Molecular Therapy最新文献

Transgene expression in stem cells is a powerful means of regulating cellular properties and differentiation into various cell types. However, existing vectors for transgene expression in stem cells suffer from limitations such as the need for genomic integration, the transient nature of gene expression, and the inability to temporally regulate transgene expression, which hinder biomedical and clinical applications. Here we report a new class of RNA virus-based vectors for scalable and integration-free transgene expression in mouse embryonic stem cells (mESCs). The vector is equipped with a small molecule-regulated riboswitch and a drug selection marker that allow temporal regulation of transgene expression and stable maintenance of the vector in proliferating stem cells. We demonstrated the utility of the vector by maintaining the pluripotency of mESCs in a differentiation induction medium by expressing Nanog and inducing myogenic differentiation by triggering Myod1 expression, without altering the mESC genome.

Pancreatic cancer (PC) is one of the most lethal digestive system tumors. Claudin18.2 is highly expressed in PC tissue and could serve as a suitable target for CAR-T therapy. In the present study, we reported the utilization of tEGFR-expressing claudin18.2-targeted CAR-T cells to treat 3 patients with advanced PC. Intriguingly, all 3 patients achieved disease remission after CAR-T cell infusion, with 1 complete remission (CR) and 2 partial remissions (PR). However, gastric mucosal injury was observed, which was recognized as on-target off-tumor toxicity (OTOT) and may be due to the expression of claudin18.2 on normal gastric tissues. To control the severe OTOT in patient 3, cyclophosphamide and cetuximab were administered to deplete CAR-T cells, and they successfully controlled OTOT. Single cell transcriptome and TCR sequencing revealed the objective alterations of CAR-T cell clones after cetuximab treatment. Collectively, the present study showed the robust anti-tumor activity of claudin18.2-targeted CAR-T cells against PC, and reported the feasibility of antibody-dependent safety switch strategy to control the OTOT caused by CAR-T cells in patients. Our study may pave the way for the development of a novel strategy to treat patients with advanced PC in the future.

Dorsal root ganglion (DRG) toxicity has been consistently reported as a potential safety concern after delivery of adeno-associated viruses (AAVs) containing gene-replacement vectors but has yet to be reported for RNAi-based vectors. Here, we report DRG toxicity after AAV intra-CSF delivery of an RNAi expression construct-artificial microRNA targeting superoxide dismutase 1 (SOD1)-in non-human primates (NHPs) and provide evidence that this can be recapitulated within mice. Histopathology evaluation showed that NHPs and mice develop DRG toxicity after AAV delivery, including DRG neuron degeneration and necrosis and nerve-fiber degeneration that were associated with increases in cerebrospinal fluid (CSF) and serum phosphorylated neurofilament heavy chain (pNF-H). RNA-sequencing analysis of DRGs showed that dysregulated pathways were preserved between NHPs and mice, including increases in innate/adaptive immune responses and decreases in mitochondrial- and neuronal-related genes, following AAV treatment. Finally, endogenous miR-21-5p was upregulated in DRGs of AAV-treated NHPs and mice. Increases in miR-21-5p were also identified within the CSF of NHPs, which significantly correlated with pNF-H, implicating miR-21-5p as a potential biomarker of DRG toxicity in conjunction with other molecular analytes. This work highlights the importance of assessing safety concerns related to DRG toxicity when developing RNAi-based AAV vectors for therapeutic purposes.

The demand for RNA-based therapeutics is increasing globally. However, their use is hampered by the lack of safe and effective delivery vehicles. Here, we developed technologies for highly efficient delivery of RNA cargo into programmable extracellular vesicle-mimetic nanovesicles (EMNVs) by fabricating hybrid EMNV-liposomes (Hybs). Tissue targeting is endowed by highly efficient genetic platforms based on truncated CD63 (ΔCD63) or PTGFRN proteins. For the first time we reveal their efficiency in functionalizing EMNVs, resulting in >10-fold enhancement of nanoparticle internalization in vitro and >2-fold in vivo. RNA delivery using Hybs demonstrated efficiency of >85% in human and mouse cell lines. Comparative analysis of EMNVs and Hyb lysosome colocalization and stability suggested that Hybs enter the lysosomal compartment and escape over time, whereas EMNVs primarily avoid it. Finally, we used these technologies to generate liver-targeting Hybs loaded with therapeutic small interfering RNA and demonstrated the robust efficiency of this system in vitro and in vivo. These technologies can be adapted for manufacturing a wide range of next-generation vehicles for highly efficient, safe delivery of RNA into desired organs and tissues for therapeutic and prophylactic applications.

