Taeyoung Koo

Clinical Trials Using Antisense Oligonucleotides in Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by mutations in the DMD gene, affecting 1 in 3500 newborn males. Complete loss of muscle dystrophin protein causes progressive muscle weakness and heart and respiratory failure, leading to premature death. Antisense oligonucleotides (AONs) that bind to complementary sequences of the dystrophin pre-mRNA to induce skipping of the targeted exon by modulating pre-mRNA splicing are promising therapeutic agents for DMD. Such AONs can restore the open reading frame of the DMD gene and produce internally deleted, yet partially functional dystrophin protein isoforms in skeletal muscle. Within the last few years, clinical trials using AONs have made considerable progress demonstrating the restoration of functional dystrophin protein and acceptable safety profiles following both local and systemic delivery in DMD patients. However, improvement of AON delivery and efficacy, along with the development of multiple AONs to treat as many DMD patients as possible needs to be addressed for this approach to fulfill its potential. Here, we review the recent progress made in clinical trials using AONs to treat DMD and discuss the current challenges to the development of AON-based therapy for DMD.

Measuring and Reducing Off-Target Activities of Programmable Nucleases Including CRISPR-Cas9.

Programmable nucleases, which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) repurposed from the type II clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system are now widely used for genome editing in higher eukaryotic cells and whole organisms, revolutionising almost every discipline in biological research, medicine, and biotechnology. All of these nucleases, however, induce off-target mutations at sites homologous in sequence with on-target sites, limiting their utility in many applications including gene or cell therapy. In this review, we compare methods for detecting nuclease off-target mutations. We also review methods for profiling genome-wide off-target effects and discuss how to reduce or avoid off-target mutations.

In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni

Several CRISPR-Cas9 orthologues have been used for genome editing. Here, we present the smallest Cas9 orthologue characterized to date, derived from Campylobacter jejuni (CjCas9), for efficient genome editing in vivo. After determining protospacer-adjacent motif (PAM) sequences and optimizing single-guide RNA (sgRNA) length, we package the CjCas9 gene, its sgRNA sequence, and a marker gene in an all-in-one adeno-associated virus (AAV) vector and produce the resulting virus at a high titer. CjCas9 is highly specific, cleaving only a limited number of sites in the human or mouse genome. CjCas9, delivered via AAV, induces targeted mutations at high frequencies in mouse muscle cells or retinal pigment epithelium (RPE) cells. Furthermore, CjCas9 targeted to the Vegfa or Hif1a gene in RPE cells reduces the size of laser-induced choroidal neovascularization, suggesting that in vivo genome editing with CjCas9 is a new option for the treatment of age-related macular degeneration.

Chromosomal context and epigenetic mechanisms control the efficacy of genome editing by rare-cutting designer endonucleases

The ability to specifically engineer the genome of living cells at precise locations using rare-cutting designer endonucleases has broad implications for biotechnology and medicine, particularly for functional genomics, transgenics and gene therapy. However, the potential impact of chromosomal context and epigenetics on designer endonuclease-mediated genome editing is poorly understood. To address this question, we conducted a comprehensive analysis on the efficacy of 37 endonucleases derived from the quintessential I-CreI meganuclease that were specifically designed to cleave 39 different genomic targets. The analysis revealed that the efficiency of targeted mutagenesis at a given chromosomal locus is predictive of that of homologous gene targeting. Consequently, a strong genome-wide correlation was apparent between the efficiency of targeted mutagenesis (≤0.1% to ∼6%) with that of homologous gene targeting (≤0.1% to ∼15%). In contrast, the efficiency of targeted mutagenesis or homologous gene targeting at a given chromosomal locus does not correlate with the activity of individual endonucleases on transiently transfected substrates. Finally, we demonstrate that chromatin accessibility modulates the efficacy of rare-cutting endonucleases, accounting for strong position effects. Thus, chromosomal context and epigenetic mechanisms may play a major role in the efficiency rare-cutting endonuclease-induced genome engineering.

