Citations

AbstractBackgroundMutation of the m. 8993 T > G ATP6 subunit of ATP synthase causes a maternally inherited Leigh Syndrome (LS), a rapidly fatal encephalomyelopathy in childhood called and related Neuropathy, Ataxia and Retinitis Pigmentosa (NARP) in adults. There is no cure, and relevant animal models are few. Here we describe a novel ATP6 mouse model of LS and preclinical efficacy of a gene therapy approach to neutralize the ATP6 mutation by competition with a mitochondrial targeted adeno‐associated virus serotype 9 (AAV9) vector containing wild‐type ATP6.MethodsLS transgenic mice with an ATP6 mutation were developed by microinjecting AAV2 modified by a COX8 mitochondrial targeting sequence (MTS) into mouse blastocysts to deliver the human ATP6 gene with a T > G mutation at position 8993 of the mitochondrial genome, responsible for clinical LS DNA, protein, and metabolic assays confirmed functional gene delivery and in depth physiological/clinical assessments supported germline transmission of an LS phenotype. Rescue of LS in this model was evaluated after intravenous injection of mito‐targeted AAV9 containing wild‐type ATP6 followed by reevaluation of phenotypes.ResultsLS transgenic mice expressed mutant ATP6 protein in multiple relevant tissues including brain, eye, liver. Mutant ATP6 incorporated into Complex V and conferred decreased ATP synthase efficiency. Mice exhibited hallmarks of the human disease with one or multiple systemic symptoms including early death, paralysis, hunching, vision loss and seizures over six generations. Necropsy revealed spongiform encephalopathy, retinal degeneration, and cardiomegaly. Rescue by mitochondrially directed AAV9 containing wild type ATP6 after disease onset conferred prolonged survival and reduced paralysis. When administered prior to disease onset all treated ATP6 mice survived with evidence of globally improved function.ConclusionsThe results are consistent with efficient delivery and expression of foreign genes in mitochondria by AAV vectors directed by MTS and support further development of such strategies via intravenous injection of mitochondrial targeted AAV9 to deliver corrective genes.

AbstractBackgroundEosinophilic esophagitis (EoE) is a chronic allergic disease associated with type 2 inflammation and epithelial barrier dysfunction. The etiology is unknown, however, genetic heritability studies suggest environmental factors play a key role in pathogenesis. Detergents, such as sodium dodecyl sulfate (SDS), are common ingredients in household products such as dish soap and toothpaste. We hypothesized detergent exposure decreases epithelial barrier function and induces esophageal inflammation.MethodsImmortalized esophageal epithelial cells (EPC2) were cultured in air‐liquid interface (ALI) and exposed to SDS. Barrier function/activity was assessed by transepithelial electrical resistance (TEER), FITC‐dextran flux, and RT‐PCR. Additionally, SDS‐treated mouse esophageal organoids were evaluated for morphology. To investigate the effects of SDS in vivo, mice were treated with 0.5% SDS in drinking water for 14 days. Esophagi were assessed by gross morphology, histopathology, protein expression, and bulk RNA sequencing.ResultsWhen EPC2 cells were exposed to SDS (5 μg/ml) for 96 h, TEER decreased (p = 0.03), and FITC‐dextran flux increased (p = 0.0002). mRNA expression of IL‐33 increased 4.5‐fold (p = 0.02) at 6 h and DSG1 decreased (p < 0.0001) by 72 h. Disrupted epithelial integrity was noted in SDS‐treated esophageal organoids. When mice were exposed to SDS, they showed increased esophageal width, chemokine, and metalloprotease levels. Mice treated with SDS also showed increased IL‐33 protein expression, basal zone hyperplasia, CD4+ cell infiltration, and esophageal eosinophilia. RNA sequencing revealed upregulation of immune response pathway genes.ConclusionExposure to SDS decreases esophageal barrier integrity, stimulates IL‐33 production, and promotes epithelial hyperplasia and tissue eosinophilia. Detergents may be a key environmental trigger in EoE pathogenesis.

