Prevention of Duchenne Muscular Dystrophy by CRISPR/Cas Therapeutic Genome Editing

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dc.contributor.advisorSadek, Hesham A.en
dc.contributor.committeeMemberOlson, Eric N.en
dc.contributor.committeeMemberMendell, Joshua T.en
dc.contributor.committeeMemberChen, Elizabethen
dc.creatorZhang, Yuen
dc.creator.orcid0000-0003-3384-8887
dc.date.accessioned2020-06-02T21:07:31Z
dc.date.available2020-06-02T21:07:31Z
dc.date.created2020-05
dc.date.issued2020-01-13
dc.date.submittedMay 2020
dc.date.updated2020-06-02T21:07:31Z
dc.description.abstractSkeletal muscle is one of the largest tissues in the human body and hence muscle diseases caused by genetic mutations have a profound and systemic impact on human health. Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disorder, caused by mutations in the DMD gene on the X chromosome, which consists of 79 exons encoding dystrophin protein. Patients with DMD develop progressive muscle weakness and cardiomyopathy, and ultimately succumb to respiratory and cardiac failure in their mid-20s. The dystrophin gene was identified three decades ago and mutations in the DMD gene are well-characterized. However, there is no effective treatment for this debilitating disease. The CRISPR/Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) was first discovered as an adaptive immune system in bacteria and archaea for defending against phage infection. Recently, the CRISPR/Cas system has been applied for mammalian genome editing because it provides site-specific DNA double-stranded breaks with simplicity and precision. In this study, I demonstrate the feasibility of using CRISPR/Cpf1 to correct a DMD exon 48-50 out-of-frame deletion mutation in cardiomyocytes derived from patient induced pluripotent stem cells by exon skipping and exon reframing strategies. Next, I precisely correct a Dmd exon 23 nonsense mutation in mdx mouse by CRISPR/Cpf1-mediated germline editing. Furthermore, I apply CRISPR/Cas9-mediated post-natal genome editing to correct a Dmd exon 44 out-of-frame deletion mutation in a DMD mouse model. Finally, I develop an effective strategy to improve CRISPR/Cas9-mediated in vivo genome editing by packaging Cas9 nuclease in conventional single-stranded AAV and CRISPR single guide RNAs in double-stranded self-complementary AAV. This strategy significantly reduces the amount of AAV vector needed for therapeutic genome editing and enhances dystrophin restoration after delivery into a mouse model of DMD harboring an exon 44 deletion. These findings represent an important advancement toward therapeutic translation of genome editing technology for permeant correction of Duchenne muscular dystrophy.en
dc.format.mimetypeapplication/pdfen
dc.identifier.oclc1156324550
dc.identifier.urihttps://hdl.handle.net/2152.5/8320
dc.language.isoenen
dc.subjectCRISPR-Cas Systemsen
dc.subjectGene Editingen
dc.subjectGenetic Therapyen
dc.subjectMuscular Dystrophiesen
dc.subjectMuscular Dystrophy, Duchenneen
dc.subjectMyocytes, Cardiacen
dc.titlePrevention of Duchenne Muscular Dystrophy by CRISPR/Cas Therapeutic Genome Editingen
dc.typeThesisen
dc.type.materialtexten
thesis.degree.departmentGraduate School of Biomedical Sciencesen
thesis.degree.disciplineGenetics and Developmenten
thesis.degree.grantorUT Southwestern Medical Centeren
thesis.degree.levelDoctoralen
thesis.degree.nameDoctor of Philosophyen

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