ADP-overexpressing adenovirus elicits enhanced cytopathic effect by induction of apoptosis

Replication-competent adenoviruses (Ads) are emerging as a promising new modality for treatment of cancer. Selective replication of viral agents in tumor may lead to improved efficacy over nonreplicating Ads due to their inherent ability to multiply, lyse, and spread to surrounding cells. We have previously shown that an E1B 55 kDa-deleted adenovirus (YKL-1) exhibits tumor-specific replication and cell lysis, but its cytolytic effects were reduced in comparison to the wild-type adenovirus. To increase the oncolytic potency of YKL-1, we have reintroduced the Ad death protein (ADP) gene under the control of either a CMV or an MLP promoter at the E3 region of YKL-1, generating YKL-cADP and YKL-mADP Ads, respectively. ADP is an 11.6 kDa protein encoded by the E3 transcription unit, and is required to kill adenovirus-infected cells efficiently. However, to date, the mechanism by which ADP mediates cell death has not been clearly defined. In this study, we report that ADP-overexpressing Ad markedly enhanced cytolytic effect (up to 100-fold) against all tumor cell lines tested, but did not increase cytopathic effect in normal skin fibroblast, BJ. Moreover, plaque size formed by YKL-cADP was substantially larger than that of YKL-1, indicating an enhancement in cell lysis. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay and Annexin-V/PI double staining indicate that ADP-mediated cytotoxicity was largely driven by apoptosis. Finally, YKL-cADP adenovirus also showed superior antitumor effect than YKL-1 and YKL-mADP in C33A cervical and Hep3B hepatoma xenograft tumor models. Taken together, these lines of evidence demonstrate that the newly generated adenovirus expressing ADP under the CMV promoter induces efficient but tumor-selective cell lysis, which is critical for adding therapeutic value to replicating adenovirus for its use in cancer gene therapy.

Gene Correction of a Duchenne Muscular Dystrophy Mutation by Meganuclease-Enhanced Exon Knock-In

Duchenne muscular dystrophy (DMD) is a severe inherited, muscle-wasting disorder caused by mutations in the DMD gene. Gene therapy development for DMD has concentrated on vector-based DMD minigene transfer, cell-based gene therapy using genetically modified adult muscle stem cells or healthy wild-type donor cells, and antisense oligonucleotide-induced exon-skipping therapy to restore the reading frame of the mutated DMD gene. This study is an investigation into DMD gene targeting-mediated correction of deletions in human patient myoblasts using a target-specific meganuclease (MN) and a homologous recombination repair matrix. The MN was designed to cleave within DMD intron 44, upstream of a deletion hotspot, and integration-competent lentiviral vectors expressing the nuclease (LVcMN) were generated. MN western blotting and deep gene sequencing for LVcMN-induced non-homologous end-joining InDels (microdeletions or microinsertions) confirmed efficient MN expression and activity in transduced DMD myoblasts. A homologous repair matrix carrying exons 45-52 (RM45-52) was designed and packaged into integration-deficient lentiviral vectors (IDLVs; LVdRM45-52). After cotransduction of DMD myoblasts harboring a deletion of exons 45 to 52 with LVcMN and LVdRM45-52 vectors, targeted knock-in of the RM45-52 region in the correct location in DMD intron 44, and expression of full-length, correctly spliced wild-type dystrophin mRNA containing exons 45-52 were observed. This work demonstrates that genome surgery on human DMD gene mutations can be achieved by MN-induced locus-specific genome cleavage and homologous recombination knock-in of deleted exons. The feasibility of human DMD gene repair in patient myoblasts has exciting therapeutic potential.

Long-term functional adeno-associated virus-microdystrophin expression in the dystrophic CXMDj dog.

Duchenne muscular dystrophy (DMD) is a severe, inherited, muscle‐wasting disorder caused by mutations in the dystrophin gene. Preclinical studies of adeno‐associated virus gene therapy for DMD have been described in mouse and dog models of this disease. However, low and transient expression of microdystrophin in dystrophic dogs and a lack of long‐term microdystrophin expression associated with a CD8+ T‐cell response in DMD patients suggests that the development of improved microdystrophin genes and delivery strategies is essential for successful clinical trials in DMD patients.

