Molecular Therapy最新文献

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 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.

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.

Gene therapy with adeno-associated virus (AAV) vectors requires knowledge of their tropism within the body. Here we analyze the tropism of 10 naturally occurring AAV serotypes (AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, and AAVrh74) following systemic delivery into male and female mice. A transgene-expressing ZsGreen and Cre recombinase was used to identify transduction in a cell-dependent manner based on fluorescence. Cre-driven activation of tdTomato fluorescence offered superior sensitivity for transduced cells. All serotypes except AAV3B and AAV4 had high liver tropism. Fluorescence activation revealed transduction of unexpected tissues, including adrenals, testes, and ovaries. Rare transduced cells within tissues were also readily visualized. Biodistribution of AAV genomes correlated with fluorescence, except in immune tissues. AAV4 was found to have a pan-endothelial tropism while also targeting pancreatic beta cells. This public resource enables selection of the best AAV serotypes for basic science and preclinical applications in mice.

Exemplified by successful use in COVID-19 vaccination, delivery of modified mRNA encapsulated in lipid nanoparticles (LNPs) provides a framework for treating various genetic and acquired disorders. However, LNPs that can deliver mRNA into specific lung cell types have not yet been established. Here, we sought to determine whether poly(β-amino ester)s (PBAE) or PEGylated PBAE (PBAE-PEG) in combination with 4A3-SC8/DOPE/cholesterol/DOTAP LNPs could deliver mRNA into different types of lung cells in vivo. PBAE-PEG/LNP was similar to Lipofectamine MessengerMAX followed by PBAE/LNP for mRNA transfection efficiency in HEK293T cells in vitro. PBAE-PEG/LNP administered by intravenous (IV) injection achieved 73% mRNA transfection efficiency into lung endothelial cells, while PBAE-PEG/LNP administered by intratracheal (IT) injection achieved 55% efficiency in lung alveolar type II (ATII) epithelial cells in mice in vivo. PBAE/LNP administered by IT injection were superior for specific delivery into lung airway club epithelial cells compared to PBAE-PEG/LNP. Lipofectamine MessengerMAX was inactive in vivo. 5-Methoxyuridine-modified mRNA was more efficient than unmodified mRNA in vivo but not in vitro. Our findings indicate that PBAE-PEG/LNP and PBAE/LNP can transfect multiple lung cell types in vivo, which can be applied in gene therapy targeting genetic lung diseases.

Loss-of-function mutations in the chromodomain helicase DNA-binding 8 (CHD8) gene are strongly associated with autism spectrum disorders (ASDs). Indeed, the reduction of CHD8 causes transcriptional, epigenetic, and cellular phenotypic changes correlated to disease, which can be monitored in assessing new therapeutic approaches. SINEUPs are a functional class of natural and synthetic antisense long non-coding RNAs able to stimulate the translation of sense target mRNA, with no effect on transcription. Here, we employed synthetic SINEUP-CHD8 targeting the first and third AUG of the CHD8 coding sequence to efficiently stimulate endogenous CHD8 protein production. SINEUP-CHD8 were effective in cells with reduced levels of the target protein and in patient-derived fibroblasts with CHD8 mutations. Functionally, SINEUP-CHD8 were able to revert molecular phenotypes associated with CHD8 suppression, i.e., genome-wide transcriptional dysregulation, and the reduction of H3K36me3 levels. Strikingly, in chd8-morpholino-treated and ENU mutant zebrafish embryos, SINEUP-chd8 injection confirmed the ability of SINEUP RNA to rescue the chd8-suppression-induced macrocephaly phenotype and neuronal hyperproliferation. Thus, SINEUP-CHD8 molecule(s) represent a proof-of-concept toward the development of an RNA-based therapy for neurodevelopmental syndromes with implications for, and beyond ASD, and relevant to genetic disorders caused by protein haploinsufficiency.

Tumor necrosis factor receptor-associated factor 1 (TRAF1) is a crucial signaling adaptor involved in multiple cellular events. However, its role in regulating osteoclastogenesis and energy metabolism remains unclear. Here, we report that TRAF1 promotes osteoclastogenesis and oxidative phosphorylation (OXPHOS). Employing RNA sequencing, we found that TRAF1 is markedly upregulated during osteoclastogenesis and is positively associated with osteoporosis. TRAF1 knockout inhibits osteoclastogenesis and increases bone mass in both normal and ovariectomized adult mice without affecting bone mass in childhood. Furthermore, TRAF1 promotes osteoclast OXPHOS by increasing the phosphorylation level of AKT. Mechanistically, TRAF1 functions to inhibit TRAF2-induced ubiquitination of Gβl, a known activator of AKT, and further upregulates AKT phosphorylation. Rescue experiments revealed that the inhibitory effects of TRAF1 knockout on osteoclastogenesis, OXPHOS, and bone mass are dependent on AKT. Collectively, our findings uncover a previously unrecognized function of TRAF1 in regulating osteoclastogenesis and energy metabolism, and establish a novel TRAF1-AKT-OXPHOS axis in osteoclasts.