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Signaling circuits crucial to systemic physiology are widespread, yet uncovering their molecular underpinnings remains a barrier to understanding the etiology of many metabolic disorders. Here, we identified a copper-linked signaling circuit activated by disruption of mitochondrial function in the murine liver or heart that resulted in atrophy of the spleen and thymus and caused a peripheral white blood cell deficiency. We demonstrated that the leukopenia was caused by α-fetoprotein, which required copper and the cell surface receptor CCR5 to promote white blood cell death. We further showed that α-fetoprotein expression was upregulated in several cell types upon inhibition of oxidative phosphorylation. Collectively, our data argue that α-fetoprotein may be secreted by bioenergetically stressed tissue to suppress the immune system, an effect that may explain the recurrent or chronic infections that are observed in a subset of mitochondrial diseases or in other disorders with secondary mitochondrial dysfunction.
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Cobre , Doenças Mitocondriais , Camundongos , Animais , Cobre/metabolismo , alfa-Fetoproteínas/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Terapia de ImunossupressãoRESUMO
INTRODUCTION: Performance assessment and feedback are critical factors in successful medical simulation-based training. The Dynamic Haptic Robotic Trainer (DHRT) allows residents to practice ultrasound-guided needle insertions during simulated central venous catheterization (CVC) procedures while providing detailed feedback and assessment. A study was performed to examine the effectiveness of the DHRT in training the important skills of needle tip tracking and aspiration and how these skills impact procedural complications in simulated CVC. METHODS: The DHRT data were collected for 163 residents at 2 hospitals for 6 simulated needle insertions. Users were given automated feedback on 5 performance metrics, which measure aspiration rate, arterial punctures, punctures through and through the vein, loss of access to the vein, and successful access to the vein. Aspiration rates and tip tracking rates were analyzed to determine their significance in preventing CVC complications and improving performance. RESULTS: Tip tracking rates higher than 40% were 2.3 times more likely to result in successful venous access than rates less than 10%. Similarly, aspiration rates higher than 80% were 2.6 times more likely to result in successful venous access than rates less than 10%. Proper tip tracking and aspiration both reduced mechanical complications. Resident performance improved for all metrics except tip tracking. CONCLUSIONS: Proper tip tracking and aspiration both reduced complications and increased the likelihood of success. However, the skill of tip tracking was not effectively learned through practice without feedback. Therefore, ultrasound-guided needle-based procedures, including CVC, can be improved by providing specific feedback to users on their ultrasound usage to track needle insertions.
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The majority of adenovirus (Ad) vectors are based on human Ad type 5, which is a member of Ad species C. Species C also includes the closely-related types 1, 2, 6, 57 and 89. It is known that coagulation factors bind to Ad5 hexon and play a key role in the liver tropism of Ad5 vectors, but it is unclear how coagulation factors affect vectors derived from other species C Ads. We evaluated species C Ad vectors both in vitro and following intravenous injection in mice. To assess the impact of hexon differences, we constructed chimeric Ad5 vectors that contain the hexon hypervariable regions from other species C types, including vectors with hexon mutations that decreased coagulation factor binding. After intravenous injection into mice, vectors with Ad5 or Ad6 hexon had strong liver tropism, while vectors with chimeric hexon from other Ad types had weaker liver tropism due to inhibition by natural antibodies and complement. In addition, we discovered a novel ability of hexon to bind prothrombin, which is the most abundant coagulation factor in blood, and we found striking differences in the affinity of Ads for human, mouse and bovine coagulation factors. When compared to Ad5, vectors with non-Ad5 species C hexons had considerably higher affinity for both human and mouse prothrombin. Most of the vectors tested were strongly dependent on coagulation factors for liver transduction, but vectors with chimeric Ad6 hexon showed much less dependence on coagulation factors than other vectors. We found that in vitro neutralization experiments with mouse serum predicted in vivo behavior of Ad5 vectors, but in vitro experiments did not predict the in vivo behavior of vectors based on other Ad types. In sum, hexons from different human Ad species C viruses confer diverse properties on vectors, including differing abilities to target the liver.