Delivery of AAV2/9-Microdystrophin Genes Incorporating Helix 1 of the Coiled-Coil Motif in the C-Terminal Domain of Dystrophin Improves Muscle Pathology and Restores the Level of α1-Syntrophin and α-Dystrobrevin in Skeletal Muscles of mdx Mice

Duchenne muscular dystrophy is a severe X-linked inherited muscle wasting disorder caused by mutations in the dystrophin gene. Adeno-associated virus (AAV) vectors have been extensively used to deliver genes efficiently for dystrophin expression in skeletal muscles. To overcome limited packaging capacity of AAV vectors (<5 kb), truncated recombinant microdystrophin genes with deletions of most of rod and carboxyl-terminal (CT) domains of dystrophin have been developed. We have previously shown the efficiency of mRNA sequence-optimized microdystrophin (ΔR4-23/ΔCT, called MD1) with deletion of spectrin-like repeat domain 4 to 23 and CT domain in ameliorating the pathology of dystrophic mdx mice. However, the CT domain of dystrophin is thought to recruit part of the dystrophin-associated protein complex, which acts as a mediator of signaling between extracellular matrix and cytoskeleton in muscle fibers. In this study, we extended the ΔR4-23/ΔCT microdystrophin by incorporating helix 1 of the coiled-coil motif in the CT domain of dystrophin (MD2), which contains the α1-syntrophin and α-dystrobrevin binding sites. Intramuscular injection of AAV2/9 expressing CT domain-extended microdystrophin showed efficient dystrophin expression in tibialis anterior muscles of mdx mice. The presence of the CT domain of dystrophin in MD2 increased the recruitment of α1-syntrophin and α-dystrobrevin at the sarcolemma and significantly improved the muscle resistance to lengthening contraction-induced muscle damage in the mdx mice compared with MD1. These results suggest that the incorporation of helix 1 of the coiled-coil motif in the CT domain of dystrophin to the microdystrophins will substantially improve their efficiency in restoring muscle function in patients with Duchenne muscular dystrophy.

Abnormal splicing switch of DMD's penultimate exon compromises muscle fibre maintenance in myotonic dystrophy

Myotonic Dystrophy type 1 (DM1) is a dominant neuromuscular disease caused by nuclear-retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors resulting in alternative splicing misregulation and muscular dysfunction. Here we show that the abnormal splicing of DMD exon 78 found in dystrophic muscles of DM1 patients is due to the functional loss of MBNL1 and leads to the re-expression of an embryonic dystrophin in place of the adult isoform. Forced expression of embryonic dystrophin in zebrafish using an exon-skipping approach severely impairs the mobility and muscle architecture. Moreover, reproducing Dmd exon 78 missplicing switch in mice induces muscle fibre remodelling and ultrastructural abnormalities including ringed fibres, sarcoplasmic masses or Z-band disorganization, which are characteristic features of dystrophic DM1 skeletal muscles. Thus, we propose that splicing misregulation of DMD exon 78 compromises muscle fibre maintenance and contributes to the progressive dystrophic process in DM1.

Genome surgery using Cas9 ribonucleoproteins for the treatment of age-related macular degeneration

RNA-guided genome surgery using CRISPR-Cas9 nucleases has shown promise for the treatment of diverse genetic diseases. Yet, the potential of such nucleases for therapeutic applications in nongenetic diseases is largely unexplored. Here, we focus on age-related macular degeneration (AMD), a leading cause of blindness in adults, which is associated with retinal overexpression of, rather than mutations in, the VEGFA gene. Subretinal injection of preassembled, Vegfa gene-specific Cas9 ribonucleoproteins (RNPs) into the adult mouse eye gave rise to mutagenesis at the target site in the retinal pigment epithelium. Furthermore, Cas9 RNPs effectively reduced the area of laser-induced choroidal neovascularization (CNV) in a mouse model of AMD. Genome-wide profiling of Cas9 off-target effects via Digenome-seq showed that off-target mutations were rarely induced in the human genome. Because Cas9 RNPs can function immediately after in vivo delivery and are rapidly degraded by endogenous proteases, their activities are unlikely to be hampered by antibody- and cell-mediated adaptive immune systems. Our results demonstrate that in vivo genome editing with Cas9 RNPs has the potential for the local treatment for nongenetic degenerative diseases, expanding the scope of RNA-guided genome surgery to a new dimension.