RESUMEN
Glaucoma is the leading cause of irreversible blindness worldwide. Therapies for glaucoma are directed toward reducing intraocular pressure (IOP), the leading risk factor and only reliable therapeutic target via topical medications or with procedural intervention including laser or surgery. Though topical therapeutics are typically first line, less than 50% of patients take drops as prescribed. Sustained release technologies that decrease IOP for extended periods of time are being examined for clinical use. We recently identified Stanniocalcin-1, a naturally occurring hormone, as an IOP-lowering agent. Here, we show that a single injection into the anterior chamber of mice with an adeno-associated viral vector containing the transgene of stanniocalcin-1 results in diffuse and sustained expression of the protein and produces IOP reduction for up to 6 months. As the treatment effect begins to wane, IOP-lowering can be rescued with a repeat injection. Aqueous humor dynamic studies revealed an increase in outflow facility as the mechanism of action. This first-in-class therapeutic approach has the potential to improve care and reduce the rates of vision loss in the 80 million people worldwide currently affected by glaucoma.
Asunto(s)
Glaucoma , Hipotensión Ocular , Animales , Glaucoma/tratamiento farmacológico , Glaucoma/genética , Glicoproteínas , Humanos , Presión Intraocular , Ratones , Tonometría Ocular , TransgenesRESUMEN
Expansion of CTG trinucleotide repeats (TNR) in the transcription factor 4 (TCF4) gene is highly associated with Fuchs Endothelial Corneal Dystrophy (FECD). Due to limitations in the availability of DNA from diseased corneal endothelium, sizing of CTG repeats in FECD patients has typically been determined using DNA samples isolated from peripheral blood leukocytes. However, it is non-feasible to extract enough DNA from surgically isolated FECD corneal endothelial tissue to determine repeat length based on current technology. To circumvent this issue, total RNA was isolated from FECD corneal endothelium and sequenced using long-read sequencing. Southern blotting of DNA samples isolated from primary cultures of corneal endothelium from these same affected individuals was also assessed. Both long read sequencing and Southern blot analysis showed significantly longer CTG TNR expansion (>1000 repeats) in the corneal endothelium from FECD patients than those characterized in leukocytes from the same individuals (<90 repeats). Our findings suggest that the TCF4 CTG repeat expansions in the FECD corneal endothelium are much longer than those found in leukocytes.
Asunto(s)
ADN/genética , Endotelio Corneal/patología , Distrofia Endotelial de Fuchs/patología , Leucocitos/patología , Factor de Transcripción 4/genética , Expansión de Repetición de Trinucleótido , Anciano , Anciano de 80 o más Años , Southern Blotting , Niño , ADN/análisis , Endotelio Corneal/metabolismo , Femenino , Distrofia Endotelial de Fuchs/epidemiología , Distrofia Endotelial de Fuchs/genética , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Leucocitos/metabolismo , Masculino , Persona de Mediana Edad , Reacción en Cadena de la PolimerasaRESUMEN
Primary cultures of human corneal endothelial cells (HCECs) are an important model system for studying the pathophysiology of corneal endothelium. The purpose of this study was to identify and validate an optimal primary culture model of normal and Fuchs endothelial corneal dystrophy (FECD) endothelial cells by comparing cell morphology and marker expression under different media conditions to in vivo donor tissues. Primary and immortalized HCECs, isolated from normal and FECD donors, were cultured in proliferation media (Joyce, M4, Bartakova) alone or sequentially with maturation media (F99, Stabilization 1, M5). CD56, CD73 and CD166 expressions were quantified in confluent and matured cell lines by flow cytometry. HCECs that were allowed to proliferate in Joyce's medium followed by maturation in low-mitogen containing media yielded cells with similar morphology to corneal endothelial tissues. Elevated expression of CD56 and CD166 and low expression of CD73 correlated with regular, hexagonal-like HCEC morphology. CD56:CD73 > 2.5 was most consistent with normal HCEC morphology and mimicked corneal endothelial tissue. Immortalization of normal HCECs by hTERT transduction showed morphology and CD56:CD73 ratios similar to parental cell lines. HCECs established from FECD donors showed reduced CD56:CD73 ratios compared to normal HCECs which coincided with reduced uniformity and regularity of cell monolayers. Overall, a dual media system with Joyce's medium for proliferation and a low-mitogen media for maturation, provided normal cultures with regular, hexagonal-like cell morphologies consistent with corneal endothelial cells in vivo. CD56:CD73 expression ratio >2.5 was predictive of in vivo-like cellular morphology.