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Adenovírus Humanos , Protrombina , Adenoviridae , Adenovírus Humanos/genética , Animais , Proteínas do Capsídeo/metabolismo , Bovinos , Vetores Genéticos , Humanos , Camundongos , Protrombina/genética , Protrombina/metabolismo , Transdução GenéticaRESUMO
MeCP2 is associated with Rett syndrome (RTT), MECP2 duplication syndrome, and a number of conditions with isolated features of these diseases, including autism, intellectual disability, and motor dysfunction. MeCP2 is known to broadly bind methylated DNA, but the precise molecular mechanism driving disease pathogenesis remains to be determined. Using proximity-dependent biotinylation (BioID), we identified a transcription factor 20 (TCF20) complex that interacts with MeCP2 at the chromatin interface. Importantly, RTT-causing mutations in MECP2 disrupt this interaction. TCF20 and MeCP2 are highly coexpressed in neurons and coregulate the expression of key neuronal genes. Reducing Tcf20 partially rescued the behavioral deficits caused by MECP2 overexpression, demonstrating a functional relationship between MeCP2 and TCF20 in MECP2 duplication syndrome pathogenesis. We identified a patient exhibiting RTT-like neurological features with a missense mutation in the PHF14 subunit of the TCF20 complex that abolishes the MeCP2-PHF14-TCF20 interaction. Our data demonstrate the critical role of the MeCP2-TCF20 complex for brain function.
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Proteína 2 de Ligação a Metil-CpG/metabolismo , Complexos Multiproteicos/metabolismo , Transtornos do Neurodesenvolvimento/etiologia , Transtornos do Neurodesenvolvimento/metabolismo , Fatores de Transcrição/metabolismo , Alelos , Animais , Biomarcadores , Encéfalo/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Biológicos , Mutação , Neurônios/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Sinapses/metabolismo , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Coronavirus disease 2019 (COVID-19) can lead to hypoxemic respiratory failure resulting in prolonged mechanical ventilation. Typically, tracheostomy is considered in patients who remain ventilator dependent beyond 2 weeks. However, in the setting of this novel respiratory virus, the safety and benefits of tracheostomy are not well-defined. Our aim is to describe our experience with percutaneous tracheostomy in patients with COVID-19. MATERIALS AND METHODS: This is a single center retrospective descriptive study. We reviewed comorbidities and outcomes in patients with respiratory failure due to COVID-19 who underwent percutaneous tracheostomy at our institution from April 2020 to September 2020. In addition, we provide details of our attempt to minimize aerosolization by using a modified protocol with brief periods of planned apnea. RESULTS: A total of 24 patients underwent percutaneous tracheostomy during the study. The average body mass index was 33.0±10.0. At 30 days posttracheostomy 17 (71%) patients still had the tracheostomy tube and 14 (58%) remained ventilator dependent. There were 3 (13%) who died within 30 days. At the time of data analysis in November 2020, 9 (38%) patients had died and 7 (29%) had been decannulated. None of the providers who participated in the procedure experienced signs or symptoms of COVID-19 infection. CONCLUSION: Percutaneous tracheostomy in prolonged respiratory failure due to COVID-19 appears to be safe to perform at the bedside for both the patient and health care providers in the appropriate clinical context. Morbid obesity did not limit the ability to perform percutaneous tracheostomy in COVID-19 patients.
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COVID-19 , Insuficiência Respiratória , COVID-19/complicações , Humanos , Insuficiência Respiratória/etiologia , Insuficiência Respiratória/terapia , Estudos Retrospectivos , SARS-CoV-2 , Traqueostomia/efeitos adversos , Traqueostomia/métodosRESUMO
BACKGROUND: This study compares surgical residents' knowledge acquisition of ultrasound-guided Internal Jugular Central Venous Catheterization (US-IJCVC) between in-person and online procedural training cohorts before receiving independent in-person Dynamic Haptic Robotic Simulation training. METHODS: Three surgical residency procedural training cohorts, two in-person (N = 26) and one online (N = 14), were compared based on their performance on a 24-item US-IJCVC evaluation checklist completed by an expert physician completed after training. Pre- and post-training US-IJCVC knowledge was also compared for the online cohort. RESULTS: No significant change in the pass rates on the US-IJCVC checklist was found between in-person and online cohorts (p = 0.208). There were differences in the Economy of Time and Motion between in-person and online cohorts (p < 0.005). The online cohort had significant increases in US-IJCVC knowledge pre-to post-training (p < 0.008). CONCLUSION: Online training with independent simulation practice was as effective as in-person training for US-IJCVC.
