Functional rescue of dystrophin deficiency in mice caused by frameshift mutations using Campylobacter jejuni Cas9

Duchenne muscular dystrophy (DMD) is a fatal, X-linked muscle wasting disease caused by mutations in the DMD gene. In 51% of DMD cases, a reading frame is disrupted because of deletion of several exons. Here, we show that CjCas9 derived from Campylobacter jejuni can be used as a gene editing tool to correct an out-of-frame Dmd exon in Dmd knockout mice. Herein, we used Cas9 derived from S. pyogenes to generate Dmd knockout (KO) mice with a frameshift mutation in Dmd gene. Then, we expressed CjCas9, its single-guide RNA, and the eGFP gene in the tibialis anterior muscle of the Dmd KO mice using an all-in-one adeno-associated virus (AAV) vector. CjCas9 cleaved the target site in the Dmd gene efficiently in vivo and induced small insertions or deletions at the target site. This treatment resulted in conversion of the disrupted Dmd reading frame from out-of-frame to in-frame, leading to the expression of dystrophin in the sarcolemma. Importantly, muscle strength was enhanced in the CjCas9-treated muscles, without off-target mutations, indicating high efficiency and specificity of CjCas9. This work suggests that in vivo DMD frame correction, mediated by CjCas9 has great potential for the treatment of DMD and other neuromuscular diseases

An ex vivo gene therapy approach to treat muscular dystrophy using inducible pluripotent stem cells.

Duchenne muscular dystrophy is a progressive and incurable neuromuscular disease caused by genetic and biochemical defects of the dystrophin-glycoprotein complex. Here we show the regenerative potential of myogenic progenitors derived from corrected dystrophic induced pluripotent stem cells generated from fibroblasts of mice lacking both dystrophin and utrophin. We correct the phenotype of dystrophic induced pluripotent stem cells using a Sleeping Beauty transposon system carrying the micro-utrophin gene, differentiate these cells into skeletal muscle progenitors and transplant them back into dystrophic mice. Engrafted muscles displayed large numbers of micro-utrophin-positive myofibers, with biochemically restored dystrophin-glycoprotein complex and improved contractile strength. The transplanted cells seed the satellite cell compartment, responded properly to injury and exhibit neuromuscular synapses. We also detect muscle engraftment after systemic delivery of these corrected progenitors. These results represent an important advance towards the future treatment of muscular dystrophies using genetically corrected autologous induced pluripotent stem cells

The lack of the Celf2a splicing factor converts a Duchenne genotype into a Becker phenotype

Substitutions, deletions and duplications in the dystrophin gene lead to either the severe Duchenne muscular dystrophy (DMD) or mild Becker muscular dystrophy depending on whether out-of-frame or in-frame transcripts are produced. We identified a DMD case (GSΔ44) where the correlation between genotype and phenotype is not respected, even if carrying a typical Duchenne mutation (exon 44 deletion) a Becker-like phenotype was observed. Here we report that in this patient, partial restoration of an in-frame transcript occurs by natural skipping of exon 45 and that this is due to the lack of Celf2a, a splicing factor that interacts with exon 45 in the dystrophin pre-mRNA. Several experiments are presented that demonstrate the central role of Celf2a in controlling exon 45 splicing; our data point to this factor as a potential target for the improvement of those DMD therapeutic treatments, which requires exon 45 skipping

MAR-Mediated Dystrophin Expression in Mesoangioblasts for Duchenne Muscular Dystrophy Cell Therapy

A cornerstone of autologous cell therapy for Duchenne muscular dystrophy is the engineering of suitable cells to express dystrophin in a stable fashion upon differentiation to muscle fibers. Most viral transduction methods are typically restricted to the expression of truncated recombinant dystrophin derivatives and by the risk of insertional mutagenesis, while non-viral vectors often suffer from inefficient transfer, expression and/or silencing

Nerve growth factor improves the muscle regeneration capacity of muscle stem cells in dystrophic muscle.

