RESUMO
Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by a mutation or deletion of the maternally inherited UBE3A allele. In neurons, the paternally inherited UBE3A allele is silenced in cis by a long non-coding RNA called UBE3A-ATS. Here, as part of a systematic screen, we found that Cas9 can be used to activate ('unsilence') paternal Ube3a in cultured mouse and human neurons when targeted to Snord115 genes, which are small nucleolar RNAs that are clustered in the 3' region of Ube3a-ATS. A short Cas9 variant and guide RNA that target about 75 Snord115 genes were packaged into an adeno-associated virus and administered to a mouse model of AS during the embryonic and early postnatal stages, when the therapeutic benefit of restoring Ube3a is predicted to be greatest1,2. This early treatment unsilenced paternal Ube3a throughout the brain for at least 17 months and rescued anatomical and behavioural phenotypes in AS mice. Genomic integration of the adeno-associated virus vector into Cas9 target sites caused premature termination of Ube3a-ATS at the vector-derived polyA cassette, or when integrated in the reverse orientation, by transcriptional collision with the vector-derived Cas9 transcript. Our study shows that targeted genomic integration of a gene therapy vector can restore the function of paternally inherited UBE3A throughout life, providing a path towards a disease-modifying treatment for a syndromic neurodevelopmental disorder.
Assuntos
Síndrome de Angelman/genética , Síndrome de Angelman/terapia , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Edição de Genes , Terapia Genética/métodos , RNA Longo não Codificante/genética , Ubiquitina-Proteína Ligases/genética , Animais , Proteína 9 Associada à CRISPR/genética , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Inativação Gênica , Vetores Genéticos/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Sistema Nervoso/metabolismo , Herança Paterna/genética , Fenótipo , RNA Guia de Cinetoplastídeos/genéticaRESUMO
ABSTRACT: Facial grimacing is used to quantify spontaneous pain in mice and other mammals, but scoring relies on humans with different levels of proficiency. Here, we developed a cloud-based software platform called PainFace (http://painface.net) that uses machine learning to detect 4 facial action units of the mouse grimace scale (orbitals, nose, ears, whiskers) and score facial grimaces of black-coated C57BL/6 male and female mice on a 0 to 8 scale. Platform accuracy was validated in 2 different laboratories, with 3 conditions that evoke grimacing-laparotomy surgery, bilateral hindpaw injection of carrageenan, and intraplantar injection of formalin. PainFace can generate up to 1 grimace score per second from a standard 30 frames/s video, making it possible to quantify facial grimacing over time, and operates at a speed that scales with computing power. By analyzing the frequency distribution of grimace scores, we found that mice spent 7x more time in a "high grimace" state following laparotomy surgery relative to sham surgery controls. Our study shows that PainFace reproducibly quantifies facial grimaces indicative of nonevoked spontaneous pain and enables laboratories to standardize and scale-up facial grimace analyses.