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Duchenne muscular dystrophy (DMD) is caused by genetic mutations that result in the absence of dystrophin protein expression. Oligonucleotide-induced exon skipping can restore the dystrophin reading frame and protein production. However, this requires continuous drug administration and may not generate complete skipping of the targeted exon. In this study, we apply genome editing with zinc finger nucleases (ZFNs) to permanently remove essential splicing sequences in exon 51 of the dystrophin gene and thereby exclude exon 51 from the resulting dystrophin transcript. This approach can restore the dystrophin reading frame in ~13% of DMD patient mutations. Transfection of two ZFNs targeted to sites flanking the exon 51 splice acceptor into DMD patient myoblasts led to deletion of this genomic sequence. A clonal population was isolated with this deletion and following differentiation we confirmed loss of exon 51 from the dystrophin mRNA transcript and restoration of dystrophin protein expression. Furthermore, transplantation of corrected cells into immunodeficient mice resulted in human dystrophin expression localized to the sarcolemmal membrane. Finally, we quantified ZFN toxicity in human cells and mutagenesis at predicted off-target sites. This study demonstrates a powerful method to restore the dystrophin reading frame and protein expression by permanently deleting exons.

Technologies for engineering synthetic transcription factors have enabled many advances in medical and scientific research. In contrast to existing methods based on engineering of DNA-binding proteins, we created a Cas9-based transactivator that is targeted to DNA sequences by guide RNA molecules. Coexpression of this transactivator and combinations of guide RNAs in human cells induced specific expression of endogenous target genes, demonstrating a simple and versatile approach for RNA-guided gene activation.

Genome editing with engineered nucleases has recently emerged as an approach to correct genetic mutations by enhancing homologous recombination with a DNA repair template. However, many genetic diseases, such as Duchenne muscular dystrophy (DMD), can be treated simply by correcting a disrupted reading frame. We show that genome editing with transcription activator-like effector nucleases (TALENs), without a repair template, can efficiently correct the reading frame and restore the expression of a functional dystrophin protein that is mutated in DMD. TALENs were engineered to mediate highly efficient gene editing at exon 51 of the dystrophin gene. This led to restoration of dystrophin protein expression in cells from Duchenne patients, including skeletal myoblasts and dermal fibroblasts that were reprogrammed to the myogenic lineage by MyoD. Finally, exome sequencing of cells with targeted modifications of the dystrophin locus showed no TALEN-mediated off-target changes to the protein-coding regions of the genome, as predicted by in silico target site analysis. This strategy integrates the rapid and robust assembly of active TALENs with an efficient gene-editing method for the correction of genetic diseases caused by mutations in non-essential coding regions that cause frameshifts or premature stop codons.

Mammalian genes are regulated by the cooperative and synergistic actions of many transcription factors. In this study we recapitulate this complex regulation in human cells by targeting endogenous gene promoters, including regions of closed chromatin upstream of silenced genes, with combinations of engineered transcription activator–like effectors (TALEs). These combinations of TALE transcription factors induced substantial gene activation and allowed tuning of gene expression levels that will broadly enable synthetic biology, gene therapy and biotechnology.

Feline infectious peritonitis is a lethal disease of felids caused by systemic infection with a feline coronavirus. Here, we report identification and analysis of the feline homologue to the human lectin DC-SIGN and show that it is a coreceptor for virulent strains of serotype 1 and serotype 2 feline coronaviruses.

Duchenne muscular dystrophy (DMD) is a devastating disease affecting about 1 out of 5000 male births and caused by mutations in the dystrophin gene. Genome editing has the potential to restore expression of a modified dystrophin gene from the native locus to modulate disease progression. In this study, adeno-associated virus was used to deliver the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to the mdx mousemodel of DMD to remove the mutated exon 23 from the dystrophin gene. This includes local and systemic delivery to adult mice and systemic delivery to neonatal mice. Exon 23 deletion by CRISPR-Cas9 resulted in expression of the modified dystrophin gene, partial recovery of functional dystrophin protein in skeletal myofibers and cardiac muscle, improvement of muscle biochemistry, and significant enhancement of muscle force.This work establishes CRISPR-Cas9-based genome editing as a potential therapy to treat DMD., Muscular Dystrophy Association (Award MDA277360), National Institutes of Health (U.S.) (Grant 5DP1-MH100706), National Institutes of Health (U.S.) (Grant R01DK097768)

The development of a facile genome engineering technology based on transcription activator-like effector nucleases (TALENs) has led to significant advances in diverse areas of science and medicine. In this review, we provide a broad overview of the development of TALENs and the use of this technology in basic science, biotechnology, and biomedical applications. This includes the discovery of DNA recognition by TALEs, engineering new TALE proteins to diverse targets, general advances in nuclease-based editing strategies, and challenges that are specific to various applications of the TALEN technology. We review examples of applying TALENs for studying gene function and regulation, generating disease models, and developing gene therapies. The current status of genome editing and future directions for other uses of these technologies are also discussed.

