and extended the median survival. Our data highlight therapeutic potential of the lead exon-skipping ASO in treating HCC.

Uncontrollable bleeding and tissue defects caused by trauma are significant clinical issues. Apoptotic vesicles (apoVs) derived from mesenchymal stem cells (MSCs) have shown promise for hemostasis and tissue regeneration, but their clinical safety and efficacy remain unverified. We investigated the procoagulant and regenerative function of lyophilized MSC-derived apoVs (MSC-apoVs) using in vitro experiments and in vivo rat models. In addition, we conducted a double-blind, randomized, self-controlled clinical trial to evaluate the safety and efficiency of lyophilized MSC-apoVs for hemostasis and bone regeneration following extraction of impacted mandibular third molars. We show that lyophilized MSC-apoVs maintain their procoagulant and regenerative functions after storage at 4°C for 3 months and upregulate tripartite motif containing 71 to activate the extracellular signal-regulated kinase signaling pathway. Furthermore, among the 43 enrolled subjects, 39 patients completed all follow-ups and 4 patients were lost to contact. All 39 patients tolerated MSC-apoVs well, with no serious adverse events or abnormal blood test results observed. The MSC-apoV group exhibited shortened hemostatic time and accelerated alveolar bone regeneration compared with the control group. This is the first clinical study to demonstrate that apoVs are safe, well tolerated, and effective as a cell-free biological therapy for hemostasis and bone regeneration.

Chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of hematologic malignancies. However, it continues to encounter significant obstacles, including treatment relapse and limited efficacy in solid tumors. While effector T cells exhibit robust cytotoxicity, central memory T cells and stem cell-like T cells are essential for in vivo expansion, long-term survival, and persistence. Strategies such as genetic engineering to enhance CAR-T cell efficacy and durability are often accompanied by increased safety risks, which not only raise regulatory approval thresholds but also escalate CAR-T production costs. In contrast, optimizing ex vivo manufacturing conditions represents a more straightforward and practical approach, offering the potential for rapid application to commercially approved CAR-T products and enhancement of their clinical outcomes. This review examines several factors that have been shown to improve T cell memory phenotype and in vivo cytotoxic activity, including cytokines, electrolytes, signaling pathway inhibitors, metabolic modulators, and epigenetic agents. The insights provided will guide the optimization of CAR-T cell industrial production. Furthermore, considerations for selecting appropriate conditions are discussed, balancing effectiveness, cost-efficiency, safety, and regulatory compliance while addressing current challenges in the field.

Aging is a major risk factor for pathologies including sarcopenia, osteoporosis, and cognitive decline, which bring suffering, disability, and elevated economic and social costs. Therefore, new therapies are needed to achieve healthy aging. The protein Klotho (KL) has emerged as a promising anti-aging molecule due to its pleiotropic actions modulating insulin, insulin-like growth factor-1, and Wnt signaling pathways and reducing inflammatory and oxidative stress. Here, we explored the anti-aging potential of the secreted isoform of this protein on the non-pathological aging progression of wild-type mice. The delivery of an adeno-associated virus serotype 9 (AAV9) coding for secreted KL (s-KL) efficiently increased the concentration of s-KL in serum, resulting in a 20% increase in lifespan. Notably, KL treatment improved physical fitness, related to a reduction in muscle fibrosis and an increase in muscular regenerative capacity. KL treatment also improved bone microstructural parameters associated with osteoporosis. Finally, s-KL-treated mice exhibited increased cellular markers of adult neurogenesis and immune response, with transcriptomic analysis revealing induced phagocytosis and immune cell activity in the aged hippocampus. These results show the potential of elevating s-KL expression to simultaneously reduce the age-associated degeneration in multiple organs, increasing both life and health span.