Abstract Mutations in mitochondrial genes are primary causes of multiple inherited disorders, especially affecting tissues with high aerobic energy demands such as the eye, brain, heart, and skeletal muscles. Such mitochondrial diseases are untreatable, have poor prognoses, and relevant animal models are few. Here we microinjected into the mouse blastocyst an adeno-associated virus (AAV) that we redirected to mitochondria to deliver the mutant human adenosine triphosphate (ATP) synthase subunit 6 gene (m.8993T>G) responsible for Maternally Inherited Leigh Syndrome, a neurologic disease renowned for causing rapidly fatal encephalomyelopathy in childhood or Neuropathy, Ataxia and Retinitis Pigmentosa in adults. Mice bred over six generations exhibited hallmarks of the human disease with one or multiple systemic symptoms including early death, paralysis, hunching, vision loss and seizures. Necropsy revealed spongiform encephalopathy, retinal degeneration, and cardiomegaly. Intravenous injection of a mitochondrially directed AAV serotype 9 vector containing wild-type ATP6 after disease onset, conferred prolonged survival and reduced paralysis. When administered prior to disease onset, all treated mice survived, and visual and motor function improved. These results support further development of gene therapy strategies involving intravenous delivery of mito-targeted AAV9.

Abstract Quiescin sulfhydryl oxidase 1 (QSOX1) is an enzyme overexpressed by many different tumor types. QSOX1 catalyzes the formation of disulfide bonds in proteins. Because short hairpin knockdowns (KD) of QSOX1 have been shown to suppress tumor growth and invasion in vitro and in vivo, we hypothesized that chemical compounds inhibiting QSOX1 enzymatic activity would also suppress tumor growth, invasion, and metastasis. High throughput screening using a QSOX1-based enzymatic assay revealed multiple potential QSOX1 inhibitors. One of the inhibitors, known as “SBI-183,” suppresses tumor cell growth in a Matrigel-based spheroid assay and inhibits invasion in a modified Boyden chamber, but does not affect viability of nonmalignant cells. Oral administration of SBI-183 inhibits tumor growth in 2 independent human xenograft mouse models of renal cell carcinoma. We conclude that SBI-183 warrants further exploration as a useful tool for understanding QSOX1 biology and as a potential novel anticancer agent in tumors that overexpress QSOX1.

Abstract Motion and uncertainty in radiotherapy is traditionally handled via margins. The clinical target volume (CTV) is expanded to a larger planning target volume (PTV), which is irradiated to the prescribed dose. However, the PTV concept has several limitations, especially in proton therapy. Therefore, robust and probabilistic optimization methods have been developed that directly incorporate motion and uncertainty into treatment plan optimization for intensity modulated radiotherapy (IMRT) and intensity modulated proton therapy (IMPT). Thereby, the explicit definition of a PTV becomes obsolete and treatment plan optimization is directly based on the CTV. Initial work focused on random and systematic setup errors in IMRT. Later, inter-fraction prostate motion and intra-fraction lung motion became a research focus. Over the past ten years, IMPT has emerged as a new application for robust planning methods. In proton therapy, range or setup errors may lead to dose degradation and misalignment of dose contributions from different beams – a problem that cannot generally be addressed by margins. Therefore, IMPT has led to the first implementations of robust planning methods in commercial planning systems, making these methods available for clinical use. This paper first summarizes the limitations of the PTV concept. Subsequently, robust optimization methods are introduced and their applications in IMRT and IMPT planning are reviewed.

Summary The transcription factor forkhead box N4 (Foxn4) is a key regulator in a variety of biological processes during development. In particular, Foxn4 plays an essential role in the genesis of horizontal and amacrine neurons from neural progenitors in the vertebrate retina. Although the functions of Foxn4 have been well established, the transcriptional regulation of Foxn4 expression during progenitor cell differentiation remains unclear. Here, we report that an evolutionarily conserved 129 bp noncoding DNA fragment (Foxn4CR4.2 or CR4.2), located ∼26 kb upstream of Foxn4 transcription start site, functions as a cis-element for Foxn4 regulation. CR4.2 directs gene expression in Foxn4-positive cells, primarily in progenitors, differentiating horizontal and amacrine cells. We further determined that the gene regulatory activity of CR4.2 is modulated by Meis1 binding motif, which is bound and activated by Meis1 transcription factor. Deletion of the Meis1 binding motif or knockdown of Meis1 expression abolishes the gene regulatory activity of CR4.2. In addition, knockdown of Meis1 expression diminishes the endogenous Foxn4 expression and affects cell lineage development. Together, we demonstrate that CR4.2 and its interacting Meis1 transcription factor play important roles in regulating Foxn4 expression during early retinogenesis. These findings provide new insights into molecular mechanisms that govern gene regulation in retinal progenitors and specific cell lineage development.