ABSTRACT
The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) system represents a powerful gene-editing tool and could enable treatment of blinding diseases of the retina. As a peptide of bacterial origin, we investigated the immunogenic potential of Cas9 in models of retinal immunocompetent cells: human microglia (IMhu) and ARPE-19 cells. Transfection with Streptococcus pyogenes-Cas9 expression plasmids (SpCas9 plasmid) induced Cas9 protein expression in both cell lines. However, only ARPE-19 cells, not IMhu cells, responded with pro-inflammatory immune responses as evidenced by the upregulation of IL-8, IL-6, and the cellular activation markers HLA-ABC and CD54 (ICAM). These pro-inflammatory responses were also induced through transfection with equally sized non-coding control plasmids. Moreover, viability rates of ARPE-19 cells were reduced after transfection with both the SpCas9 plasmids and the control plasmids. Although these results demonstrate cell type-specific responses to the DNA plasmid vector, they show no evidence of an immunogenic effect due to the presence of Cas9 in models of human retinal pigment epithelial and microglia cells. These findings add another layer of confidence in the immunological safety of potential future Cas9-mediated retinal gene therapies.
Subject(s)
Bacterial Proteins , CRISPR-Cas Systems , Bacterial Proteins/metabolism , Epithelial Cells/metabolism , Gene Editing/methods , Humans , Immunity , Plasmids/genetics , Retinal PigmentsABSTRACT
Retinal gene therapy has recently been at the cutting edge of clinical development in the diverse field of genetic therapies. The retina is an attractive target for genetic therapies such as gene editing due to the distinctive anatomical and immunological features of the eye, known as immune privilege, so that inherited retinal diseases (IRDs) have been studied in several clinical studies. Thus, rapid strides are being made toward developing targeted treatments for IRDs. Gene editing in the retina faces a group of heterogenous challenges, including editing efficiencies, off-target effects, the anatomy of the target organ, immune responses, inactivation, and identifying optimal application methods. As clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) based technologies are at the forefront of current gene editing advances, their specific editing efficiency challenges and potential off-target effects were assessed. The immune privilege of the eye reduces the likelihood of systemic immune responses following retinal gene therapy, but possible immune responses must not be discounted. Immune responses to gene editing in the retina may be humoral or cell mediated, with immunologically active cells, including microglia, implicated in facilitating possible immune responses to gene editing. Immunogenicity of gene therapeutics may also lead to the inactivation of edited cells, reducing potential therapeutic benefits. This review outlines the broad spectrum of potential challenges currently facing retinal gene editing, with the goal of facilitating further advances in the safety and efficacy of gene editing therapies.
Subject(s)
Gene Editing , Retinal Diseases , CRISPR-Cas Systems/genetics , Gene Editing/methods , Genetic Therapy/methods , Humans , Retina , Retinal Diseases/geneticsABSTRACT
El objetivo de este trabajo fue estudiar el efecto de la exposición a plaguicidas en las funciones cognitivas y motoras de 42 niños de una zona rural con mayor probabilidad de exposición a plaguicidas organofosforados y carbámicos (Yuto, Jujuy). Los resultados de las pruebas realizadas para estudiar el efecto fueron comparados con los resultados obtenidos con 29 niños con menor exposición a plaguicidas (León, Jujuy). A todos los participantes se les realizó historia clínica, evaluación médica, revisión de antecedentes, pruebas neuroconductuales, de motricidad gruesa y equilibrio, y medición de biomarcadores (colinesterasa plasmática y eritrocitaria). Se realizó una historia clínica neurológica estándar con las siguientes pruebas: Subtest de Dígitos y Símbolos, Subtest de Recuerdo de Dígitos, Subtest de Laberinto del WISC-III de Wechsler. En la prueba de Laberintos realizada en el presente estudio se observan diferencias entre las localidades y se comprobó una relación lineal inversa entre los años de residencia y la puntuación de la prueba. Se observó reducción en las actividades de acetilcolinesterasas en los niños pertenecientes a Yuto. No se observó una correlación signifca-tiva entre los niveles de actividad de las enzimas y el rendimiento en la prueba, para ninguna de las dos localidades. Los resultados señalaron la probabilidad de que la exposición crónica a bajas dosis de plaguicidas pudiera estar dañando algunas funciones cognitivas y que esto dependería de la duración de la exposición. Por falta de correlación entre las actividades de las enzimas y los resultados adversos en las pruebas neuroconductuales de motricidad y de equilibrio, no fue evidente una asociación entre ambos.