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Cateterismo Venoso Central , Internato e Residência , Treinamento por Simulação , Competência Clínica , Educação de Pós-Graduação em Medicina , HumanosRESUMO
Hartmann's procedure involves resecting the rectosigmoid colon, closure of the distal rectal stump, and forming an end colostomy for complicated left colon diverticulitis or malignancy. Recovery from the initial operation can, in a second stage, be followed by a reversal stage with the restoration of bowel continuity. This study aimed to assess the reversal rate and its correlation with demographic data, ASA grade, and length of hospital stay. All patients who underwent Hartmann's emergency procedure from 2014 to 2018 at Lewisham and Greenwich hospital were enrolled in this retrospective study. Data was collected from the inpatient electronic files and NELA (UK National Laparotomy Audit). 118 patients were included in the study, with 57.6% females and a median age of patients of 69 years (range 35-91). Findings of the study indicate that the most common indications for Hartmann's procedure were diverticular complications 60% (n=71) and benign perforated sigmoid or rectosigmoid cancer 16% (n=19). The average length of hospital stay was 24 days (range n=2 - 212 days). The reversal rate was 34.9% (41/118 cases). No significant difference was observed between gender and length of hospital stay in relation to the reversal rate while there was a significant correlation between age and ASA grade in relation to reversal rate; the calculated P values were recorded as (<0.000) and (<0.009) respectively. Our results show that the highest reversal rate was observed in younger and fitter (I-II) ASA grade patients. The most common medical complication from reversal of Hartmann's procedure was an anastomotic leak (n=6, 16.7%). Reversal rate of Hartmann's procedure was 34.9%. The average timeframe for reversal was within 18-20 months. There was a significant correlation between age and ASA grade in relation to reversal rate.
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Colostomia , Complicações Pós-Operatórias , Adulto , Idoso , Idoso de 80 Anos ou mais , Anastomose Cirúrgica , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reto/cirurgia , Reoperação , Estudos Retrospectivos , Resultado do TratamentoRESUMO
Previous studies have shown that nitric oxide (NO) supplements may prevent bone loss and fractures in preclinical models of estrogen deficiency. However, the mechanisms by which NO modulates bone anabolism remain largely unclear. Argininosuccinate lyase (ASL) is the only mammalian enzyme capable of synthesizing arginine, the sole precursor for nitric oxide synthase-dependent (NOS-dependent) NO synthesis. Moreover, ASL is also required for channeling extracellular arginine to NOS for NO production. ASL deficiency (ASLD) is thus a model to study cell-autonomous, NOS-dependent NO deficiency. Here, we report that loss of ASL led to decreased NO production and impairment of osteoblast differentiation. Mechanistically, the bone phenotype was at least in part driven by the loss of NO-mediated activation of the glycolysis pathway in osteoblasts that led to decreased osteoblast differentiation and function. Heterozygous deletion of caveolin 1, a negative regulator of NO synthesis, restored NO production, osteoblast differentiation, glycolysis, and bone mass in a hypomorphic mouse model of ASLD. The translational significance of these preclinical studies was further reiterated by studies conducted in induced pluripotent stem cells from an individual with ASLD. Taken together, our findings suggest that ASLD is a unique genetic model for studying NO-dependent osteoblast function and that the NO/glycolysis pathway may be a new target to modulate bone anabolism.
