RESUMO
Hematopoietic stem cell transplantation (HSCT) is the only approved treatment for presymptomatic infantile globoid cell leukodystrophy (GLD [Krabbe disease]). However, correction of disease is not complete, and outcomes remain poor. Herein we evaluated HSCT, intravenous (IV) adeno-associated virus rh10 vector (AAVrh10) gene therapy, and combination HSCT + IV AAVrh10 in the canine model of GLD. While HSCT alone resulted in no increase in survival as compared with untreated GLD dogs (â¼16 weeks of age), combination HSCT + IV AAVrh10 at a dose of 4E13 genome copies (gc)/kg resulted in delayed disease progression and increased survival beyond 1 year of age. A 5-fold increase in AAVrh10 dose to 2E14 gc/kg, in combination with HSCT, normalized neurological dysfunction up to 2 years of age. IV AAVrh10 alone resulted in an average survival to 41.2 weeks of age. In the peripheral nervous system, IV AAVrh10 alone or in addition to HSCT normalized nerve conduction velocity, improved ultrastructure, and normalized GALC enzyme activity and psychosine concentration. In the central nervous system, only combination therapy at the highest dose was able to restore galactosylceramidase activity and psychosine concentrations to within the normal range. These data have now guided clinical translation of systemic AAV gene therapy as an addition to HSCT (NCT04693598, NCT05739643).
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Transplante de Células-Tronco Hematopoéticas , Leucodistrofia de Células Globoides , Cães , Animais , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/terapia , Galactosilceramidase/genética , Psicosina , Transplante de Células-Tronco Hematopoéticas/métodos , Terapia Genética/métodos , Modelos Animais de DoençasRESUMO
Based on the observation that humans have variable responses of gene expression with the same dose of an adeno-associated vector, we hypothesized that there are deleterious variants in genes coding for processes required for adeno-associated virus (AAV)-mediated gene transfer/expression that may hamper or enhance the effectiveness of AAV-mediated gene therapy. To assess this hypothesis, we evaluated 69,442 whole genome sequences from three populations (European, African/African American, and Qatari) for predicted deleterious variants in 62 genes known to play a role in AAV-mediated gene transfer/expression. The analysis identified 5,564 potentially deleterious mutations of which 27 were classified as common based on an allele frequency ≥1% in at least one population studied. Many of these deleterious variants are predicated to prevent while others enhance effective AAV gene transfer/expression, and several are linked to known hereditary disorders. The data support the hypothesis that, like other drugs, human genetic variability contributes to the person-to-person effectiveness of AAV gene therapy and the screening for genetic variability should be considered as part of future clinical trials.
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Late infantile Batten disease (CLN2 disease) is an autosomal recessive, neurodegenerative lysosomal storage disease caused by mutations in the CLN2 gene encoding tripeptidyl peptidase 1 (TPP1). We tested intraparenchymal delivery of AAVrh.10hCLN2, a nonhuman serotype rh.10 adeno-associated virus vector encoding human CLN2, in a nonrandomized trial consisting of two arms assessed over 18 months: AAVrh.10hCLN2-treated cohort of 8 children with mild to moderate disease and an untreated, Weill Cornell natural history cohort consisting of 12 children. The treated cohort was also compared to an untreated European natural history cohort of CLN2 disease. The vector was administered through six burr holes directly to 12 sites in the brain without immunosuppression. In an additional safety assessment under a separate protocol, five children with severe CLN2 disease were treated with AAVrh.10hCLN2. The therapy was associated with a variety of expected adverse events, none causing long-term disability. Induction of systemic anti-AAVrh.10 immunity was mild. After therapy, the treated cohort had a 1.3- to 2.6-fold increase in cerebral spinal fluid TPP1. There was a slower loss of gray matter volume in four of seven children by MRI and a 42.4 and 47.5% reduction in the rate of decline of motor and language function, compared to Weill Cornell natural history cohort (P < 0.04) and European natural history cohort (P < 0.0001), respectively. Intraparenchymal brain administration of AAVrh.10hCLN2 slowed the progression of disease in children with CLN2 disease. However, improvements in vector design and delivery strategies will be necessary to halt disease progression using gene therapy.