Neuromuscular diseases are a diverse range of conditions that include myopathic and neuropathic disorders related to muscular dysfunction. Inherited neuromuscular diseases are the result of a broad spectrum of genetic mutations, including point mutations, insertions and deletions, chromosomal rearrangements, epigenetic aberrations, and repeat expansions or contractions. Targeted genome editing is a promising method to correct the inherited mutations underlying these disorders. Over the last decade there have been many significant advances in engineering targeted DNA-binding proteins to manipulate specific sequences of complex genomes. These genome editing tools are rapidly becoming viable therapeutics that will allow the targeted addition, exchange, or removal of almost any genetic sequence in the human genome. In this chapter, selected neuromuscular diseases representing inherited myopathies or neuropathies are discussed. The genome editing tools available to create targeted genetic modifications are reviewed. Promising cell- and gene-based therapies are introduced in the context of the treatment of neuromuscular disorders in combination with genome editing therapies. Finally, specific examples of how genome editing may be applied to correct the genetic basis of particular neuromuscular disorders are presented and discussed.

Genetic reprogramming holds great potential for disease modeling, drug screening, and regenerative medicine. Genetic reprogramming of mammalian cells is typically achieved by forced expression of natural transcription factors that control master gene networks and cell lineage specification. However, in many instances, the natural transcription factors do not induce a sufficiently robust response to completely reprogram cell phenotype. In this study, we demonstrate that protein engineering of the master transcription factor MyoD can enhance the conversion of human dermal fibroblasts and adult stem cells to a skeletal myocyte phenotype. Fusion of potent transcriptional activation domains to MyoD led to increased myogenic gene expression, myofiber formation, cell fusion, and global reprogramming of the myogenic gene network. This work supports a general strategy for synthetically enhancing the direct conversion between cell types that can be applied in both synthetic biology and regenerative medicine.

Duchenne muscular dystrophy (DMD) is a monogenic and fatal genetic disorder characterized by muscle wasting, loss of ambulation, and death by the age of 20 due to the loss of functional dystrophin – an essential musculoskeletal protein. Gene therapy has held promise for treating monogenetic diseases, however, delivery barriers and the requirements for costly repetitive treatments are a few of the challenges of technologies in the therapeutic pipeline. Recently, the CRISPR/Cas9 system has been adapted for genome editing to make specific modifications in the host genome. This technology would enable permanent genome corrections and a potentially curative approach to DMD.Previously we adapted CRISPR/Cas9 for targeted deletions of regions of the human dystrophin gene that restore expression of a functional protein (Ousterout et al., Nature Communications (in press)). The delivery of Cas9 with single guide RNAs (sgRNAs) targeting the introns surrounding exons 51 and 45-55 created double strand breaks which are repaired through non-homologous end joining to create genomic deletions of these regions. These deletions restored the reading frame and recovered dystrophin expression in cultured patient-derived myoblasts. Therapeutically, this approach could be used to convert the fatal DMD phenotype to the milder phenotype associated with partial dystrophin function characteristic of Becker muscular dystrophy.Here we utilize adeno-associated virus (AAV) to deliver the CRISPR/Cas9 system to the mdx mouse model of DMD. AAV vectors were designed to package and deliver genes encoding the Staphylococcus aureus Cas9 (SaCas9), due to its smaller size (3.2 kb) compared to the widely used Cas9 from Streptococcus pyogenes. Two single guide RNAs (sgRNAs) were designed to target sites flanking exon 23 of the dystrophin gene and validated to direct SaCas9-mediated deletion of this exon, which contains a premature stop codon in this model. Mice were anesthetized and AAV1 containing the SaCas9 and sgRNA expression cassettes was injected into the gastrocnemius muscle of 8 week old mice. At 4 and 8 weeks post-injection, the gastrocnemius was harvested and genomic DNA and mRNA were extracted. PCR of the genomic DNA demonstrated the expected ~1200 bp deletion of exon 23. Further, RT-PCR of the extracted mRNA showed removal of the 216 bp encoding exon 23. Furthermore, sgRNAs compatible with SaCas9 have been developed and validated for the deletion of human exon 51.This work demonstrates proof-of-principle of CRISPR/Cas9-mediated genome editing in skeletal muscle in an adult mammal, opening up new possibilities for gene therapy and the study of gene function. Optimization of delivery and activity is underway to improve the therapeutic potential of this approach. Additional work to characterize functional improvement in the mouse hind limb is also ongoing. This work establishes CRISPR/Cas9-mediated genome editing as a potential therapeutic approach to DMD.