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Acidúria Argininossuccínica/metabolismo , Osso e Ossos/metabolismo , Diferenciação Celular , Glicólise , Ácido Nítrico/metabolismo , Osteoblastos/metabolismo , Adolescente , Adulto , Animais , Acidúria Argininossuccínica/genética , Acidúria Argininossuccínica/patologia , Osso e Ossos/patologia , Criança , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Osteoblastos/patologiaRESUMO
BACKGROUND: In the setting of the COVID pandemic, many patients falling ill with acute respiratory distress syndrome eventually require prone positioning for gas exchange. Traditionally, central venous catheters are inserted with patient in the supine or Trendelenburg position. However, when a patient cannot tolerate supine position and the need for central venous access is urgent, catheter placement may be considered with the patient in the prone position. CASE SUMMARY: A 69-year-old male with rapidly declining respiratory status secondary to COVID pneumonia quickly developed acute respiratory distress syndrome, was rapidly intubated, and then placed in the prone position. Patient could not tolerate the supine position even briefly and required a central venous catheter insertion for continuous renal replacement therapy. We kept the patient in the prone position and successfully inserted a central venous catheter in such position with real-time ultrasound guidance and using micropuncture technique. CONCLUSION: In the setting of the COVID pandemic, many cases of acute respiratory distress syndrome require patients to be prone in order to improve gas exchange. In the most severe situations, these patients would not be able to tolerate rotating back to the supine position but would still require central venous catheter insertion urgently. We demonstrated feasibility of central venous catheter insertion in the prone position in these severely ill patients.
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COVID-19/terapia , Cateterismo Venoso Central/métodos , Posicionamento do Paciente/métodos , Decúbito Ventral , Síndrome do Desconforto Respiratório/terapia , Ultrassonografia de Intervenção/métodos , Idoso , Humanos , Intubação Intratraqueal , Masculino , Punções , SARS-CoV-2RESUMO
In this study, we developed a single helper-dependent adenovirus (HDAd) to deliver all of the components (donor DNA, CRISPR-associated protein 9 [Cas9], and guide RNA [gRNA]) needed to achieve high-efficiency gene targeting and homology-directed repair in transduced cells. We show that these "all-in-one" HDAds are up to 117-fold more efficient at gene targeting than donor HDAds that do not express CRISPR/Cas9 in human induced pluripotent stem cells (iPSCs). The vast majority (>90%) of targeted recombinants had only one allele targeted, and this was accompanied by high-frequency indel formation in the non-targeted allele at the site of Cas9 cleavage. These indels varied in size and nature, and included large deletions of â¼8 kb. The remaining minority of recombinants had both alleles targeted (so-called bi-allelic targeting). These all-in-one HDAds represent an important platform for accomplishing and expanding the utility of homology-directed repair, especially for difficult-to-transfect cells and for in vivo applications.
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Axon initial segments (AISs) generate action potentials and regulate the polarized distribution of proteins, lipids, and organelles in neurons. While the mechanisms of AIS Na+ and K+ channel clustering are understood, the molecular mechanisms that stabilize the AIS and control neuronal polarity remain obscure. Here, we use proximity biotinylation and mass spectrometry to identify the AIS proteome. We target the biotin-ligase BirA* to the AIS by generating fusion proteins of BirA* with NF186, Ndel1, and Trim46; these chimeras map the molecular organization of AIS intracellular membrane, cytosolic, and microtubule compartments. Our experiments reveal a diverse set of biotinylated proteins not previously reported at the AIS. We show many are located at the AIS, interact with known AIS proteins, and their loss disrupts AIS structure and function. Our results provide conceptual insights and a resource for AIS molecular organization, the mechanisms of AIS stability, and polarized trafficking in neurons.