Assuntos
Dependovirus , Lipofuscinoses Ceroides Neuronais , Aminopeptidases/genética , Encéfalo , Criança , Dependovirus/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Terapia Genética , Humanos , Imageamento por Ressonância Magnética , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/terapia , Tripeptidil-Peptidase 1RESUMO
[This corrects the article DOI: 10.1371/journal.pone.0156834.].
RESUMO
BACKGROUND: The prevalence of type 2 diabetes (T2D) is increasing in the Middle East. However, the genetic risk factors for T2D in the Middle Eastern populations are not known, as the majority of studies of genetic risk for T2D are in Europeans and Asians. METHODS: All subjects were ≥3 generation Qataris. Cases with T2D (n = 1,124) and controls (n = 590) were randomly recruited and assigned to the 3 known Qatari genetic subpopulations [Bedouin (Q1), Persian/South Asian (Q2) and African (Q3)]. Subjects underwent genotyping for 37 single nucleotide polymorphisms (SNPs) in 29 genes known to be associated with T2D in Europeans and/or Asian populations, and an additional 27 tag SNPs related to these susceptibility loci. Pre-study power analysis suggested that with the known incidence of T2D in adult Qataris (22%), the study population size would be sufficient to detect significant differences if the SNPs were risk factors among Qataris, assuming that the odds ratio (OR) for T2D SNPs in Qatari's is greater than or equal to the SNP with highest known OR in other populations. RESULTS: Haplotype analysis demonstrated that Qatari haplotypes in the region of known T2D risk alleles in Q1 and Q2 genetic subpopulations were similar to European haplotypes. After Benjamini-Hochberg adjustment for multiple testing, only two SNPs (rs7903146 and rs4506565), both associated with transcription factor 7-like 2 (TCF7L2), achieved statistical significance in the whole study population. When T2D subjects and control subjects were assigned to the known 3 Qatari subpopulations, and analyzed individually and with the Q1 and Q2 genetic subpopulations combined, one of these SNPs (rs4506565) was also significant in the admixed group. No other SNPs associated with T2D in all Qataris or individual genetic subpopulations. CONCLUSIONS: With the caveats of the power analysis, the European/Asian T2D SNPs do not contribute significantly to the high prevalence of T2D in the Qatari population, suggesting that the genetic risks for T2D are likely different in Qataris compared to Europeans and Asians.
Assuntos
Diabetes Mellitus Tipo 2/etnologia , Diabetes Mellitus Tipo 2/genética , Polimorfismo de Nucleotídeo Único , Adulto , Idoso , Alelos , Povo Asiático , Estudos de Casos e Controles , Frequência do Gene , Genoma , Genótipo , Haplótipos , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Catar/epidemiologia , Fatores de Risco , Inquéritos e Questionários , População BrancaRESUMO
The median survival of glioblastoma multiforme (GBM) is approximately 1 year. Following surgical removal, systemic therapies are limited by the blood-brain barrier. To circumvent this, we developed a method to modify neurons with the genetic sequence for therapeutic monoclonal antibodies using adeno-associated virus (AAV) gene transfer vectors, directing persistent, local expression in the tumor milieu. The human U87MG GBM cell line or patient-derived early passage GBM cells were administered to the striatum of NOD/SCID immunodeficient mice. AAVrh.10BevMab, an AAVrh.10-based vector coding for bevacizumab (Avastin), an anti-human vascular endothelial growth factor (VEGF) monoclonal antibody, was delivered to the area of the GBM xenograft. Localized expression of bevacizumab was demonstrated by quantitative PCR, ELISA and western blotting. Immunohistochemistry showed that bevacizumab was expressed in neurons. Concurrent administration of AAVrh.10BevMab with the U87MG tumor reduced tumor blood vessel density and tumor volume, and increased survival. Administration of AAVrh.10BevMab 1 week after U87MG xenograft reduced growth and increased survival. Studies with patient-derived early passage GBM primary cells showed a reduction in primary tumor burden with an increased survival. These data support the strategy of AAV-mediated central nervous system gene therapy to treat GBM, overcoming the blood-brain barrier through local, persistent delivery of an anti-angiogenesis monoclonal antibody.
