Molecular Therapy最新文献

Optogenetic therapy is a promising vision restoration method where light-sensitive opsins are introduced to the surviving inner retina following photoreceptor degeneration. The cell type targeted for opsin expression will likely influence the quality of restored vision. However, a like-for-like preclinical comparison of visual responses evoked following equivalent opsin expression in the two major targets, ON bipolar (ON BCs) or retinal ganglion cells (RGCs), is absent. We address this deficit by comparing stimulus-response characteristics at single-unit resolution in the retina and dorsal lateral geniculate nucleus of retinally degenerate mice genetically engineered to express the opsin ReaChR in Grm6- or Brn3c-expressing cells (ON BC vs. RGCs, respectively). For both targeting strategies, we find ReaChR-evoked responses have equivalent sensitivity and can encode contrast across different background irradiances. Compared with ON BCs, targeting RGCs decreased response reproducibility and resulted in more stereotyped responses with reduced diversity in response polarity, contrast sensitivity, and temporal frequency tuning. Recording ReaChR-driven responses in visually intact retinas confirmed that RGC-targeted ReaChR expression disrupts visual feature selectivity of individual RGCs. Our data show that, while both approaches restore visual responses with impressive fidelity, ON BC targeting produces a richer visual code closer to that of wild-type mice.

Natural killer (NK) cells are an appealing off-the-shelf, allogeneic cellular therapy due to their cytotoxic profile. However, their activity against solid tumors remains suboptimal in part due to the upregulation of NK-inhibitory ligands, such as HLA-E, within the tumor microenvironment. Here, we utilize CRISPR-Cas9 to disrupt the KLRC1 gene (encoding the HLA-E-binding NKG2A receptor) and perform non-viral insertion of a GD2-targeting chimeric antigen receptor (CAR) within NK cells isolated from human peripheral blood. Genome editing with CRISPR-Cas9 ribonucleoprotein complexes yields efficient genomic disruption of the KLRC1 gene with 98% knockout efficiency and specific knockin of the GD2 CAR transgene as high as 23%, with minimal off-target activity as shown by CHANGE-seq, in-out PCR, amplicon sequencing, and long-read whole-genome sequencing. KLRC1-GD2 CAR NK cells display high viability and proliferation, as well as precise cellular targeting and potency against GD2⁺ human tumor cells. Notably, KLRC1-GD2 CAR NK cells overcome HLA-E-based inhibition in vitro against HLA-E-expressing, GD2⁺ melanoma cells. Using a single-step, virus-free genome editing workflow, this study demonstrates the feasibility of precisely disrupting inhibitory signaling within NK cells via CRISPR-Cas9 while expressing a CAR to generate potent allogeneic cell therapies against HLA-E⁺ solid tumors.

KRAS mutations are instrumental in the development and progression of pancreatic ductal adenocarcinoma (PDAC). Nevertheless, the efficacy of direct targeting of KRAS mutations to inhibit tumor development remains doubtful. It is therefore necessary to gain a deeper insight into the mechanism in which KRAS mutations influence the effectiveness of clinical treatments. In this study, KRAS^G12D protein was detected in cancer-associated fibroblasts (CAFs) from clinical samples of pancreatic ductal adenocarcinoma (PDAC). In vitro experiments demonstrated that KRAS^G12D protein in CAFs was not expressed from its own mutant gene but was derived from the ingestion of tumor cell-derived extracellular vesicles (EVs). The presence of KRAS^G12D protein in CAFs resulted in enhanced proliferation and migration. Furthermore, KRAS^G12D-containing CAFs were observed to promote tumor chemoresistance to gemcitabine treatment both in vitro and in vivo. Application of a KRAS mutation-specific inhibitor, MRTX1133, has been demonstrated to reverse chemoresistance in PDAC. Moreover, clinical data suggest that patients with KRAS mutations have poorer prognosis following adjuvant chemotherapy. These findings elucidate the mechanism by which oncogenic KRAS mutations promote cancer chemoresistance and remodel tumor environment in a non-autonomous manner, suggesting a novel strategy for targeting KRAS mutations to enhance chemosensitivity in PDAC.

Antibody-mediated rejection (AMR) remains a major complication after solid organ transplantation (SOT). Current treatment options are inefficient and result in drastic impairment of the general immunity. To selectively eliminate responsible alloreactive B cells characterized by anti-donor-HLA B cell receptors (BCRs), we generated T cells overcoming rejection by antibodies (CORA-Ts) engineered with a novel chimeric receptor comprising a truncated donor-HLA molecule as antigen recognition domain. As proof-of-concept, CORA receptors based on HLA-A∗02 were developed. In co-cultures with anti-HLA-A∗02 B cell lines, CORA-Ts were specifically activated, released pro-inflammatory mediators, and exhibited strong cytotoxicity resulting in an effective reduction of anti-HLA-A∗02 antibody release. Significant reduction of growth of an anti-HLA-A∗02 B cell line could be confirmed using an in vivo mouse model. Modification of the CORA receptor effectively abrogated T cell binding, thereby avoiding T cell sensitization. Additionally, using CRISPR-Cas9-mediated knockout of the FKBP12 gene, CORA-Ts were able to resist immunosuppressive treatment with tacrolimus, thereby allowing high efficiency in transplant patients. Our results demonstrate that CORA-Ts are able to specifically eliminate alloreactive, anti-HLA B cells, thus selectively preventing anti-HLA antibody release even under immunosuppressive conditions. This suggests CORA-Ts as potent approach to combat AMR and improve long-term graft survival in SOT patients while preserving their overall B cell immunity.

The development of efficient and targeted methods for delivering DNA in vivo has long been a major focus of research. In this study, we introduce a gene delivery approach admitted by small metabolites (gDAM) for the efficient and targeted delivery of naked DNA into astrocytes in the adult brains of mice. gDAM uses a straightforward combination of DNA and small metabolites, including glycine, L-proline, L-serine, L-histidine, D-alanine, Gly-Gly, and Gly-Gly-Gly, to achieve astrocyte-specific delivery of naked DNA, resulting in transient and robust gene expression in these cells. Using gDAM, we successfully co-deliver the PiggyBac transposon and the CRISPR-Cas9 system to induce long-term overexpression of the oncogene EGFRvIII and knockout of tumor suppressor genes Nf1, Pten, and Trp53 in astrocytes, leading to the development of astrocyte-derived gliomas in immunocompetent mice. Furthermore, gDAM facilitates the delivery of naked DNA to peripheral glioma astrocytes. The overexpression of interferon-β and granulocyte-macrophage colony-stimulating factor in these peripheral glioma astrocytes significantly prolongs the overall survival of mice bearing 73C glioma cells. This approach offers a new perspective on developing gene delivery systems that specifically target astrocytes to meet the varied needs of both research and gene therapy. The innovative strategy behind gDAM is expected to provide fresh inspiration in the quest for DNA delivery to other tissues, such as skeletal muscle and skin.