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Segmento Inicial do Axônio/metabolismo , Proteoma/metabolismo , Animais , Axônios , Biotinilação , Humanos , Espectrometria de Massas , Camundongos , Neurônios/metabolismo , Transporte Proteico , Ratos , Ratos Sprague-DawleyRESUMO
Prolonged expression of CRISPR/Cas9 raises concerns about off-target cleavage, cytotoxicity, and immune responses. To address these issues, we have developed a system to produce helper-dependent adenoviruses that express CRISPR/Cas9 to direct cleavage of the vectors' own genome after transduction of target cells. To prevent self-cleavage during vector production, it was necessary to downregulate Cas9 mRNA as well as inhibit Cas9 protein activity. Cas9 mRNA downregulation was achieved by inserting the target sequences for the helper-virus-encoded miRNA, mivaRNAI, and producer-cell-encoded miRNAs, hsa-miR183-5p, and hsa-miR218-5p, into the 3' UTR of the HDAd-encoded Cas9 expression cassette. Cas9 protein activity was inhibited by expressing anti-CRISPR proteins AcrIIA2 and AcrAII4 from both the producer cells and the helper virus. After purification, these helper-dependent adenoviruses will perform CRISPR/Cas9-mediated self-cleavage in the transduced target cells, thereby limiting the duration of Cas9 expression and thus represent an important platform for improving the safety of gene editing by CRISPR/Cas9.
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The urea cycle and glutamine synthetase (GS) are the two main pathways for waste nitrogen removal and their deficiency results in hyperammonemia. Here, we investigated the efficacy of liver-specific GS overexpression for therapy of hyperammonemia. To achieve hepatic GS overexpression, we generated a helper-dependent adenoviral (HDAd) vector expressing the murine GS under the control of a liver-specific expression cassette (HDAd-GS). Compared to mice injected with a control vector expressing an unrelated reporter gene (HDAd-alpha-fetoprotein), wild-type mice with increased hepatic GS showed reduced blood ammonia levels and a concomitant increase of blood glutamine after intraperitoneal injections of ammonium chloride, whereas blood urea was unaffected. Moreover, injection of HDAd-GS reduced blood ammonia levels at baseline and protected against acute hyperammonemia following ammonia challenge in a mouse model with conditional hepatic deficiency of carbamoyl phosphate synthetase 1 (Cps1), the initial and rate-limiting step of ureagenesis. In summary, we found that upregulation of hepatic GS reduced hyperammonemia in wild-type and Cps1-deficient mice, thus confirming a key role of GS in ammonia detoxification. These results suggest that hepatic GS augmentation therapy has potential for treatment of both primary and secondary forms of hyperammonemia.
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Amônia/metabolismo , Terapia Genética/métodos , Glutamato-Amônia Ligase/genética , Hiperamonemia/genética , Hiperamonemia/terapia , Fígado/metabolismo , Amônia/toxicidade , Animais , Carbamoil-Fosfato Sintase (Amônia)/genética , Carbamoil-Fosfato Sintase (Amônia)/metabolismo , Doença da Deficiência da Carbamoil-Fosfato Sintase I/genética , Doença da Deficiência da Carbamoil-Fosfato Sintase I/metabolismo , Doença da Deficiência da Carbamoil-Fosfato Sintase I/terapia , Modelos Animais de Doenças , Feminino , Técnicas de Transferência de Genes , Glutamato-Amônia Ligase/metabolismo , Hiperamonemia/metabolismo , Hiperamonemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Especificidade de Órgãos/genéticaRESUMO
Atherosclerosis, a disease of blood vessels, is driven by cholesterol accumulation and inflammation. Gene therapy that removes cholesterol from blood vessels and decreases inflammation is a promising approach for prevention and treatment of atherosclerosis. In previous work, we reported that helper-dependent adenoviral (HDAd) overexpression of apolipoprotein A-I (apoAI) in endothelial cells (ECs) increases cholesterol efflux in vitro and reduces atherosclerosis in vivo. However, the effect of HDAdApoAI on atherosclerosis is partial. To improve this therapy, we considered concurrent overexpression of ATP-binding cassette subfamily A, member 1 (ABCA1), a protein that is required for apoAI-mediated cholesterol efflux. Before attempting combined apoAI/ABCA1 gene therapy, we tested whether an HDAd that expresses ABCA1 (HDAdABCA1) increases EC cholesterol efflux, whether increased cholesterol efflux alters normal EC physiology, and whether ABCA1 overexpression in ECs has anti-inflammatory effects. HDAdABCA1 increased EC ABCA1 protein (â¼3-fold; p < 0.001) and apoAI-mediated cholesterol efflux (2.3-fold; p = 0.007). Under basal culture conditions, ABCA1 overexpression did not alter EC proliferation, metabolism, migration, apoptosis, nitric oxide production, or inflammatory gene expression. However, in serum-starved, apoAI-treated EC, ABCA1 overexpression had anti-inflammatory effects: decreased inflammatory gene expression (â¼50%; p ≤ 0.02 for interleukin [IL]-6, tumor necrosis factor [TNF]-α, and vascular cell adhesion protein-1); reduced lipid-raft Toll-like receptor 4 (80%; p = 0.001); and a trend towards increased nitric oxide production (â¼55%; p = 0.1). In ECs stimulated with lipopolysaccharide, ABCA1 overexpression markedly decreased inflammatory gene expression (â¼90% for IL-6 and TNF-α; p < 0.001). Therefore, EC ABCA1 overexpression has no toxic effects and counteracts the two key drivers of atherosclerosis: cholesterol accumulation and inflammation. In vivo testing of HDAdABCA1 is warranted.