Assuntos
Anticorpos Monoclonais Humanizados/genética , Expressão Gênica , Glioblastoma/genética , Glioblastoma/terapia , Neovascularização Patológica/terapia , Neurônios/metabolismo , Animais , Bevacizumab , Encéfalo/metabolismo , Encéfalo/patologia , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Glioblastoma/mortalidade , Glioblastoma/patologia , Humanos , Imageamento por Ressonância Magnética , Camundongos , Transdução Genética , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Aging involves multiple biologically complex processes characterized by a decline in cellular homeostasis over time leading to a loss and impairment of physiological integrity and function. Specific cellular hallmarks of aging include abnormal gene expression patterns, shortened telomeres and associated biological dysfunction. Like all organs, the lung demonstrates both physiological and structural changes with age that result in a progressive decrease in lung function in healthy individuals. Cigarette smoking accelerates lung function decline over time, suggesting smoking accelerates aging of the lung. Based on this data, we hypothesized that cigarette smoking accelerates the aging of the small airway epithelium, the cells that take the initial brunt of inhaled toxins from the cigarette smoke and one of the primary sites of pathology associated with cigarette smoking. METHODS: Using the sensitive molecular parameters of aging-related gene expression and telomere length, the aging process of the small airway epithelium was assessed in age matched healthy nonsmokers and healthy smokers with no physical manifestation of lung disease or abnormalities in lung function. RESULTS: Analysis of a 73 gene aging signature demonstrated that smoking significantly dysregulates 18 aging-related genes in the small airway epithelium. In an independent cohort of male subjects, smoking significantly reduced telomere length in the small airway epithelium of smokers by 14% compared to nonsmokers. CONCLUSION: These data provide biologic evidence that smoking accelerates aging of the small airway epithelium.
Assuntos
Senescência Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Mucosa Respiratória/efeitos dos fármacos , Fumaça/efeitos adversos , Fumar/efeitos adversos , Adulto , Estudos de Casos e Controles , Senescência Celular/genética , Células Epiteliais/química , Células Epiteliais/patologia , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/análise , Mucosa Respiratória/química , Mucosa Respiratória/patologia , Fumar/genética , Fumar/patologia , Telômero/genética , Encurtamento do TelômeroRESUMO
Genome-wide association studies (GWAS) and candidate gene studies have identified a number of risk loci associated with the smoking-related disease COPD, a disorder that originates in the airway epithelium. Since airway basal cell (BC) stem/progenitor cells exhibit the earliest abnormalities associated with smoking (hyperplasia, squamous metaplasia), we hypothesized that smoker BC have a dysregulated transcriptome, enriched, in part, at known GWAS/candidate gene loci. Massive parallel RNA sequencing was used to compare the transcriptome of BC purified from the airway epithelium of healthy nonsmokers (nâ=â10) and healthy smokers (nâ=â7). The chromosomal location of the differentially expressed genes was compared to loci identified by GWAS to confer risk for COPD. Smoker BC have 676 genes differentially expressed compared to nonsmoker BC, dominated by smoking up-regulation. Strikingly, 166 (25%) of these genes are located on chromosome 19, with 13 localized to 19q13.2 (p<10â»4 compared to chance), including 4 genes (NFKBIB, LTBP4, EGLN2 and TGFB1) associated with risk for COPD. These observations provide the first direct connection between known genetic risks for smoking-related lung disease and airway BC, the population of lung cells that undergo the earliest changes associated with smoking.