Researchers have attempted to use gene- and cell-based therapies to restore dystrophin and alleviate the muscle weakness that results from Duchenne muscular dystrophy (DMD). Our research group has isolated populations of muscle-derived stem cells (MDSCs) from the postnatal skeletal muscle of mice. In comparison with satellite cells, MDSCs display an improved transplantation capacity in dystrophic mdx muscle that we attribute to their ability to undergo long-term proliferation, self-renewal, and multipotent differentiation, including differentiation toward endothelial and neuronal lineages. Here we tested whether the use of nerve growth factor (NGF) improves the transplantation efficiency of MDSCs. We used two methods of in vitro NGF stimulation: retroviral transduction of MDSCs with a CL-NGF vector and direct stimulation of MDSCs with NGF protein. Neither method of NGF treatment changed the marker profile or proliferation behavior of the MDSCs, but direct stimulation with NGF protein significantly reduced the in vitro differentiation ability of the cells. NGF stimulation also significantly enhanced the engraftment efficiency of MDSCs transplanted within the dystrophic muscle of mdx mice, resulting in the regeneration of numerous dystrophin-positive muscle fibers. These findings highlight the importance of NGF as a modulatory molecule, the study of which will broaden our understanding of its biologic role in the regeneration and repair of skeletal muscle by musclederived cells

<i>piggyBac </i>transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes.status: publishe

Artificial restoration of the linkage between laminin and dystroglycan ameliorates the disease progression of MDC1A muscular dystrophy at all stages

Laminin-α2 deficient congenital muscular dystrophy, classified as MDC1A, is a severe progressive muscle-wasting disease that leads to death in early childhood. MDC1A is caused by mutations in lama2, the gene encoding the laminin-α2 chain being part of laminin-2, the main laminin isoform present in the extracellular matrix of muscles and peripheral nerves. Via selfpolymerization, laminin-2 forms the primary laminin scaffold and binds with high affinity to α- dystroglycan on the cell surface, providing a connection to the cytoskeleton via the transmembranous protein β-dystroglycan. Deficiency in laminin-α2 leads to absence of laminin-2 and to upregulation of laminin-8, a laminin isoform that cannot self-polymerize and does not bind to α-dystroglycan. Therefore, in laminin α2-deficient muscle the chain of proteins linking the intracellular contractile apparatus via the plasma membrane to the extracellular matrix is interrupted. Consequently, muscle fibers loose their stability and degenerate what finally leads to a progressive muscle wasting. In previous studies, we have shown that a miniaturized form of the extracellular matrix protein agrin, which is not related to the disease-causing lama2 gene and was designed to contain highaffinity binding sites for the laminins and for α-dystroglycan, was sufficient to markedly improve muscle function and overall health in the dyW-/- mouse model of MDC1A. In a follow-up study we provided additional evidence that mini-agrin, both increases the tolerance to mechanical load but also improves the regeneration capacity of the dystrophic muscle. We now report on our progress towards further testing the use of this approach for the treatment of MDC1A. To test whether mini-agrin application after onset of the disease would still ameliorate the dystrophic symptoms, we have established the inducible tetracycline-regulated “tet-off” expression system in dyW-/- mice to temporally control mini-agrin expression in skeletal muscles. We show that mini-agrin slows down the progression of the dystrophy when applied at birth or in advanced stages of the disease. However, the extent of the amelioration depends on the dystrophic condition of the muscle at the time of mini-agrin application. Thus, the earlier miniagrin is applied, the higher is the profit of its beneficial properties. In addition to gene therapeutical approaches, the increase of endogenous agrin expression levels in skeletal muscles by pharmacologically active compounds would be a safe and promising strategy for the treatment of MDC1A. To evaluate the potential and pave the way to further expand on the development of such a treatment, we determined whether full-length agrin ameliorates the dystrophic phenotype to a comparable extent as it was observed by application of mini-agrin. We provide evidence that constitutive overexpression of chick full-length agrin in dyW-/- muscle ameliorates the dystrophic phenotype, although not as pronounced as mini-agrin does. In conclusion, our results are conceptual proof that linkage of laminin to the muscle fiber membrane is a means to treat MDC1A at any stage of the disease. Our findings definitely encourage to further expanding on this therapeutic concept, especially in combination with treatment using functionally different approaches. Moreover, these experiments set the basis for further developing clinically feasible and relevant application methods such as gene therapy4 and/or the screening of small molecules able to upregulate production of agrin in muscle

