Courtney S. Young

A Single CRISPR-Cas9 Deletion Strategy that Targets the Majority of DMD Patients Restores Dystrophin Function in hiPSC-Derived Muscle Cells

Mutations in DMD disrupt the reading frame, prevent dystrophin translation, and cause Duchenne muscular dystrophy (DMD). Here we describe a CRISPR/Cas9 platform applicable to 60% of DMD patient mutations. We applied the platform to DMD-derived hiPSCs where successful deletion and non-homologous end joining of up to 725 kb reframed the DMD gene. This is the largest CRISPR/Cas9-mediated deletion shown to date in DMD. Use of hiPSCs allowed evaluation of dystrophin in disease-relevant cell types. Cardiomyocytes and skeletal muscle myotubes derived from reframed hiPSC clonal lines had restored dystrophin protein. The internally deleted dystrophin was functional as demonstrated by improved membrane integrity and restoration of the dystrophin glycoprotein complex in vitro and in vivo. Furthermore, miR31 was reduced upon reframing, similar to observations in Becker muscular dystrophy. This work demonstrates the feasibility of using a single CRISPR pair to correct the reading frame for the majority of DMD patients.

CRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ cells

Targeted genome editing technology can correct the sickle cell disease mutation of the β-globin gene in hematopoietic stem cells. This correction supports production of red blood cells that synthesize normal hemoglobin proteins. Here, we demonstrate that Transcription Activator-Like Effector Nucleases (TALENs) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system can target DNA sequences around the sickle-cell mutation in the β-globin gene for site-specific cleavage and facilitate precise correction when a homologous donor template is codelivered. Several pairs of TALENs and multiple CRISPR guide RNAs were evaluated for both on-target and off-target cleavage rates. Delivery of the CRISPR/Cas9 components to CD34+ cells led to over 18% gene modification in vitro. Additionally, we demonstrate the correction of the sickle cell disease mutation in bone marrow derived CD34+ hematopoietic stem and progenitor cells from sickle cell disease patients, leading to the production of wild-type hemoglobin. These results demonstrate correction of the sickle mutation in patient-derived CD34+ cells using CRISPR/Cas9 technology.

Exon Skipping Therapy.

Exondys 51 is the first therapy for Duchenne muscular dystrophy (DMD) to have been granted accelerated approval by the FDA. Approval was granted based on using dystrophin expression as a surrogate marker. Exondys 51 targets DMD exon 51 for skipping to restore the reading frame for 13% of Duchenne patients.

Creation of a Novel Humanized Dystrophic Mouse Model of Duchenne Muscular Dystrophy and Application of a CRISPR/Cas9 Gene Editing Therapy

Duchenne muscular dystrophy is caused by mutations in DMD which disrupt the reading frame. Therapeutic strategies that restore DMDs reading frame, such as exon skipping and CRISPR/Cas9, need to be tested in the context of the human DMD sequence in vivo. We have developed a novel dystrophic mouse model by using CRISPR/Cas9 to delete exon 45 in the human DMD gene in hDMD mice, which places DMD out-of-frame. We have utilized this model to demonstrate that our clinically-relevant CRISPR/Cas9 platform, which targets deletion of human DMD exons 45-55, can be directly applied in vivo to restore dystrophin.

“Of Mice and Measures”: A Project to Improve How We Advance Duchenne Muscular Dystrophy Therapies to the Clinic

A new line of dystrophic mdx mice on the DBA/2J (D2) background has emerged as a candidate to study the efficacy of therapeutic approaches for Duchenne muscular dystrophy (DMD). These mice harbor genetic polymorphisms that appear to increase the severity of the dystropathology, with disease modifiers that also occur in DMD patients, making them attractive for efficacy studies and drug development. This workshop aimed at collecting and consolidating available data on the pathological features and the natural history of these new D2/mdx mice, for comparison with classic mdx mice and controls, and to identify gaps in information and their potential value. The overall aim is to establish guidance on how to best use the D2/mdx mouse model in preclinical studies.