Assuntos
Biomarcadores/metabolismo , Cromossomos Humanos Par 19/genética , Células Endoteliais/metabolismo , Perfilação da Expressão Gênica , Loci Gênicos , Doença Pulmonar Obstrutiva Crônica/genética , Mucosa Respiratória/metabolismo , Fumar/efeitos adversos , Adulto , Variações do Número de Cópias de DNA/genética , Metilação de DNA , Estudo de Associação Genômica Ampla , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Doença Pulmonar Obstrutiva Crônica/induzido quimicamente , Doença Pulmonar Obstrutiva Crônica/patologia , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/patologia , Fumar/genéticaRESUMO
Juvenile neuronal ceroid lipofuscinosis (JNCL or CLN3 disease) is an autosomal recessive lysosomal storage disease resulting from mutations in the CLN3 gene that encodes a lysosomal membrane protein. The disease primarily affects the brain with widespread intralysosomal accumulation of autofluorescent material and fibrillary gliosis, as well as the loss of specific neuronal populations. As an experimental treatment for the CNS manifestations of JNCL, we have developed a serotype rh.10 adeno-associated virus vector expressing the human CLN3 cDNA (AAVrh.10hCLN3). We hypothesized that administration of AAVrh.10hCLN3 to the Cln3(Δex7/8) knock-in mouse model of JNCL would reverse the lysosomal storage defect, as well as have a therapeutic effect on gliosis and neuron loss. Newborn Cln3(Δex7/8) mice were administered 3 × 10(10) genome copies of AAVrh.10hCLN3 to the brain, with control groups including untreated Cln3(Δex7/8) mice and wild-type littermate mice. After 18 months, CLN3 transgene expression was detected in various locations throughout the brain, particularly in the hippocampus and deep anterior cortical regions. Changes in the CNS neuronal lysosomal accumulation of storage material were assessed by immunodetection of subunit C of ATP synthase, luxol fast blue staining, and periodic acid-Schiff staining. For all parameters, Cln3(Δex7/8) mice exhibited abnormal lysosomal accumulation, but AAVrh.10hCLN3 administration resulted in significant reductions in storage material burden. There was also a significant decrease in gliosis in AAVrh.10hCLN3-treated Cln3(Δex7/8) mice, and a trend toward improved neuron counts, compared with their untreated counterparts. These data demonstrate that AAVrh.10 delivery of a wild-type cDNA to the CNS is not harmful and instead provides a partial correction of the neurological lysosomal storage defect of a disease caused by a lysosomal membrane protein, indicating that this may be an effective therapeutic strategy for JNCL and other diseases in this category.
Assuntos
Dependovirus/genética , Expressão Gênica , Vetores Genéticos/genética , Glicoproteínas de Membrana/genética , Chaperonas Moleculares/genética , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/terapia , Animais , Animais Recém-Nascidos , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Vetores Genéticos/administração & dosagem , Vetores Genéticos/efeitos adversos , Humanos , Imuno-Histoquímica , Injeções , Interneurônios/metabolismo , Lisossomos/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Chaperonas Moleculares/metabolismo , Neuroglia/imunologia , Neuroglia/metabolismo , Neurônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , TransgenesRESUMO
MOTIVATION: Identification of expression Quantitative Trait Loci (eQTL), the genetic loci that contribute to heritable variation in gene expression, can be obstructed by factors that produce variation in expression profiles if these factors are unmeasured or hidden from direct analysis. METHODS: We have developed a method for Hidden Expression Factor analysis (HEFT) that identifies individual and pleiotropic effects of eQTL in the presence of hidden factors. The HEFT model is a combined multivariate regression and factor analysis, where the complete likelihood of the model is used to derive a ridge estimator for simultaneous factor learning and detection of eQTL. HEFT requires no pre-estimation of hidden factor effects; it provides P-values and is extremely fast, requiring just a few hours to complete an eQTL analysis of thousands of expression variables when analyzing hundreds of thousands of single nucleotide polymorphisms on a standard 8 core 2.6 G desktop. RESULTS: By analyzing simulated data, we demonstrate that HEFT can correct for an unknown number of hidden factors and significantly outperforms all related hidden factor methods for eQTL analysis when there are eQTL with univariate and multivariate (pleiotropic) effects. To demonstrate a real-world application, we applied HEFT to identify eQTL affecting gene expression in the human lung for a study that included presumptive hidden factors. HEFT identified all of the cis-eQTL found by other hidden factor methods and 91 additional cis-eQTL. HEFT also identified a number of eQTLs with direct relevance to lung disease that could not be found without a hidden factor analysis, including cis-eQTL for GTF2H1 and MTRR, genes that have been independently associated with lung cancer. AVAILABILITY: Software is available at http://mezeylab.cb.bscb.cornell.edu/Software.aspx. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Perfilação da Expressão Gênica/métodos , Locos de Características Quantitativas , Expressão Gênica , Humanos , Pulmão/metabolismo , Polimorfismo de Nucleotídeo Único , Análise de Regressão , SoftwareRESUMO