Asunto(s)
Técnicas de Cultivo de Célula , Células Endoteliales/patología , Distrofia Endotelial de Fuchs/patología , Proliferación Celular , Medios de Cultivo , Endotelio Corneal , HumanosRESUMEN
Purpose: To evaluate pharmacokinetic parameters and ocular hypotensive effects of cromakalim prodrug 1 (CKLP1) in normotensive large animal models. Methods: Optimal CKLP1 concentration was determined by dose response and utilized in short- (5-8 days) and long-term (60 days) evaluation in hound dogs (n = 5) and African Green Monkeys (n = 5). Blood pressure was recorded 3-5 times per week with a tail cuff. Concentrations of CKLP1 and the parent compound levcromakalim were assessed in hound dog plasma and select tissues by LC-MS/MS after bilateral ocular treatment with CKLP1 for 8 days. Pharmacokinetic parameters were calculated from days 1, 4, and 8 data. After necropsy, histology was assessed in 43 tissue samples from each animal. Results: In hound dogs and African Green monkeys, 10 mM CKLP1 (optimal concentration) significantly lowered intraocular pressure (IOP) by 18.9% ± 1.1% and 16.7% ± 6.7%, respectively, compared with control eyes (P < 0.05). During treatment, no significant change in systolic or diastolic blood pressure was observed in either species (P > 0.1). Average values for half-life of CKLP1 was 295.3 ± 140.4 min, Cmax, 10.5 ± 1.6 ng/mL, and area under the concentration vs. time curve (AUClast) 5261.4 ± 918.9 ng·min/mL. For levcromakalim, average values of half-life were 96.2 ± 27 min, Cmax 1.2 ± 0.2 ng/mL, and AUClast 281.2 ± 110.8 ng·min/mL. No significant pathology was identified. Conclusions: CKLP1 lowered IOP in hound dogs and African green monkeys with no effect on systemic blood pressure. Ocular topical treatment of CKLP1 showed excellent tolerability even after extended treatment periods.
Asunto(s)
Antihipertensivos/farmacocinética , Cromakalim/farmacocinética , Presión Intraocular/efectos de los fármacos , Canales KATP/efectos de los fármacos , Administración Oftálmica , Administración Tópica , Animales , Antihipertensivos/administración & dosificación , Antihipertensivos/farmacología , Área Bajo la Curva , Autopsia/métodos , Presión Sanguínea/efectos de los fármacos , Chlorocebus aethiops , Cromakalim/administración & dosificación , Cromakalim/farmacología , Perros , Relación Dosis-Respuesta a Droga , Femenino , Semivida , Modelos Animales , Primates , ProfármacosRESUMEN
Purpose: To characterize inheritance, penetrance, and trinucleotide repeat expansion stability in Fuchs endothelial corneal dystrophy (FECD). Methods: One thousand unrelated and related subjects with and without FECD were prospectively recruited. CTG18.1 repeat length (CTG18.1L) was determined via short tandem repeat assay and Southern blotting of leukocyte DNA. Multivariable logistic regression and generalized estimating equation models were employed. Results: There were 546 unrelated FECD cases (67.6% female; 70 ± 10 years) and 235 controls (63.8% female; 73 ± 8 years; all ≥ 50 years). CTG18.1 expansion (CTG18.1exp+) was observed in 424 (77.7%) cases and 18 (7.7%) controls (P = 2.48 × 10-44). CTG18.1 expansion was associated with FECD severity (P = 5.62 × 10-7). The family arm of the study included 331 members from 112 FECD-affected families; 87 families were CTG18.1exp+. Autosomal dominant inheritance with variable expression of FECD was observed, regardless of expansion status. FECD penetrance of CTG18.1 expansion increased with age, ranging from 44.4% in the youngest (19-46 years) to 86.2% in the oldest (64-91 years) age quartiles. Among 62 parent-offspring transmissions of CTG18.1exp+, 48 (77.4%) had a change in CTG18.1L ≤ 10 repeats, and eight (12.9%) were ≥50 repeats, including five large expansions (â¼1000-2000 repeats) that contracted. Among 44 offspring who did not inherit the CTG18.1exp+ allele, eight (18.2%) exhibited FECD. Conclusions: CTG18.1 expansion was highly associated with FECD but demonstrated incomplete penetrance. CTG18.1L instability occurred in a minority of parent-offspring transmissions, with large expansions exhibiting contraction. The observation of FECD without CTG18.1 expansion among family members in CTG18.1exp+ families highlights the complexity of the relationship between the FECD phenotype and CTG18.1 expansion.
