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.

A reporter mouse for optical imaging of inflammation in mdx muscles.

BACKGROUND: Duchenne muscular dystrophy (DMD) is due to mutations in the gene coding for human DMD; DMD is characterized by progressive muscle degeneration, inflammation, fat accumulation, and fibrosis. The mdx mouse model of DMD lacks dystrophin protein and undergoes a predictable disease course. While this model has been a valuable resource for pre-clinical studies aiming to test therapeutic compounds, its utility is compromised by a lack of reliable biochemical tools to quantifiably assay muscle disease. Additionally, there are few non-invasive assays available to researchers for measuring early indicators of disease progression in mdx mice. METHODS: Mdx mice were crossed to knock-in mice expressing luciferase from the Cox2 promoter. These reporter mice (Cox2 (FLuc/+) DMD (-/-) ) were created to serve as a tool for researchers to evaluate muscle inflammation. Luciferase expression was assayed by immunohistochemistry to insure that it correlated with muscle lesions. The luciferase signal was quantified by optical imaging and luciferase assays to verify that the signal correlated with muscle damage. As proof of principle, Cox2 (FLuc/+) DMD (-/-) mice were also treated with prednisolone to validate that a reduction in luciferase signal correlated with prednisone treatment. RESULTS: In this investigation, a novel reporter mouse (Cox2 (FLuc/+) DMD (-/-) mice) was created and validated for non-invasive quantification of muscle inflammation in vivo. In this dystrophic mouse, luciferase is expressed from cyclooxygenase 2 (Cox2) expressing cells and bioluminescence is detected by optical imaging. Bioluminescence is significantly enhanced in damaged muscle of exercised Cox2 (FLuc/+) DMD (-/-) mice compared to non-exercised Cox2 (FLuc/+) DMD (+/+) mice. Moreover, the Cox2 bioluminescent signal is reduced in Cox2 (FLuc/+) DMD (-/-) mice in response to a course of steroid treatment. Reduction in bioluminescence is detectable prior to measurable therapy-elicited improvements in muscle strength, as assessed by traditional means. Biochemical assay of luciferase provides a second means to quantify muscle inflammation. CONCLUSIONS: The Cox2 (FLuc/+) DMD (-/-) mouse is a novel tool to evaluate the therapeutic benefits of drugs intended to target inflammatory aspects of dystrophic pathology. This mouse model will be a useful adjunct to traditional outcome measures in assessing potential therapeutic compounds.

Interhelical packing modulates conformational flexibility in the lactose permease of Escherichia coli.

A key to obtaining an X-ray structure of the lactose permease of Escherichia coli (LacY) (Abramson, J., Smirnova, I., Kasho, V., Verner, G., Kaback, H. R., and Iwata, S. (2003) Science 301, 549-716) was the use of a mutant in which Cys154 (helix V) is replaced with Gly. LacY containing this mutation strongly favors an inward-facing conformation, which binds ligand with high affinity, but catalyzes little transport and exhibits few if any of the ligand-dependent conformational changes observed with wild-type LacY. The X-ray structure demonstrates that helix V crosses helix I in the approximate middle of the membrane in such a manner that Cys154 lies close to Gly24 (helix I). Therefore, it seems likely that replacing Cys154 with Gly may lead to tighter packing between helices I and V, thereby resulting in the phenotype observed. Consistently, replacement of Gly24 with Cys in the C154G mutant rescues significant transport activity, and the mutant exhibits properties similar to wild-type LacY with respect to substrate binding and thermostability. However, the only other replacements that rescue transport to any extent whatsoever are Val and Asp, both of which are much less effective than Cys. The results suggest that, although helix packing probably plays an important role with respect to the properties of the C154G mutant, the ability of Cys at position 24 to rescue transport activity of C154G is more complicated than simple replacement of bulk between positions 24 and 154. Rather, activity is dependent on more subtle interactions between the helices, and mutations that disrupt interactions between helix IV and loop 6-7 or between helices II and IV also rescue transport in the C154G mutant.

Expression, purification, and initial characterization of a recombinant form of plant PEP-carboxylase kinase from CAM-induced Mesembryanthemum crystallinum with enhanced solubility in Escherichia coli.

Plant phosphoenolpyruvate-carboxylase kinase (PEPC-kinase [PpcK]) is the smallest Ser/Thr kinase identified to date, having a molecular mass of approximately 32,000. This novel, monomeric kinase is dedicated to the phosphorylation of plant PEPC, thereby regulating this target enzyme's activity and allosteric properties. Although several recombinant, non-fusion PpcK proteins have been produced recently in Escherichia coli, these are plagued by their high degree of insolubility. Here, we report the use of the native, E. coli NusA protein and a related E. coli expression vector (pET-43a(+) [Novagen]) for enhancing the solubility of this recalcitrant Ser/Thr kinase at least 10-fold by its production as a dual 6xHis-tagged NusA/McPpcK1 fusion protein, which accounts for approximately 10% of the soluble protein fraction from induced cells. Capture of this fusion protein from the centrifuged cell extract by immobilized metal (Ni(2+)) affinity-chromatography, its "on-bead" cleavage by thrombin, and subsequent elution yielded milligram quantities of a "free," approximately 36-kDa form of PpcK for further purification by fast-protein liquid chromatography on blue dextran-agarose or preparative SDS-PAGE. Steady-state kinetic analysis of the former, active preparation revealed that this dedicated kinase discriminates against neither various isoforms of plant PEPC nor certain mutant forms of recombinant C(4) PEPC. Alternatively, the latter, electrophoretically homogeneous sample of the approximately 36-kDa polypeptide was used as antigen for polyclonal-antibody production in rabbits. The antibodies against the recombinant McPpcK1 from Mesembryanthemum crystallinum cross-reacted on Western blots with an enriched preparation of the maize-leaf kinase, but not with the parent crude extract, thus directly documenting this protein's extremely low abundance in vivo. However, these antibodies were effective in immunoprecipitating 32P-based PpcK activity from crude, desalted extracts of maize leaves and soybean root-nodules.