Apolipoprotein E, a protein component of blood lipid particles, plays an important role in lipid transport. Different mutations in the apolipoprotein E gene have been associated with various clinical phenotypes. In an initiated study of Qataris, we observed that 17% of the African-derived genetic subgroup were heterozygotes for a rare Arg145Cys (R145C) variant that functions as a dominant trait with incomplete penetrance associated with type III hyperlipoproteinemia. On the basis of this observation, we hypothesized that the R145C polymorphism might be common in African-derived populations. The prevalence of the R145C variant was assessed worldwide in the "1000 Genomes Project" and in 1,012 whites and 1,226 African-Americans in New York, New York. The 1000 Genomes Project data demonstrated that the R145C polymorphism is rare in non-African-derived populations but present in 5% to 12% of Sub-Saharan African-derived populations. The R145C polymorphism was also rare in New York whites (1 of 1,012, 0.1%); however, strikingly, 53 of the 1,226 New York African-Americans (4.3%) were R145C heterozygotes. The lipid profiles of the Qatari and New York R145C heterozygotes were compared with those of controls. The Qatari R145C subjects had higher triglyceride levels than the Qatari controls (p <0.007) and the New York African-American R145C subjects had an average of 52% greater fasting triglyceride levels than the New York African-American controls (p <0.002). From these observations, likely millions of people worldwide derived from Sub-Saharan Africans are apolipoprotein E R145C. In conclusion, although larger epidemiologic studies are necessary to determine the long-term consequences of this polymorphism, the available evidence suggests it is a common cause of a mild triglyceride dyslipidemia.
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Apolipoproteínas E/genética , Negro ou Afro-Americano , DNA/genética , Dislipidemias/genética , Polimorfismo Genético , Alelos , Apolipoproteínas E/sangue , Dislipidemias/sangue , Dislipidemias/etnologia , Predisposição Genética para Doença , Genótipo , Humanos , New York/epidemiologia , Reação em Cadeia da Polimerase , Prevalência , Fatores de RiscoRESUMO
Exome sequencing of families of related individuals has been highly successful in identifying genetic polymorphisms responsible for Mendelian disorders. Here, we demonstrate the value of the reverse approach, where we use exome sequencing of a sample of unrelated individuals to analyze allele frequencies of known causal mutations for Mendelian diseases. We sequenced the exomes of 100 individuals representing the three major genetic subgroups of the Qatari population (Q1 Bedouin, Q2 Persian-South Asian, Q3 African) and identified 37 variants in 33 genes with effects on 36 clinically significant Mendelian diseases. These include variants not present in 1000 Genomes and variants at high frequency when compared with 1000 Genomes populations. Several of these Mendelian variants were only segregating in one Qatari subpopulation, where the observed subpopulation specificity trends were confirmed in an independent population of 386 Qataris. Premarital genetic screening in Qatar tests for only four out of the 37, such that this study provides a set of Mendelian disease variants with potential impact on the epidemiological profile of the population that could be incorporated into the testing program if further experimental and clinical characterization confirms high penetrance.
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Cromossomos Humanos/genética , Doenças Genéticas Inatas/genética , Predisposição Genética para Doença , Testes Genéticos , Variação Genética , Análise de Sequência de DNA , Bases de Dados Genéticas , Exoma , Feminino , Frequência do Gene , Doenças Genéticas Inatas/epidemiologia , Humanos , Masculino , Prevalência , Catar/epidemiologiaRESUMO
Alpha-1 antitrypsin (α1AT) deficiency is a common autosomal recessive disorder characterized by a marked reduction in serum α1AT levels, lung and liver disease. α1AT is mainly produced and secreted by hepatocytes, with its primary function to protect the lung against the proteolytic activity of neutrophil elastase. Serum α1AT levels <11 µM are associated with progressive destruction of lung parenchyma and early-onset of panacinar emphysema in the age range 35-45. The current approved treatment for α1AT deficiency is a costly protein augmentation therapy requiring weekly intravenous infusion of purified α1AT from pooled human plasma. Gene therapy offers the advantage of a single vector administration, eliminating the burden of the repeated purified protein infusions, with the consequent reduced overall drug cost and improved compliance. We have developed a novel, highly efficient gene therapy approach for α1AT deficiency based on the administration of AAVrh.10hα1AT, an adeno-associated viral vector serotype rh.10 coding for normal M-type human α1AT via the intrapleural route. On the basis of prior murine studies, this approach provides sustained α1AT proximal to the lung with a highly efficient vector. In support of a clinical trial for this approach, we carried out a study to assess the safety of intrapleural administration of AAVrh.10hα1AT to 280 mice and 36 nonhuman primates. The data demonstrate that this approach is safe, with no toxicity issues. Importantly, there was persistent expression of the human α1AT mRNA in chest cavity cells for the duration of the study (6 months in mice and 1 year in nonhuman primates). Together, these data support the initiation of a clinical trial of intrapleural human AAVrh.10hα1AT for the treatment of α1AT deficiency.