Asunto(s)
Distrofia Endotelial de Fuchs/genética , Factor de Transcripción 4/genética , Expansión de Repetición de Trinucleótido/genética , Adulto , Anciano , Anciano de 80 o más Años , Southern Blotting , ADN/genética , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Patrón de Herencia , Masculino , Repeticiones de Microsatélite , Persona de Mediana Edad , Linaje , Penetrancia , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Estudios Prospectivos , Adulto JovenRESUMEN
Prostaglandin F2 alpha (PGF2α) analogues such as latanoprost are common first-line intraocular pressure (IOP) lowering medications. However, their clinical use is limited in some patient populations due to minimal or no IOP lowering response or side effects. In searching for a more targeted approach for IOP reduction, our lab recently identified Stanniocalcin-1 (STC-1) as a molecule that was required for latanoprost-mediated IOP reduction and also acted as a stand-alone IOP lowering agent. In order to determine whether latanoprost and STC-1 were equivalent and/or additive for IOP reduction, we treated C57BL/6J mice with one or a combination of these agents and measured IOP. Importance of the FP receptor for latanoprost- and STC-1-mediated IOP reduction was examined in C57BL/6J mice utilizing the pharmacologic FP receptor inhibitor AL-8810 as well as FP receptor knockout mice generated in our laboratory. Latanoprost-free acid (LFA) and STC-1 reduced IOP to a similar degree and were non-additive in C57BL/6J mice. As expected, the IOP lowering effects of LFA were abrogated by pharmacologic inhibition of the FP receptor with AL-8810 and in FP receptor knockout mice. In contrast, STC-1 maintained IOP-lowering effects in the presence of AL-8810 and also in FP receptor knockout mice. These results suggest that LFA and STC-1 show equivalent and non-additive IOP reduction in C57BL/6J mice and that unlike LFA, STC-1-mediated IOP reduction occurs independent of the FP receptor.
Asunto(s)
Glicoproteínas/fisiología , Presión Intraocular/efectos de los fármacos , Latanoprost/metabolismo , Receptores de Prostaglandina/fisiología , Animales , Dinoprost/análogos & derivados , Dinoprost/farmacología , Femenino , Genotipo , Homocigoto , Latanoprost/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Prostaglandina/genética , Transducción de SeñalRESUMEN
Elevated intraocular pressure is the only treatable risk factor for glaucoma, an eye disease that is the leading cause of irreversible blindness worldwide. We have identified cromakalim prodrug 1 (CKLP1), a novel water-soluble ATP-sensitive potassium channel opener, as a new ocular hypotensive agent. To evaluate the pharmacokinetic and safety profile of CKLP1 and its parent compound levcromakalim, Dutch-belted pigmented rabbits were treated intravenously (0.25 mg/kg) or topically (10 mM; 4.1 mg/ml) with CKLP1. Body fluids (blood, aqueous and vitreous humor) were collected at multiple time points and evaluated for the presence of CKLP1 and levcromakalim using a liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) based assay. Histology of tissues isolated from Dutch-belted pigmented rabbits treated once daily for 90 days was evaluated in a masked manner by a certified veterinary pathologist. The estimated plasma parameters following intravenous administration of 0.25 mg/kg of CKLP1 showed CKLP1 had a terminal half-life of 61.8 ± 55.2 min, Tmax of 19.8 ± 23.0 min and Cmax of 1968.5 ± 831.0 ng/ml. Levcromakalim had a plasma terminal half-life of 85.0 ± 37.0 min, Tmax of 61.0 ± 32.0 min and Cmax of 10.6 ± 1.2 ng/ml. Topical CKLP1 treatment in the eye showed low levels (<0.3 ng/mL) of levcromakalim in aqueous and vitreous humor, and trace amounts of CKLP1 and levcromakalim in the plasma. No observable histological changes were noted in selected tissues that were examined following topical application of CKLP1 for 90 consecutive days. These results suggest that CKPL1 is converted to levcromakalim in the eye and likely to some extent in the systemic circulation.
