RESUMO
Clustered regularly interspaced short palindromic repeat (CRISPR) has arisen as a frontrunner for efficient genome engineering. However, the potentially broad therapeutic implications are largely unexplored. Here, to investigate the therapeutic potential of CRISPR/Cas9 in a diverse set of genetic disorders, we establish a pipeline that uses readily obtainable cells from affected individuals. We show that an adapted version of CRISPR/Cas9 increases the amount of utrophin, a known disease modifier in Duchenne muscular dystrophy (DMD). Furthermore, we demonstrate preferential elimination of the dominant-negative FGFR3 c.1138G>A allele in fibroblasts of an individual affected by achondroplasia. Using a previously undescribed approach involving single guide RNA, we successfully removed large genome rearrangement in primary cells of an individual with an X chromosome duplication including MECP2. Moreover, removal of a duplication of DMD exons 18-30 in myotubes of an individual affected by DMD produced full-length dystrophin. Our findings establish the far-reaching therapeutic utility of CRISPR/Cas9, which can be tailored to target numerous inherited disorders.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Doenças Genéticas Inatas/terapia , Alelos , Expressão Gênica , Doenças Genéticas Inatas/genética , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapiaRESUMO
Becker Muscular Dystrophy (BMD) is a rare X-linked recessive neuromuscular disorder frequently caused by in-frame deletions in the DMD gene that result in the production of a truncated, yet functional, dystrophin protein. The consequences of BMD-causing in-frame deletions on the organism are difficult to predict, especially in regard to long-term prognosis. Here, we employed CRISPR-Cas9 to generate a new Dmd del52-55 mouse model by deleting exons 52-55, resulting in a BMD-like in-frame deletion. To delineate the long-term effects of this deletion, we studied these mice over 52 weeks by performing histology and echocardiography analyses and assessing motor functions. Our results suggest that a truncated dystrophin is sufficient to maintain wildtype-like muscle and heart histology and functions in young mice. However, the truncated protein appears insufficient to maintain normal muscle homeostasis and protect against exercise-induced damage at 52 weeks. To further delineate the effects of this exon52-55 in-frame deletion, we performed RNA-Seq pre- and post-exercise and identified several differentially expressed pathways that reflect the abnormal muscle phenotype observed at 52 weeks in the BMD model.
RESUMO
Developing safe and effective strategies to deliver biomolecules such as oligonucleotides and proteins into cells has grown in importance over recent years, with an increasing demand for non-viral methods that enable clinical translation. Here, we investigate uniquely configured oligo-urethane nanoparticles based on synthetic chemistries that minimize the release of pro-inflammatory biomarkers from immune cells, show low cytotoxicity in a broad range of cells, and efficiently deliver oligonucleotides and proteins into mammalian cells. The mechanism of cell uptake for the self-assembled oligo-urethane nanoparticles was shown to be directed by caveolae-dependent endocytosis in murine myoblasts (C2C12) cells. Inhibiting caveolae functions with genistein and methyl-ß-cyclodextrin limited nanoparticle internalization. The nanoparticles showed a very high delivery efficiency for the genetic material (a 47-base oligonucleotide) (â¼80% incorporation into cells) as well as the purified protein (full length firefly luciferase, 67 kDa) into human embryonic kidney (HEK293T) cells. Luciferase enzyme activity in HEK293T cells demonstrated that intact and functional proteins could be delivered and showed a significant extension of activity retention up to 24 h, well beyond the 2 h half-life of the free enzyme. This study introduces a novel self-assembled oligo-urethane nanoparticle delivery platform with very low associated production costs, enabled by their scalable chemistry (the benchwork cost is $ 0.152/mg vs $ 974.6/mg for typical lipid carriers) that has potential to deliver both oligonucleotides and proteins for biomedical purposes.
Assuntos
Materiais Biocompatíveis/química , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Oligonucleotídeos/química , Animais , Materiais Biocompatíveis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células HEK293 , Humanos , Luciferases/metabolismo , Teste de Materiais , Camundongos , Estrutura Molecular , Oligonucleotídeos/genética , Oligonucleotídeos/farmacologiaRESUMO
Tandem duplication mutations are increasingly found to be the direct cause of many rare heritable diseases, accounting for up to 10% of cases. Unfortunately, animal models recapitulating such mutations are scarce, limiting our ability to study them and develop genome editing therapies. Here, we describe the generation of a novel duplication mouse model, harboring a multi-exonic tandem duplication in the Dmd gene which recapitulates a human mutation. Duplication correction of this mouse was achieved by implementing a single-guide RNA (sgRNA) CRISPR/Cas9 approach. This strategy precisely removed a duplication mutation in vivo, restored full-length dystrophin expression, and was accompanied by improvements in both histopathological and clinical phenotypes. We conclude that CRISPR/Cas9 represents a powerful tool to accurately model and treat tandem duplication mutations. Our findings will open new avenues of research for exploring the study and therapeutics of duplication disorders.