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Dependovirus/genética , Terapia Genética , Vetores Genéticos/administração & dosagem , Deficiência de alfa 1-Antitripsina/terapia , alfa 1-Antitripsina/genética , Animais , Vetores Genéticos/efeitos adversos , Humanos , Injeções , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Primatas , alfa 1-Antitripsina/metabolismoRESUMO
BACKGROUND: Late infantile neuronal ceroid lipofuscinosis (LINCL), one form of Batten's disease is a progressive neurodegenerative disorder resulting from a CLN2 gene mutation. The spectrum of ophthalmic manifestations of LINCL and the relationship with neurological function has not been previously described. METHODS: Patients underwent ophthalmic evaluations, including anterior segment and dilated exams, optical coherence tomography, fluorescein and indocyanine green angiography. Patients were also assessed with the LINCL Neurological Severity Scale. Ophthalmic findings were categorized into one of five severity scores, and the association of the extent of ocular disease with neurological function was assessed. RESULTS: Fifty eyes of 25 patients were included. The mean age at the time of exam was 4.9 years (range 2.5 to 8.1). The mean ophthalmic severity score was 2.6 (range 1 to 5). The mean neurological severity score was 6.1 (range 2 to 11). Significantly more severe ophthalmic manifestations were observed among older patients (p<0.005) and patients with more severe neurological findings (p<0.03). A direct correlation was found between the Ophthalmic Severity Scale and the Weill Cornell Neurological Scale (p<0.002). A direct association was also found between age and the ophthalmic manifestations (p<0.0002), with older children having more severe ophthalmic manifestations. CONCLUSIONS: Ophthalmic manifestations of LINCL correlate closely with the degree of neurological function and the age of the patient. The newly established LINCL Ophthalmic Scale may serve as an objective marker of LINCL severity and disease progression, and may be valuable in the evaluation of novel therapeutic strategies for LINCL, including gene therapy.
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Oftalmopatias/etiologia , Oftalmopatias/patologia , Oftalmopatias/fisiopatologia , Lipofuscinoses Ceroides Neuronais/complicações , Lipofuscinoses Ceroides Neuronais/patologia , Lipofuscinoses Ceroides Neuronais/fisiopatologia , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Aminopeptidases/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Oftalmopatias/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Lipofuscinoses Ceroides Neuronais/genética , Serina Proteases/genética , Índice de Gravidade de Doença , Tripeptidil-Peptidase 1RESUMO
Activation of the human embryonic stem cell (hESC) signature genes has been observed in various epithelial cancers. In this study, we found that the hESC signature is selectively induced in the airway basal stem/progenitor cell population of healthy smokers (BC-S), with a pattern similar to that activated in all major types of human lung cancer. We further identified a subset of 6 BC-S hESC genes, whose coherent overexpression in lung adenocarcinoma (AdCa) was associated with reduced lung function, poorer differentiation grade, more advanced tumor stage, remarkably shorter survival, and higher frequency of TP53 mutations. BC-S shared with hESC and a considerable subset of lung carcinomas a common TP53 inactivation molecular pattern which strongly correlated with the BC-S hESC gene expression. These data provide transcriptome-based evidence that smoking-induced reprogramming of airway BC toward the hESC-like phenotype might represent a common early molecular event in the development of aggressive lung carcinomas in humans.