Asunto(s)
Cromakalim/farmacología , Cromakalim/farmacocinética , Profármacos/farmacología , Profármacos/farmacocinética , Administración Intravenosa , Administración Tópica , Animales , Humor Acuoso/efectos de los fármacos , Humor Acuoso/metabolismo , Cromatografía Liquida , Córnea/citología , Córnea/efectos de los fármacos , Cromakalim/administración & dosificación , Cromakalim/sangre , Ojo/citología , Ojo/efectos de los fármacos , Ojo/metabolismo , Femenino , Espectrometría de Masas , Profármacos/uso terapéutico , Conejos , Cuerpo Vítreo/efectos de los fármacos , Cuerpo Vítreo/metabolismoRESUMEN
Purpose: The strongest genetic association with Fuchs' endothelial corneal dystrophy (FECD) is the presence of an intronic (CTG·CAG)n trinucleotide repeat (TNR) expansion in the transcription factor 4 (TCF4) gene. Repeat-associated non-ATG (RAN) translation, an unconventional protein translation mechanism that does not require an initiating ATG, has been described in many TNR expansion diseases, including myotonic dystrophy type 1 (DM1). Given the similarities between DM1 and FECD, we wished to determine whether RAN translation occurs in FECD. Methods: Antibodies against peptides in the C-terminus of putative RAN translation products from TCF4 were raised and validated by Western blotting and immunofluorescence (IF). CTG·CAG repeats of various lengths in the context of the TCF4 gene were cloned in frame with a 3× FLAG tag and transfected in human cells. IF with antipeptide and anti-FLAG antibodies, as well as cytotoxicity and cell proliferation assays, were performed in these transfected cells. Corneal endothelium derived from patients with FECD was probed with validated antibodies by IF. Results: CTG·CAG repeats in the context of the TCF4 gene are transcribed and translated via non-ATG initiation in transfected cells and confer toxicity to an immortalized corneal endothelial cell line. An antipeptide antibody raised against the C-terminus of the TCF4 poly-cysteine frame recognized RAN translation products by IF in cells transfected with CTG·CAG repeats and in FECD corneal endothelium. Conclusions: Expanded CTG·CAG repeats in the context of the third intron of TCF4 are transcribed and translated via non-ATG initiation, providing evidence for RAN translation in corneal endothelium of patients with FECD.
Asunto(s)
Distrofia Endotelial de Fuchs/genética , Biosíntesis de Proteínas , Factor de Transcripción 4/genética , Expansión de Repetición de Trinucleótido/genética , Western Blotting , Proliferación Celular , Células Cultivadas , Endotelio Corneal/metabolismo , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Expresión Génica/fisiología , Humanos , Intrones , Reacción en Cadena en Tiempo Real de la Polimerasa , TransfecciónRESUMEN
Purpose: Cromakalim prodrug 1 (CKLP1) is a water-soluble ATP-sensitive potassium channel opener that has shown ocular hypotensive properties in ex vivo and in vivo experimental models. To determine its mechanism of action, we assessed the effect of CKLP1 on aqueous humor dynamics and in combination therapy with existing ocular hypotensive agents. Methods: Outflow facility was assessed in C57BL/6 mice by ex vivo eye perfusions and by in vivo constant flow infusion following CKLP1 treatment. Human anterior segments with no trabecular meshwork were evaluated for effect on pressure following CKLP1 treatment. CKLP1 alone and in combination with latanoprost, timolol, and Rho kinase inhibitor Y27632 were evaluated for effect on intraocular pressure in C57BL/6 mice and Dutch-belted pigmented rabbits. Results: CKLP1 lowered episcleral venous pressure (control: 8.9 ± 0.1 mm Hg versus treated: 6.2 ± 0.1 mm Hg, P < 0.0001) but had no detectable effect on outflow facility, aqueous humor flow rate, or uveoscleral outflow. Treatment with CKLP1 in human anterior segments without the trabecular meshwork resulted in a 50% ± 9% decrease in pressure, suggesting an effect on the distal portion of the conventional outflow pathway. CKLP1 worked additively with latanoprost, timolol, and Y27632 to lower IOP, presumably owing to combined effects on different aspects of aqueous humor dynamics. Conclusions: CKLP1 lowered intraocular pressure by reducing episcleral venous pressure and lowering distal outflow resistance in the conventional outflow pathway. Owing to this unique mechanism of action, CKLP1 works in an additive manner to lower intraocular pressure with latanoprost, timolol, and Rho kinase inhibitor Y27632.