Primary hyperoxaluria type 1 (PH1) is a severe genetic metabolic disorder caused by mutations in the AGXT gene, leading to defects in enzymes crucial for glyoxylate metabolism. PH1 is characterized by severe, potentially life-threatening manifestations due to excessive oxalate accumulation, which leads to calcium oxalate crystal deposits in the kidneys and, ultimately, renal failure and systemic oxalosis. Existing substrate reduction therapies, such as inhibition of liver-specific glycolate oxidase (GO) encoded by HAO1 using siRNA or CRISPR-Cas9 delivered by adeno-associated virus, either require repeated dosing or have raised safety concerns. To address these limitations, our study employed lipid nanoparticles (LNPs) for CRISPR-Cas9 delivery to rapidly generate a PH1 mouse model and validate the therapeutic efficacy of LNP-CRISPR-Cas9 targeting the Hao1 gene. The LNP-CRISPR-Cas9 system exhibited efficient editing of the Hao1 gene, significantly reducing GO expression and lowering urinary oxalate levels in treated PH1 mice. Notably, these effects persisted for 12 months with no significant off-target effects, liver-induced toxicity, or substantial immune responses, highlighting the approach's safety and specificity. Furthermore, the developed humanized mouse model validated the efficacy of our therapeutic strategy. These findings support LNP-CRISPR-Cas9 targeting HAO1 as a promising and safer alternative for PH1 treatment with a single administration.

Diabetic kidney disease (DKD) is the leading cause of end-stage kidney diseases resulting in enormous socio-economic burden. Accumulated evidence has indicated that C-reactive protein (CRP) exacerbates DKD by enhancing renal inflammation and fibrosis through TGF-β/Smad3 signaling. NLRP3 inflammasome is the key sensor contributing to renal inflammation. However, whether CRP enhances inflammation in DKD via NLRP3 inflammasome-related pathway remains unknown. In this study, we demonstrate that CRP promotes DKD via Smad3-mediated NLRP3 inflammasome activation as mice overexpressing human CRP gene exhibits accelerated renal inflammation in diabetic kidneys, which is associated with the activation of Smad3 and NLRP3 inflammasomes. In contrast, blockade of CPR signaling with a neutralizing anti-CD32 antibody attenuates CRP-induced activation of Smad3 and NLRP3 in vitro. Importantly, genetic deletion or pharmacological inhibition of Smad3 also mitigates CRP-induced activation of NLRP3 in diabetic kidneys or in high glucose-treated cells. Mechanistically, we reveal that Smad3 binds to the NLRP3 gene promoter, which is enhanced by CRP. Taken together, we conclude that CRP induces renal inflammation in DKD via a Smad3-NLRP3 inflammasome-dependent mechanism. Thus, targeting CRP or Smad3-NLRP3 pathways may be a new therapeutic potential for DKD.

Glioblastoma (GB), the most aggressive tumor of the central nervous system (CNS), has poor patient outcomes with limited effective treatments available. MicroRNA-21 (miR-21(a)) is a known oncogene, abundantly expressed in many cancer types. miR-21(a) promotes GB progression, and lack of miR-21(a) reduces the tumorigenic potential. Here, we propose a single adeno-associated virus (AAV) vector strategy targeting mmu-miR-21a using the Staphylococcus aureus Cas9 ortholog (SaCas9) guided by a single-guide RNA (sgRNA). Our results demonstrate that AAV8 is a well-suited AAV serotype to express SaCas9-KKH/sgRNA at the tumor site in an orthotopic GB model. The SaCas9-KKH induced a genomic deletion, resulting in lowered mmu-miR-21a levels in the brain, leading to reduced tumor growth and improved overall survival. In this study, we demonstrated that disruption of genomic mmu-miR-21a with a single AAV vector influenced glioma development, resulting in beneficial anti-tumor outcomes in GB-bearing mice.

Despite a dramatic increase in ischemic stroke incidence worldwide, effective therapies for attenuating sequelae of cerebral infarction are lacking. This study investigates the use of human mesenchymal stem cells (hMSCs) induced toward glia-like cells (ghMSCs) to ameliorate chronic sequelae resulting from cerebral infarction. Transcriptome analysis demonstrated that ghMSCs exhibited astrocytic characteristics, and assessments conducted ex vivo using organotypic brain slice cultures demonstrated that ghMSCs exhibited superior neuroregenerative and neuroprotective activity against ischemic damage compared to hMSCs. The observed beneficial effects of ghMSCs were diminished by pre-treatment with a CXCR2 antagonist, indicating a direct role for CXCR2 signaling. Studies conducted in rats subjected to cerebral infarction demonstrated that ghMSCs restored neurobehavioral functions and reduced chronic brain infarction in a dose-dependent manner when transplanted at the subacute-to-chronic phase. These beneficial impacts were also inhibited by a CXCR2 antagonist. Molecular analyses confirmed that increased neuroplasticity contributed to ghMSCs' neuroregenerative effects. These data indicate that ghMSCs hold promise for treating refractory sequelae resulting from cerebral infarction by enhancing neuroplasticity and identify CXCR2 signaling as an important mediator of ghMSCs' mechanism of action.