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Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Pulmão/patologia , Fumar/genética , Fumar/patologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Animais , Linhagem Celular Tumoral , Epitélio/metabolismo , Epitélio/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Pulmão/metabolismo , Camundongos , Análise Multivariada , Fenótipo , Modelos de Riscos Proporcionais , Análise de Sobrevida , Proteína Supressora de Tumor p53/metabolismoRESUMO
The small airway epithelium (SAE), the first site of smoking-induced lung pathology, exhibits genome-wide changes in gene expression in response to cigarette smoking. Based on the increasing evidence that the epigenome can respond to external stimuli in a rapid manner, we assessed the SAE of smokers for genome-wide DNA methylation changes compared with nonsmokers, and whether changes in SAE DNA methylation were linked to the transcriptional output of these cells. Using genome-wide methylation analysis of SAE DNA of nonsmokers and smokers, the data identified 204 unique genes differentially methylated in SAE DNA of smokers compared with nonsmokers, with 67% of the regions with differential methylation occurring within 2 kb of the transcriptional start site. Among the genes with differential methylation were those related to metabolism, transcription, signal transduction and transport. For the differentially methylated genes, 35 exhibited a correlation with gene expression, 54% with an inverse correlation of DNA methylation with gene expression and 46% a direct correlation. These observations provide evidence that cigarette smoking alters the DNA methylation patterning of the SAE and that, for some genes, these changes are associated with the smoking-related changes in gene expression.
Assuntos
Epigênese Genética , Epitélio/metabolismo , Mucosa Respiratória/metabolismo , Fumar/genética , Adulto , Estudos de Casos e Controles , Metilação de DNA/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Mucosa Respiratória/efeitos dos fármacos , Fumar/efeitos adversos , Fumar/metabolismo , Sítio de Iniciação de Transcrição , Adulto JovemRESUMO
BACKGROUND: Ciliated cells play a central role in cleansing the airways of inhaled contaminants. They are derived from basal cells that include the airway stem/progenitor cells. In animal models, the transcription factor FOXJ1 has been shown to induce differentiation to the ciliated cell lineage, and the RFX transcription factor-family has been shown to be necessary for, but not sufficient to induce, correct cilia development. METHODS: To test the hypothesis that FOXJ1 and RFX3 cooperatively induce expression of ciliated genes in the differentiation process of basal progenitor cells toward a ciliated cell linage in the human airway epithelium, primary human airway basal cells were assessed under conditions of in vitro differentiation induced by plasmid-mediated gene transfer of FOXJ1 and/or RFX3. TaqMan PCR was used to quantify mRNA levels of basal, secretory, and cilia-associated genes. RESULTS: Basal cells, when cultured in air-liquid interface, differentiated into a ciliated epithelium, expressing FOXJ1 and RFX3. Transfection of FOXJ1 into resting basal cells activated promoters and induced expression of ciliated cell genes as well as both FOXJ1 and RFX3, but not basal cell genes. Transfection of RFX3 induced expression of RFX3 but not FOXJ1, nor the expression of cilia-related genes. The combination of FOXJ1 + RFX3 enhanced ciliated gene promoter activity and mRNA expression beyond that due to FOXJ1 alone. Corroborating immunoprecipitation studies demonstrated an interaction between FOXJ1 and RFX3. CONCLUSION: FOXJ1 is an important regulator of cilia gene expression during ciliated cell differentiation, with RFX3 as a transcriptional co-activator to FOXJ1, helping to induce the expression of cilia genes in the process of ciliated cell differentiation of basal/progenitor cells.
Assuntos
Cílios/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Mucosa Respiratória/metabolismo , Mucosa Respiratória/ultraestrutura , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Diferenciação Celular , Células Cultivadas , Cílios/ultraestrutura , Regulação da Expressão Gênica/fisiologia , Humanos , Fatores de Transcrição de Fator Regulador XRESUMO
Abstract Coronary artery disease (CAD), a leading cause of mortality, is a chronic disease in which blood flow to the myocardium is obstructed, leading to ischemia. Although coronary artery stenting and surgical bypass are successful with localized coronary lesions, patients with diffuse CAD have only pharmacologic options. In a mouse ischemic hind-limb model, AdVEGF-All6A+, an Ad5 vector expressing the cDNA/genomic hybrid of the vascular endothelial growth factor (VEGF) gene (expressing isoforms, 121, 165, and 189) mediated recovery of blood flow at a dose of two logs less than that required with a single isoform. The objective of the current study was to ascertain the safety profile of good manufacturing practice (GMP)-grade AdVEGF-All6A+ in the adult rat ischemic heart model in support of a clinical study to treat humans with diffuse CAD. AdVEGF-All6A+ (10(5), 10(6), or 10(7) particle units), a control vector (AdNull, 10(7) particle units) with no translatable expression cassette and a vehicle sham control (phosphate buffered saline [PBS]) were administered separately to the left ventricle of rats immediately following acute coronary artery ligation to initiate myocardial infarction (MI), designed to evoke an extreme ischemic myocardium in cohorts (n=5 males; n=5 females), with sacrifice at 5, 14, or 30 days. Six cohorts received no ligation but were administered AdVEGF-All6A+ vector or PBS with sacrifice at 30 or 365 days. Although there were surgical-related abnormalities among the groups, blinded evaluation of gross and histopathology, complete blood count, and serum chemistry found no significant differences between control- and vector-treated groups and no adverse effects could be attributed to AdVEGF-All6A+. No changes in serum troponin-I levels persisted beyond those associated with the MI. Gross pathology and histopathology of all major organs showed no AdVEGF-All6A+-related changes. Overall, this safety profile suggests that AdVEGF-All6A+ or AdNull administration to the myocardium meets the criteria to proceed to clinical trial.