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Transportador 1 de Cassete de Ligação de ATP/biossíntese , Apolipoproteína A-I/metabolismo , Aterosclerose , Colesterol/metabolismo , Células Endoteliais , Terapia Genética , Transportador 1 de Cassete de Ligação de ATP/genética , Adenoviridae , Animais , Apolipoproteína A-I/genética , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Aterosclerose/terapia , Bovinos , Colesterol/genética , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Vetores Genéticos , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Inflamação/terapia , CoelhosRESUMO
We describe a strategy to achieve footprintless bi-allelic homology-directed repair (HDR) using helper-dependent adenoviruses (HDAds). This approach utilizes two HDAds to deliver the donor DNA. These two HDAds are identical except for their selectable marker. One expresses the puromycin N-acetyltransferase-herpes simplex virus I thymidine kinase fusion gene (PACTk), while the other expresses the hygromycin phosphotransferase-herpes simplex virus I thymidine kinase fusion gene (HyTk). Therefore, puromycin and hygromycin double resistance can be used to select for targeted HDAd integration into both alleles. Subsequently, piggyBac-mediated excision of both PACTk and HyTk will confer resistance to gancyclovir, resulting in footprintless HDR at both alleles. However, gene-targeting frequency was not high enough to achieve simultaneous targeting at both alleles. Instead, sequential targeting, whereby the two alleles were targeted one at a time, was required in order to achieve bi-allelic HDR with HDAd.
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Carbamoyl phosphate synthetase 1 (CPS1) is a urea cycle enzyme that forms carbamoyl phosphate from bicarbonate, ammonia and ATP. Bi-allelic mutations of the CPS1 gene result in a urea cycle disorder presenting with hyperammonemia, often with reduced citrulline, and without orotic aciduria. CPS1 deficiency is particularly challenging to treat and lack of early recognition typically results in early neonatal death. Therapeutic interventions have limited efficacy and most patients develop long-term neurologic sequelae. Using transgenic techniques, we generated a conditional Cps1 knockout mouse. By loxP/Cre recombinase technology, deletion of the Cps1 locus was achieved in adult transgenic animals using a Cre recombinase-expressing adeno-associated viral vector. Within four weeks from vector injection, all animals developed hyperammonemia without orotic aciduria and died. Minimal CPS1 protein was detectable in livers. To investigate the efficacy of gene therapy for CPS deficiency following knock-down of hepatic endogenous CPS1 expression, we injected these mice with a helper-dependent adenoviral vector (HDAd) expressing the large murine CPS1 cDNA under control of the phosphoenolpyruvate carboxykinase promoter. Liver-directed HDAd-mediated gene therapy resulted in survival, normalization of plasma ammonia and glutamine, and 13% of normal Cps1 expression. A gender difference in survival suggests that female mice may require higher hepatic CPS1 expression. We conclude that this conditional murine model recapitulates the clinical and biochemical phenotype detected in human patients with CPS1 deficiency and will be useful to investigate ammonia-mediated neurotoxicity and for the development of cell- and gene-based therapeutic approaches.
