RESUMO
Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a rare neurodevelopmental disorder caused by a mutation in the X-linked CDKL5 gene. CDKL5 is a serine/threonine kinase that is critical for axon outgrowth and dendritic morphogenesis as well as synapse formation, maturation, and maintenance. This disorder is characterized by early-onset epilepsy, hypotonia, and failure to reach cognitive and motor developmental milestones. Because the disease is monogenic, delivery of the CDKL5 gene to the brain of patients should provide clinical benefit. To this end, we designed a gene therapy vector, adeno-associated virus (AAV)9.Syn.hCDKL5, in which human CDKL5 gene expression is driven by the synapsin promoter. In biodistribution studies conducted in mice, intracerebroventricular (i.c.v.) injection resulted in broader, more optimal biodistribution than did intra-cisterna magna (i.c.m.) delivery. AAV9.Syn.hCDKL5 treatment increased phosphorylation of EB2, a bona fide CDKL5 substrate, demonstrating biological activity in vivo. Our data provide proof of concept that i.c.v. delivery of AAV9.Syn.hCDKL5 to neonatal male Cdkl5 knockout mice reduces pathology and reduces aberrant behavior. Functional improvements were seen at doses of 3e11 to 5e11 vector genomes/g brain, which resulted in transfection of ≥50% of the neurons. Functional improvements were not seen at lower doses, suggesting a requirement for broad distribution for efficacy.
Assuntos
Dependovirus , Modelos Animais de Doenças , Síndromes Epilépticas , Terapia Genética , Vetores Genéticos , Proteínas Serina-Treonina Quinases , Espasmos Infantis , Animais , Terapia Genética/métodos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Camundongos , Vetores Genéticos/genética , Vetores Genéticos/administração & dosagem , Humanos , Dependovirus/genética , Síndromes Epilépticas/terapia , Síndromes Epilépticas/genética , Espasmos Infantis/terapia , Espasmos Infantis/genética , Espasmos Infantis/metabolismo , Encéfalo/metabolismo , Masculino , Fosforilação , Sinapsinas/genética , Sinapsinas/metabolismo , Deficiência Intelectual Ligada ao Cromossomo X/terapia , Deficiência Intelectual Ligada ao Cromossomo X/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Regiões Promotoras Genéticas , Distribuição Tecidual , Expressão GênicaRESUMO
BACKGROUND: The origins of life on the Earth required chemical entities to interact with their environments in ways that could respond to natural selection. The concept of interpretation, where biotic entities use signs in their environment as proxy for the existence of other items of selective value in their environment, has been proposed on theoretical grounds to be relevant to the origins and early evolution of life. However this concept has not been demonstrated empirically. RESULTS: Here, we present data that certain catalytic RNA sequences have properties that would enable interpretation of divalent cation levels in their environment. By assaying the responsiveness of two variants of the Tetrahymena ribozyme to the Ca(2+) ion as a sign for the more catalytically useful Mg(2+) ion, we show an empirical proof-of-principle that interpretation can be an evolvable trait in RNA, often suggested as a model system for early life. In particular we demonstrate that in vitro, the wild-type version of the Tetrahymena ribozyme is not interpretive, in that it cannot use Ca(2+) as a sign for Mg(2+). Yet a variant of this sequence containing five mutations that alter its ability to utilize the Ca(2+) ion engenders a strong interpretive characteristic in this RNA. CONCLUSIONS: We have shown that RNA molecules in a test tube can meet the minimum criteria for the evolution of interpretive behaviour in regards to their responses to divalent metal ion concentrations in their environment. Interpretation in RNA molecules provides a property entirely dependent on natural physico-chemical interactions, but capable of shaping the evolutionary trajectory of macromolecules, especially in the earliest stages of life's history.
Assuntos
Cátions Bivalentes/metabolismo , Evolução Molecular , RNA Catalítico/genética , RNA Catalítico/metabolismo , Tetrahymena/genética , Sequência de Bases , Dados de Sequência Molecular , Origem da Vida , RNA Catalítico/química , Tetrahymena/enzimologiaRESUMO
Unlike with zinc finger nuclease and transcriptional activator-like effector nuclease DNA modification technologies that rely on lead proteins, developed through expensive and time-consuming processes, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system has rapidly emerged as the most promising gene-editing technology to date for the modification of any selected DNA sequence. CRISPR is receiving tremendous fanfare due, in part, to its potential to provide a means to fundamentally alter medical genetics and especially cancer medicine. In this review, we compare key technologies of genome-editing zinc finger nucleases, transcriptional activator-like effector nucleases and CRISPR, with a focus on the race to acquire lucrative intellectual property rights, the current CRISPR patent dispute and potential repercussions on innovation and the adoption of this promising technology by the medical community.