Assuntos
Adenoviridae/genética , Vetores Genéticos/metabolismo , Isquemia Miocárdica/terapia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Análise Química do Sangue , DNA Complementar/genética , Modelos Animais de Doenças , Feminino , Terapia Genética , Vetores Genéticos/genética , Humanos , Masculino , Isquemia Miocárdica/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Troponina I/sangue , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Late infantile neuronal ceroid lipofuscinosis (LINCL), a fatal, lysosomal storage disorder caused by mutations in the CLN2 gene, results in a deficiency of tripeptidyl-peptidase I (TPP-I) activity in neurons. Our prior studies showed that delivery of the human CLN2 cDNA directly to the CNS, using an adeno-associated virus serotype 2 (AAV2) vector, is safe in children with LINCL. As a second-generation strategy, we have demonstrated that AAVrh.10hCLN2, a rhesus-derived AAV vector, mediates wide distribution of TPP-I through the CNS in a murine model. This study tests the hypothesis that direct administration of AAVrh.10hCLN2 to the CNS of rats and nonhuman primates at doses scalable to humans has an acceptable safety profile and mediates significant CLN2 expression in the CNS. A dose of 10(11) genome copies (GC) was administered bilaterally to the striatum of Sprague Dawley rats with sacrifice at 7 and 90 days with no significant impact except for mild vector-related histopathological changes at the site of vector administration. A dose of 1.8×10(12) GC of AAVrh.10hCLN2 was administered to the CNS of 8 African green monkeys. The vector-treated monkeys did not differ from controls in any safety parameter except for mild to moderate white matter edema and inflammation localized to the administration sites of the vector. There were no clinical sequelae to these localized findings. TPP-I activity was >2 SD over background in 31.7±8.1% of brain at 90 days. These findings establish the dose and safety profile for human clinical studies for the treatment of LINCL with AAVrh.10hCLN2.
Assuntos
Aminopeptidases/genética , Encéfalo/metabolismo , Dependovirus/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Expressão Gênica , Vetores Genéticos/genética , Lipofuscinoses Ceroides Neuronais/genética , Serina Proteases/genética , Aminopeptidases/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Comportamento Animal , Encéfalo/patologia , Linhagem Celular , Dependovirus/imunologia , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Feminino , Terapia Genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/efeitos adversos , Humanos , Masculino , Lipofuscinoses Ceroides Neuronais/metabolismo , Lipofuscinoses Ceroides Neuronais/terapia , Primatas , Ratos , Serina Proteases/metabolismo , Fatores de Tempo , Transdução Genética , Tripeptidil-Peptidase 1RESUMO
The Qatari population, located at the Arabian migration crossroads of African and Eurasia, is comprised of Bedouin, Persian and African genetic subgroups. By deep exome sequencing of only 7 Qataris, including individuals in each subgroup, we identified 2,750 nonsynonymous SNPs predicted to be deleterious, many of which are linked to human health, or are in genes linked to human health. Many of these SNPs were at significantly elevated deleterious allele frequency in Qataris compared to other populations worldwide. Despite the small sample size, SNP allele frequency was highly correlated with a larger Qatari sample. Together, the data demonstrate that exome sequencing of only a small number of individuals can reveal genetic variations with potential health consequences in understudied populations.