The aim of this research was to study the effects of these toxics on the cognitive and motor functions of a group of 42 children of a rural town with high percentage of exposure to organophosphate and carbamic pesticide (Yuto) and compare the results with a group of 29 children with low percentage of exposure to pesticides. In both cases a research of the patients history was done, a medical revision and a full checkup including neurobehavioral tests, motor and balance tests and biomarkers levels (plasma pseudocholinesterase and erythrocyte cholinesterase). The behavioral assessment was performed by a standard neurological clinic history, a digital and symbol subtest, a digital memory, a maze test, intelligence test WISC-III of Wechsler and gross motor and balance tests. In the group from Yuto, Pearson correlations were done between the years of residency and the neurobehavioral tests and the linear correlation between the years of residency and the acetylcholinesterase activities. In the maze test results there were differences between the two towns and it was proved an inverse lineal relation between the years of residency and the tests scores. It was also observed a reduction in the acetylcholinesterase activities both erythrocyte and plasma in the children from Yuto. Nevertheless there were not signifcant correlations between the levels of enzyme activities and the performance in the tests for neither of the towns. The results achieved did not point out that chronic exposure to low-dose pesticides can produce cognitive malfunctions, such as psychomotor problems. The lack of correlations between the activities of the acetylcholinesterase enzyme and the negative results of the neurobehavioral motor and balance tests couldn´t be associated.
Subject(s)
Humans , Male , Female , Child , Neurotoxicity Syndromes/epidemiology , Organophosphate Poisoning/complications , Organophosphorus Compounds/toxicity , Pesticides/toxicity , Argentina/epidemiology , Cholinesterases/blood , Neuropsychological TestsABSTRACT
El objetivo de este trabajo fue estudiar el efecto de la exposición a plaguicidas en las funciones cognitivas y motoras de 42 niños de una zona rural con mayor probabilidad de exposición a plaguicidas organofosforados y carbámicos (Yuto, Jujuy). Los resultados de las pruebas realizadas para estudiar el efecto fueron comparados con los resultados obtenidos con 29 niños con menor exposición a plaguicidas (León, Jujuy). A todos los participantes se les realizó historia clínica, evaluación médica, revisión de antecedentes, pruebas neuroconductuales, de motricidad gruesa y equilibrio, y medición de biomarcadores (colinesterasa plasmática y eritrocitaria). Se realizó una historia clínica neurológica estándar con las siguientes pruebas: Subtest de Dígitos y Símbolos, Subtest de Recuerdo de Dígitos, Subtest de Laberinto del WISC-III de Wechsler. En la prueba de Laberintos realizada en el presente estudio se observan diferencias entre las localidades y se comprobó una relación lineal inversa entre los años de residencia y la puntuación de la prueba. Se observó reducción en las actividades de acetilcolinesterasas en los niños pertenecientes a Yuto. No se observó una correlación signifca-tiva entre los niveles de actividad de las enzimas y el rendimiento en la prueba, para ninguna de las dos localidades. Los resultados señalaron la probabilidad de que la exposición crónica a bajas dosis de plaguicidas pudiera estar dañando algunas funciones cognitivas y que esto dependería de la duración de la exposición. Por falta de correlación entre las actividades de las enzimas y los resultados adversos en las pruebas neuroconductuales de motricidad y de equilibrio, no fue evidente una asociación entre ambos.(AU)
The aim of this research was to study the effects of these toxics on the cognitive and motor functions of a group of 42 children of a rural town with high percentage of exposure to organophosphate and carbamic pesticide (Yuto) and compare the results with a group of 29 children with low percentage of exposure to pesticides. In both cases a research of the patients history was done, a medical revision and a full checkup including neurobehavioral tests, motor and balance tests and biomarkers levels (plasma pseudocholinesterase and erythrocyte cholinesterase). The behavioral assessment was performed by a standard neurological clinic history, a digital and symbol subtest, a digital memory, a maze test, intelligence test WISC-III of Wechsler and gross motor and balance tests. In the group from Yuto, Pearson correlations were done between the years of residency and the neurobehavioral tests and the linear correlation between the years of residency and the acetylcholinesterase activities. In the maze test results there were differences between the two towns and it was proved an inverse lineal relation between the years of residency and the tests scores. It was also observed a reduction in the acetylcholinesterase activities both erythrocyte and plasma in the children from Yuto. Nevertheless there were not signifcant correlations between the levels of enzyme activities and the performance in the tests for neither of the towns. The results achieved did not point out that chronic exposure to low-dose pesticides can produce cognitive malfunctions, such as psychomotor problems. The lack of correlations between the activities of the acetylcholinesterase enzyme and the negative results of the neurobehavioral motor and balance tests couldn´t be associated.(AU)