Asunto(s)
Antihipertensivos/uso terapéutico , Humor Acuoso/fisiología , Cromakalim/uso terapéutico , Presión Intraocular/efectos de los fármacos , Profármacos/uso terapéutico , Amidas/uso terapéutico , Animales , Segmento Anterior del Ojo/efectos de los fármacos , Sinergismo Farmacológico , Quimioterapia Combinada , Femenino , Humanos , Latanoprost , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Soluciones Oftálmicas , Prostaglandinas F Sintéticas/uso terapéutico , Piridinas/uso terapéutico , Conejos , Esclerótica/irrigación sanguínea , Timolol/uso terapéutico , Tonometría Ocular , Presión Venosa/efectos de los fármacosRESUMEN
For many emerging and re-emerging infectious diseases, definitive solutions via sterilizing adaptive immunity may require years or decades to develop, if they are even possible. The innate immune system offers alternative mechanisms that do not require antigen-specific recognition or a priori knowledge of the causative agent. However, it is unclear whether effective stable innate immune system activation can be achieved without triggering harmful autoimmunity or other chronic inflammatory sequelae. Here, we show that transgenic expression of a picornavirus RNA-dependent RNA polymerase (RdRP), in the absence of other viral proteins, can profoundly reconfigure mammalian innate antiviral immunity by exposing the normally membrane-sequestered RdRP activity to sustained innate immune detection. RdRP-transgenic mice have life-long, quantitatively dramatic upregulation of 80 interferon-stimulated genes (ISGs) and show profound resistance to normally lethal viral challenge. Multiple crosses with defined knockout mice (Rag1, Mda5, Mavs, Ifnar1, Ifngr1, and Tlr3) established that the mechanism operates via MDA5 and MAVS and is fully independent of the adaptive immune system. Human cell models recapitulated the key features with striking fidelity, with the RdRP inducing an analogous ISG network and a strict block to HIV-1 infection. This RdRP-mediated antiviral mechanism does not depend on secondary structure within the RdRP mRNA but operates at the protein level and requires RdRP catalysis. Importantly, despite lifelong massive ISG elevations, RdRP mice are entirely healthy, with normal longevity. Our data reveal that a powerfully augmented MDA5-mediated activation state can be a well-tolerated mammalian innate immune system configuration. These results provide a foundation for augmenting innate immunity to achieve broad-spectrum antiviral protection.