Chimeric antigen receptor (CAR)-based immune cell therapy involves genetically engineering immune cells, such as T cells and natural killer (NK) cells, to express CARs that can specifically recognize target antigens. This modification enables T/NK cells to selectively eliminate tumor cells following adoptive transfer. One common approach to stably integrate CARs into the genome of T/NK cells is through retroviral or lentiviral vectors. However, these vectors mediate semi-random gene integration, posing risks such as oncogenic mutations, gene silencing, and variable CAR expression levels. Targeted integration of CAR genes into the specific genomic locus could overcome these limitations, but identifying the optimal integration sites to maximize the safety and efficacy of CAR-T/NK cell products remains a critical question. Improper integration sites may disturb the endogenous genes surrounding the integration sites, raising safety concerns. Additionally, regulatory elements at the integration sites, such as promoters, can influence the expression level of CAR genes, thus affecting the efficacy of CAR-T/NK cells. In this review, we summarized current strategies for selecting integration sites and promoters in the engineering of CAR-T/NK cells to achieve potent anti-tumor efficacy in preclinical studies.

Chimeric antigen receptor (CAR) T cells are effective cancer therapies, particularly in indications with high, stable, and tumor-specific antigen expression. Other settings may require improved targeting sensitivity, controllable targeting selectivity, and/or additional potency enhancements to achieve robust efficacy. Here, we describe a novel receptor architecture called RESET (rapamycin-enabled, switchable endogenous T cell receptor) that combines (1) cell surface antigen targeting, (2) small-molecule regulation, and (3) the signaling proficiency and inherent sensitivity of native T cell receptors. RESET-T cells outperformed both constitutive and drug-regulated CAR-T cells and show hallmarks of TCR activation that suggest improved fidelity to native T cell responses. Pharmacological control then increases safety through toggling T cell activation between active and resting states and may mitigate T cell exhaustion caused by continuous antigen exposure. This convergence of drug-regulated targeting and natural immune receptor signal transduction may better replicate the kinetics and physiology of a classical T cell response and potentiate more successful and safer immunotherapies.

In spite of adequate seizure control, approximately 75% of pyridoxine-dependent epilepsy (PDE) patients with ALDH7A1 mutation still suffer from intellectual disability. However, the mechanisms underlying brain dysfunction in PDE patients are still unknown even when seizure control is achieved. In this study, we show that mice with specific deletion of Aldh7a1 from astrocytes, but not neurons, exhibit PDE, and have defective dendritic spine development and cognitive impairment when seizure occurrence is well controlled. Mechanistically, ALDH7A1 deficiency leads to dysregulation of astrocyte-derived matrix gla protein (MGP), one of the vitamin K-dependent proteins, thereby impairing dendritic spine development and synaptic transmission. Notably, supplementation of menaquinone-7, a form of vitamin K, promotes MGP activation and rescues defective dendritic spine development, abnormal synaptic transmission, and cognitive impairment in Aldh7a1-deficient mice. Therefore, our findings not only unravel the important role of ALDH7A1 in astrocytes contributing to the pathogenesis of PDE, but also provide a potential therapeutic intervention to ameliorate cognitive impairment in PDE beyond pyridoxine treatment.

Triple-negative breast cancer (TNBC) remains one of the most challenging subtypes of breast cancer to treat due to a lack of effective targeted therapies. Chimeric antigen receptor (CAR)-T cells hold promise, but their efficacy in solid tumors is often limited by on-target/off-tumor toxicities. Through comprehensive bioinformatic analysis of public RNA and proteomic data, we identified zona pellucida glycoprotein 4 (ZP4) as a novel target for TNBC. ZP4 RNA and protein were detected in a subset of TNBC patient samples and patient-derived xenograft (PDX) models, with expression otherwise restricted to oocytes. We generated 89 ZP4-specific novel monoclonal antibodies and used the single-chain variable fragment (scFv) antigen binding domains from the top three candidates to engineer CAR constructs. ZP4 CAR-T cells demonstrated efficacy against ZP4-expressing TNBC cells and PDX models. Additionally, we found that variations in the scFv antigen binding domain significantly influence CAR-T cell function.