735. Functional Restoration of Dystrophin Protein in HiPSC-Derived Skeletal Myotubes and Cardiomyocytes After CRISPR/Cas9-Mediated Deletion of 530-725kb of DMD

Duchenne muscular dystrophy (DMD) is typically due to frameshifting mutations in the DMD gene encoding dystrophin. Loss of dystrophin protein results in progressive muscle degeneration and premature death. Approximately 60% of DMD patients have frameshifting mutations in a hotspot region within exons 45-55 in the rod domain of dystrophin. Genotype/phenotype assessments have revealed that in-frame deletion of exons 45-55 leads to the milder, allelic disease, Becker muscular dystrophy. This finding suggests that restoration of the reading frame by targeting exons 45-55 could treat ~60% of DMD patients to greatly reduce disease severity. We have developed a platform using clustered regularly interspaced short palindromic repeats (CRISPR) and- associated protein (Cas9) gene editing to achieve this purpose. We have utilized CRISPR/Cas9-mediated deletion and rejoining of up to 725kb to restore the reading frame in DMD human induced pluripotent stem cells (hiPSCs). This is the largest deletion shown to date in DMD. Clonal hiPSC lines containing the exon 45-55 deletion were differentiated to disease relevant types, such as cardiomyocytes and skeletal muscle myotubes, which had restored dystrophin protein. We demonstrated, for the first time, that the internally deleted dystrophin generated by CRISPR/Cas9 was functional and improved membrane integrity, reduced miR31 expression, and restored the dystrophin glycoprotein complex in vitro and after engraftment of skeletal muscle cells in vivo. This gene editing platform restores the reading frame for the majority of DMD patients and offers potential as an ex vivo correction for stem cell therapy or for use in vivo.

121. Targeted Gene Therapy in the Treatment of X-Linked Hyper IgM Syndrome

X-linked hyper-IgM syndrome results in absent IgG, IgA, IgE and normal/elevated IgM due to defects in the CD40 ligand gene. HSCT is the only curative modality, but it carries significant risks, suggesting the need for improved methods of treatment. TALENs or CRISPRs, combined with the effective delivery of a homologous donor sequence containing normal CD40L DNA, will allow for targeted integration and provide physiologic expression of the endogenous CD40L gene to provide long-term immune reconstitution. TALENs targeting the 5′ UTR of the CD40L gene lead to allelic disruption of up to 31%at the target locus inK562 cells. In order to evaluate the capacity for targeted integration of a cassette at the cut site, cells were electroporated with the TALEN pair and a donor molecule with a promoterless GFP reporter gene flanked by homology arms. Expression of the GFP reporter was evaluated in Jurkat cells, with up to 10 % detected by flow cytometry and increasing to 22 % upon PHA activation. In addition, CRISPRs targeting a patient-specific mutation in intron 3 achieved >50 % gene disruption in K562 cells. Co-electroporation with a donormodified to contain a unique restriction enzyme site demonstrated site-specific gene integration. Site-specific modification at CD40L is achievable and physiologic expression of the endogenous CD40L gene could provide a viable therapy for XHIM.

338. Evaluation of TALENs and the CRISPR/Cas9 Nuclease System To Correct the Sickle Cell Disease Mutation

Targeted genome editing technology can correct the sickle cell disease mutation of the beta-globin gene in hematopoietic stem and progenitor cells (HSCs). The correction induces production of red blood cells that synthesize normal hemoglobin proteins. Transcription Activator-Like Effector Nucleases (TALENs) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease systems have been developed to target the sickle mutation in the beta-globin gene for site-specific cleavage to facilitate precise correction of the sickle mutation by a co-delivered homologous donor template. K562 cells were electroporated with TALEN and CRISPR/Cas9 expression plasmids and, using the Surveyor Nuclease Assay (Cel-1), cleavage rates were quantified and compared between TALEN- and CRISPR/Cas9-treated cells. Of the six CRISPR/Cas9 guides tested, each of them led to target disruption of the beta-globin locus with the highest cleavage rates upwards of 35% of alleles. Of 4 distinct TALEN pairs generated, only 2 demonstrated targeted cleavage at rates nearing 10% of alleles. In addition to on-target cleavage at beta-globin, nuclease off-target cleavage at other beta-globin family genes was evaluated for each technology by Cel-1 of nucleofected K562 cells. Here the two TALEN pairs demonstrate cleavage in the highly-homologous delta-globin gene with the optimal TALEN pair cleaving 11% of alleles. In this assay, of the 6 CRISPR guides tested, none showed off-target disruption of delta-globin or any of the other beta-globin cluster genes. Of note, in each of the tested guides, at least one base differed from the target site in beta-globin to the respective sequence in delta-globin in the 10bp PAM proximal region. Further experiments are being conducted to determine the genome-wide off-target effects of each of these nucleases.Upon co-delivery of a plasmid donor template containing the corrective base at the sickle site as well as a restriction fragment length polymorphism (RFLP) for rapid assessment of targeted gene modification, both nuclease technologies led to gene modification. Gene modification rates were assayed by qPCR with primers specific to the modified base. TALENs drove gene modification rates of 18%, while the optimal CRISPR guides resulted in 37% modification in K562 cells without sorting for transfected cells. These results provide the basis for pursuing the use of the CRISPR/Cas9 nuclease system for targeted correction of the sickle mutation in human HSCs.