Asunto(s)
Genes Virales/inmunología , Inmunidad Innata/inmunología , ARN Polimerasa Dependiente del ARN/inmunología , Proteínas Virales/inmunología , Animales , Western Blotting , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunidad Innata/genética , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Picornaviridae/genética , Picornaviridae/inmunología , ARN Polimerasa Dependiente del ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Virosis/inmunología , Virosis/prevención & controlRESUMEN
Antibody-based therapeutics have now had success in the clinic. The affinity and specificity of the antibody for the target ligand determines the specificity of therapeutic delivery and off-target side effects. The discovery and optimization of high-affinity antibodies to important therapeutic targets could be significantly improved by the availability of a robust, eukaryotic display technology comparable to phage display that would overcome the protein translation limitations of microorganisms. The use of eukaryotic cells would improve the diversity of the displayed antibodies that can be screened and optimized as well as more seamlessly transition into a large-scale mammalian expression system for clinical production. In this study, we demonstrate that the replication and polypeptide display characteristics of a eukaryotic retrovirus, avian leukosis virus (ALV), offers a robust, eukaryotic version of bacteriophage display. The binding affinity of a model single-chain Fv antibody was optimized by using ALV display, improving affinity >2,000-fold, from micromolar to picomolar levels. We believe ALV display provides an extension to antibody display on microorganisms and offers virus and cell display platforms in a eukaryotic expression system. ALV display should enable an improvement in the diversity of properly processed and functional antibody variants that can be screened and affinity-optimized to improve promising antibody candidates.
Asunto(s)
Anticuerpos/metabolismo , Virus de la Leucosis Aviar/metabolismo , Técnicas de Visualización de Superficie Celular/métodos , Células Eucariotas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Pollos , Regiones Determinantes de Complementariedad , Glicoproteínas/metabolismo , Humanos , Cinética , Laminina/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Anticuerpos de Cadena Única/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Virión/metabolismo , Replicación ViralRESUMEN
Ocular hypertension arising from increased resistance to aqueous humor (AH) outflow through the trabecular meshwork is a primary risk factor for open-angle glaucoma, a leading cause of blindness. Ongoing efforts have found little about the molecular and cellular bases of increased resistance to AH outflow through the trabecular meshwork in ocular hypertension patients. To test the hypothesis that dysregulated Rho GTPase signaling and a resulting fibrotic activity within the trabecular meshwork may result in ocular hypertension, we investigated the effects of expressing a constitutively active RhoA GTPase (RhoAV14) in the AH outflow pathway in Sprague-Dawley rats by using lentiviral vector-based gene delivery. Rats expressing RhoAV14 in the iridocorneal angle exhibited a significantly elevated intraocular pressure. Elevated intraocular pressure in the RhoAV14-expressing rats was associated with fibrotic trabecular meshwork and increased levels of F-actin, phosphorylated myosin light chain, α-smooth muscle actin, collagen-1A, and total collagen in the trabecular AH outflow pathway. Most of these changes were ameliorated by topical application of Rho kinase inhibitor. Human autopsy eyes from patients with glaucoma exhibited significant increases in levels of collagen-1A and total collagen in the trabecular AH outflow pathway. Collectively, these observations indicate that increased fibrogenic activity because of dysregulated RhoA GTPase activity in the trabecular AH outflow pathway increases intraocular pressure in a Rho kinase-dependent manner.
Asunto(s)
Colágeno/biosíntesis , Proteínas del Ojo/metabolismo , Mutación Missense , Hipertensión Ocular/enzimología , Malla Trabecular/enzimología , Proteína de Unión al GTP rhoA/metabolismo , Sustitución de Aminoácidos , Animales , Colágeno/genética , Proteínas del Ojo/antagonistas & inhibidores , Proteínas del Ojo/genética , Femenino , Humanos , Hipertensión Ocular/tratamiento farmacológico , Hipertensión Ocular/genética , Hipertensión Ocular/patología , Ratas , Ratas Sprague-Dawley , Malla Trabecular/patología , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/genéticaRESUMEN
Studies of the domestic cat have contributed to many scientific advances, including the present understanding of the mammalian cerebral cortex. A practical capability for cat transgenesis is needed to realize the distinctive potential of research on this neurobehaviorally complex, accessible species for advancing human and feline health. For example, humans and cats are afflicted with pandemic AIDS lentiviruses that are susceptible to species-specific restriction factors. Here we introduced genes encoding such a factor, rhesus macaque TRIMCyp, and eGFP, into the cat germline. The method establishes gamete-targeted transgenesis for the first time in a carnivore. We observed uniformly transgenic outcomes, widespread expression, no mosaicism and no F1 silencing. TRIMCyp transgenic cat lymphocytes resisted feline immunodeficiency virus replication. This capability to experimentally manipulate the genome of an AIDS-susceptible species can be used to test the potential of restriction factors for HIV gene therapy and to build models of other infectious and noninfectious diseases.