Lentiviral vectors can be used for full-length dystrophin gene therapy

Duchenne Muscular Dystrophy (DMD) is caused by a lack of dystrophin expression in patient muscle fibres. Current DMD gene therapy strategies rely on the expression of internally deleted forms of dystrophin, missing important functional domains. Viral gene transfer of full-length dystrophin could restore wild-type functionality, although this approach is restricted by the limited capacity of recombinant viral vectors. Lentiviral vectors can package larger transgenes than adeno-associated viruses, yet lentiviral vectors remain largely unexplored for full-length dystrophin delivery. In our work, we have demonstrated that lentiviral vectors can package and deliver inserts of a similar size to dystrophin. We report a novel approach for delivering large transgenes in lentiviruses, in which we demonstrate proof-of-concept for a 'template-switching' lentiviral vector that harnesses recombination events during reverse-transcription. During this work, we discovered that a standard, unmodified lentiviral vector was efficient in delivering full-length dystrophin to target cells, within a total genomic load of more than 15,000 base pairs. We have demonstrated gene therapy with this vector by restoring dystrophin expression in DMD myoblasts, where dystrophin was expressed at the sarcolemma of myotubes after myogenic differentiation. Ultimately, our work demonstrates proof-of-concept that lentiviruses can be used for permanent full-length dystrophin gene therapy, which presents a significant advancement in developing an effective treatment for DMD

Safe, Site-specific Gene Delivery using Ultrasound and Microbubble Technology

The following study investigates the use of diagnostic ultrasound in combination with microbubbles (ultrasound contrast agents) as a physical enhancer for non-viral gene delivery. The aim of this work was firstly, to demonstrate that ultrasound exposure using settings within the range of diagnostic ultrasound, in combination with microbubbles can improve gene delivery, and secondly, to show that it is a safe, site-specific technique which mitigates the risk of tissue damage often seen with other physical enhancers of gene delivery such as, electroporation. Initially, a feasibility study was carried out to test the efficiency and safety of microbubble ultrasound (MBUS) in a reporter gene setting. Experiments using intravenous injections of a luciferase reporter gene established that MBUS is a safe, site-specific technique which improved levels of the luciferase expression in the organ targeted by MBUS. Luciferase was successfully delivered to the liver and heart, showing significantly higher levels compared to injections without MBUS, and with no detectable expression in other non-target organs. A therapeutic application of MBUS was tested using the mdx mouse, an animal model for Duchenne Muscular Dystrophy (DMD), a genetic disorder caused by the lack of functional dystrophin in muscle fibres due to premature termination of translation. The most successful treatment approach in the mdx mouse thus far had been the injection of Phosphorodiamidate Morpholino Oligomers (PMOs), which by inducing exon skipping, re-introduced dystrophin expression in most muscles in the body, with the exception of the heart. Injections of PMOs with MBUS to the heart successfully re-introduced dystrophin expression in cardiomyocytes. Furthermore, treatment parameters were investigated in more detail in order to optimize PMO delivery to the heart. Finally, an investigation into different types of commercially available microbubbles compared the efficiencies (with respect to gene delivery) of the different bubbles, in order to understand why different microbubbles show different results when used for MBUS, potentially enabling the design of microbubbles specifically for gene delivery