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Carbamoil-Fosfato Sintase (Amônia)/genética , Doença da Deficiência da Carbamoil-Fosfato Sintase I/terapia , Terapia Genética , Hiperamonemia/terapia , Amônia/metabolismo , Animais , Carbamoil-Fosfato Sintase (Amônia)/uso terapêutico , Doença da Deficiência da Carbamoil-Fosfato Sintase I/genética , Doença da Deficiência da Carbamoil-Fosfato Sintase I/metabolismo , Doença da Deficiência da Carbamoil-Fosfato Sintase I/patologia , Carbamoil-Fosfato/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica , Glutamina/metabolismo , Humanos , Hiperamonemia/genética , Hiperamonemia/metabolismo , Hiperamonemia/patologia , Fígado/enzimologia , Fígado/patologia , Masculino , Camundongos , Camundongos Knockout , Mutação , Orotato Fosforribosiltransferase/deficiência , Orotato Fosforribosiltransferase/genética , Orotidina-5'-Fosfato Descarboxilase/deficiência , Orotidina-5'-Fosfato Descarboxilase/genética , Erros Inatos do Metabolismo da Purina-Pirimidina/genética , Erros Inatos do Metabolismo da Purina-Pirimidina/patologiaRESUMO
We recently reported on an in vivo hematopoietic stem cell (HSC) gene therapy approach. It involves the subcutaneous injections of G-CSF/AMD3100 to mobilize HSCs from the bone marrow into the peripheral blood stream and the intravenous injection of an integrating helper-dependent adenovirus vector system. HSCs transduced in the periphery homed back to the bone marrow, where they persisted long-term. However, high transgene marking rates found in primitive bone marrow HSCs were not reflected in peripheral blood cells. Here, we tested small-molecule drugs to achieve selective mobilization and transduction of HSCs. We found more efficient GFP marking in bone marrow HSCs but no increased marking in the peripheral blood cells. We then used an in vivo HSC chemo-selection based on a mutant of the O6-methylguanine-DNA methyltransferase (mgmtP140K) gene that confers resistance to O6-BG/BCNU and should give stably transduced HSCs a proliferation stimulus and allow for the selective survival and expansion of progeny cells. Short-term exposure of G-CSF/AMD3100-mobilized, in vivo-transduced mice to relatively low selection drug doses resulted in stable GFP expression in up to 80% of peripheral blood cells. Overall, the further improvement of our in vivo HSC transduction approach creates the basis for a simpler HSC gene therapy.
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Helper-dependent adenoviral vectors (HDAds) possess long homology arms that mediate high-efficiency gene editing. These long homology arms may permit simultaneous introduction of multiple modifications into a large genomic region or may permit a single HDAd to correct many different individual mutations spread widely across a gene. We investigated this important potential using an HDAd bearing 13 genetic markers in the region of homology to the target CFTR locus in human iPSCs and found that all markers can be simultaneously introduced into the target locus, with the two farthest markers being 22.2 kb apart. We found that genetic markers closer to the HDAd's selectable marker are more efficiency introduced into the target locus; a marker located 208 bp from the selectable marker was introduced with 100% efficiency. However, even markers 11 kb from the selectable marker were introduced at a relatively high frequency of 21.7%. Our study also revealed extensive heteroduplex DNA formation of up to 10 kb with no bias toward vector or chromosomal repair. However, mismatches escape repair at a frequency of up to 15%, leading to a genetically mixed colony and emphasizing the need for caution, especially if the donor and target sequences are not 100% homologous.
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Helper-dependent adenoviral vectors (HDAd) are deleted of all viral genes and they can efficiently transduce a wide variety of dividing and non-dividing cells to mediate high transgene expression levels. Unlike early generation adenoviral vectors, the absence of viral genes in HDAd results in long-term transgene expression without chronic toxicity and permits a large cloning capacity of 36 kb. Moreover, HDAd genomes exist extra-chromosomally thus minimizing the risks of germline transmission and insertional mutagenesis. For these reasons, HDAd offers tremendous potential for in vivo gene therapy. This chapter reviews preclinical studies using HDAd in large animal models to assess safety and efficacy in a wide variety of gene therapy applications.