Asunto(s)
Animales Modificados Genéticamente , Gatos/genética , Gatos/virología , Técnicas de Transferencia de Gen , Virus de la Inmunodeficiencia Felina/genética , Animales , Modelos Animales de Enfermedad , Femenino , Linfocitos/inmunología , Masculino , Replicación ViralRESUMEN
Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide, with elevated intraocular pressure as an important risk factor. Increased resistance to outflow of aqueous humor through the trabecular meshwork causes elevated intraocular pressure, but the specific mechanisms are unknown. In this study, we used genome-wide SNP arrays to map the disease gene in a colony of Beagle dogs with inherited POAG to within a single 4 Mb locus on canine chromosome 20. The Beagle POAG locus is syntenic to a previously mapped human quantitative trait locus for intraocular pressure on human chromosome 19. Sequence capture and next-generation sequencing of the entire canine POAG locus revealed a total of 2,692 SNPs segregating with disease. Of the disease-segregating SNPs, 54 were within exons, 8 of which result in amino acid substitutions. The strongest candidate variant causes a glycine to arginine substitution in a highly conserved region of the metalloproteinase ADAMTS10. Western blotting revealed ADAMTS10 protein is preferentially expressed in the trabecular meshwork, supporting an effect of the variant specific to aqueous humor outflow. The Gly661Arg variant in ADAMTS10 found in the POAG Beagles suggests that altered processing of extracellular matrix and/or defects in microfibril structure or function may be involved in raising intraocular pressure, offering specific biochemical targets for future research and treatment strategies.
Asunto(s)
Proteínas ADAM/genética , Mapeo Cromosómico , Estudios de Asociación Genética , Sitios Genéticos/genética , Glaucoma de Ángulo Abierto/enzimología , Glaucoma de Ángulo Abierto/genética , Proteínas ADAM/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromosomas de los Mamíferos/genética , Modelos Animales de Enfermedad , Perros , Femenino , Ligamiento Genético , Genoma/genética , Glaucoma de Ángulo Abierto/fisiopatología , Haplotipos/genética , Humanos , Presión Intraocular/genética , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Linaje , Polimorfismo de Nucleótido Simple/genética , Sitios de Carácter Cuantitativo/genética , Malla Trabecular/metabolismo , Malla Trabecular/patologíaRESUMEN
PURPOSE: To investigate the possibility that Angiopoietin-like 7 (ANGPTL7) protein is involved in the pathogenesis of glaucoma. METHODS: Primary human trabecular meshwork (TM) cells and corneoscleral explants were stimulated with either dexamethasone (DEX) or transforming growth factor beta (TGFbeta), and ANGPTL7 protein secreted into culture medium was determined by Western blot analysis. The effect of stable overexpression of ANGPTL7 in transfected immortalized TM cell lines on collagen expression was investigated by immunocytochemistry. Localization of ANGPTL7 protein in human eyes was determined by immunohistochemistry. The concentration of ANGPTL7 protein in aqueous humor (AH) from patients with glaucoma and control patients was compared by Western blot analysis. The beagle model of primary open-angle glaucoma (POAG) was used to correlate ANGPTL7 protein levels in canine AH with disease progression. RESULTS: TGFbeta and DEX stimulated secretion of ANGPTL7 protein by TM cells and corneoscleral explants. Overexpression of ANGPTL7 by immortalized TM cell lines increased expression of type I collagen. Expression of ANGPTL7 protein was located in the corneal stroma, near the limbus, and throughout the sclera, with lower expression in the TM. In the lamina cribrosa, ANGPTL7 expression was associated with the cribriform plates. The concentration of ANGPTL7 protein was elevated in AH from patients with glaucoma and increased as disease progressed in POAG beagle dogs. CONCLUSIONS: Induction of ANGPTL7 secretion by glaucoma stimuli and increased concentration of ANGPTL7 in glaucomatous AH suggest that ANGPTL7 is overexpressed in glaucoma. Since overexpression of ANGPTL7 increases collagen expression, a potential disease mechanism, ANGPTL7 could have a pathogenic role in glaucoma, and may serve as a potential therapeutic target.