Genome editing with engineered nucleases has recently emerged as an approach to correct genetic mutations by enhancing homologous recombination with a DNA repair template. However, many genetic diseases, such as Duchenne muscular dystrophy (DMD), can be treated simply by correcting a disrupted reading frame. We show that genome editing with transcription activator-like effector nucleases (TALENs), without a repair template, can efficiently correct the reading frame and restore the expression of a functional dystrophin protein that is mutated in DMD. TALENs were engineered to mediate highly efficient gene editing at exon 51 of the dystrophin gene. This led to restoration of dystrophin protein expression in cells from Duchenne patients, including skeletal myoblasts and dermal fibroblasts that were reprogrammed to the myogenic lineage by MyoD. Finally, exome sequencing of cells with targeted modifications of the dystrophin locus showed no TALEN-mediated off-target changes to the protein-coding regions of the genome, as predicted by in silico target site analysis. This strategy integrates the rapid and robust assembly of active TALENs with an efficient gene-editing method for the correction of genetic diseases caused by mutations in non-essential coding regions that cause frameshifts or premature stop codons.

Duchenne muscular dystrophy (DMD) is a highly prevalent genetic disorder leading to muscle wasting, loss of ambulation, and premature death by the third decade of life. DMD is caused by gene deletions, duplications, or nonsense mutations leading to the loss of dystrophin, an essential musculoskeletal protein. Gene therapy has held tremendous promise for the treatment of monogenic disorders, yet an effective gene replacement therapy has been elusive. Genome editing has been established as a potential approach to correct the dystrophin gene in cultured human cells by excising non-essential exons from the dystrophin gene producing a shortened yet in-frame dystrophin protein (1). In contrast to gene replacement therapy, genome editing repairs the causative mutation in the native genomic context with the potential for permanent gene repair. Recently, we and others have demonstrated that CRISPR/Cas9 genome editing in neonatal and adult mouse models of DMD restores dystrophin expression, improves muscle biochemistry, and strengthens muscle force generation (2-4). However, further optimization of the approach for systemic gene correction is still needed.To target the dystrophin gene in the mdx mouse, the 3.2kb S. aureus Cas9 and two guide RNAs (gRNA) targeting intronic regions surrounding exon 23 were packaged into an adeno-associated virus (AAV). Double stranded breaks created by Cas9 were repaired with the relatively efficient non-homologous end joining pathway leading to excision of the nonsense mutation in exon 23. AAV vectors were injected intramuscularly into the tibialis anterior muscle in adult mice and intravenously into neonatal mice and adult mice and characterized for gene deletions, dystrophin restoration, and improvements in muscle physiology.Local correction restored overall dystrophin levels to 8% by western blot with 67% of muscle fibers positive for dystrophin by immunofluorescence. Repeated cycles of eccentric contraction showed 60% resistance to damage compared to sham-treated mice. Systemic correction was achieved through IP injection into P2 neonates with dystrophin restoration primarily in the cardiac muscle and skeletal muscle surrounding the peritoneal cavity. IV administration in adult mice restored dystrophin expression in the cardiac muscle. Improved systemic distribution and correction was achieved with intravenous administration into P2 neonates with AAV8 or AAV9 (Fig. 1Fig. 1).Figure 1Intravenously administered AAV restores dystrophin in cardiac and skeletal muscle in P2 neonates after 8 weeks of treatement. a) Cardiac muscle. b) Tibialis anterior muscle. Scale bar = 200µm, green - dystrophin, blue - DAPI.View Large Image | Download PowerPoint SlideThis study establishes CRISPR/Cas9-based gene editing as a promising approach for the treatment of DMD. Ongoing work to improve the efficiency and safety of in vivo gene editing includes the incorporation of muscle specific promoters, minimization of vector packaging, and optimization of AAV serotype.1. Ousterout et al. Nat Comm 2015. | 2. Nelson et al. Science 2015 | 3. Tabebordbar et al. Science 2015 | 4. Long et al. Science 2015

New technologies have recently emerged that enable targeted editing of genomes in diverse systems. This includes precise manipulation of gene sequences in their natural chromosomal context and addition of transgenes to specific genomic loci. This progress has been facilitated by advances in engineering targeted nucleases with programmable, site-specific DNA-binding domains, including zinc finger proteins and transcription activator-like effectors (TALEs). Recent improvements have enhanced nuclease performance, accelerated nuclease assembly, and lowered the cost of genome editing. These advances are driving new approaches to many areas of biotechnology, including biopharmaceutical production, agriculture, creation of transgenic organisms and cell lines, and studies of genome structure, regulation, and function. Genome editing is also being investigated in preclinical and clinical gene therapies for many diseases.

Engineered DNA-binding proteins that manipulate the human genome and transcriptome have enabled rapid advances in biomedical research. In particular, the RNA-guided CRISPR/Cas9 system has recently been engineered to create site-specific double-strand breaks for genome editing or to direct targeted transcriptional regulation. A unique capability of the CRISPR/Cas9 system is multiplex genome engineering by delivering a single Cas9 enzyme and two or more single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can be used to simultaneously create multiple DNA breaks or to target multiple transcriptional activators to a single promoter for synergistic enhancement of gene induction. To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells. This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types.