Search

Vector production scale-up is a major barrier in systemic adeno-associated virus (AAV) gene therapy. Many scalable manufacturing methods have been developed. However, the potency of the vectors generated by these methods has rarely been compared with vectors made by transient transfection (TT), the most commonly used method in preclinical studies. In this study, we blindly compared therapeutic efficacy of an AAV9 micro-dystrophin vector generated by the TT method and scalable herpes simplex virus (HSV) system in a Duchenne muscular dystrophy mouse model. AAV was injected intravenously at 5 × 1014 (high), 5 × 1013 (medium), or 5 × 1012 (low) viral genomes (vg)/kg. Comparable levels of micro-dystrophin expression were observed at each dose in a dose-dependent manner irrespective of the manufacturing method. Vector biodistribution was similar in mice injected with either the TT or the HSV method AAV. Evaluation of muscle degeneration/regeneration showed equivalent protection by vectors made by either method in a dose-dependent manner. Muscle function was similarly improved in a dose-dependent manner irrespective of the vector production method. No apparent toxicity was observed in any mouse. Collectively, our results suggest that the biological potency of the AAV micro-dystrophin vector made by the scalable HSV method is comparable to that made by the TT method., Graphical Abstract, Hakim and colleagues compared AAV9 micro-dystrophin vectors produced by transient transfection and HSV-based methods in the murine DMD model by systemic delivery. They found that these vectors had comparable biological potency. Their findings paved the way to translate an HSV-based AAV production system for clinical application.

Clustered regularly interspaced short palindromic repeats (CRISPR) editing is being considered as a potential gene repair therapy to treat Duchenne muscular dystrophy, a dystrophin-deficient lethal muscle disease affecting all muscles in the body. A recent preliminary study from the Olson laboratory (Amoasii et al. Science 2018;362:89–91) showed robust dystrophin restoration in a canine Duchenne muscular dystrophy model following intramuscular or intravenous delivery of the CRISPR editing machinery by adeno-associated virus serotype 9. Despite the limitation of the small sample size, short study duration, and the lack of muscle function data, the Olson lab findings have provided important proof of principle for scaling up CRISPR therapy from rodents to large mammals. Future large-scale, long-term, and comprehensive studies are warranted to establish the safety and efficacy of CRISPR editing therapy in large mammals.

Key points We developed a novel method to study sympatholysis in dogs. We showed abolishment of sarcolemmal nNOS, and reduction of total nNOS and total eNOS in the canine Duchenne muscular dystrophy (DMD) model. We showed sympatholysis in dogs involving both nNOS-derived NO-dependent and NO-independent mechanisms. We showed that the loss of sarcolemmal nNOS compromised sympatholysis in the canine DMD model. We showed that NO-independent sympatholysis was not affected in the canine DMD model. Abstract The absence of dystrophin in Duchenne muscular dystrophy (DMD) leads to the delocalization of neuronal nitric oxide synthase (nNOS) from the sarcolemma. Sarcolemmal nNOS plays an important role in sympatholysis, a process of attenuating reflex sympathetic vasoconstriction during exercise to ensure blood perfusion in working muscle. Delocalization of nNOS compromises sympatholysis resulting in functional ischaemia and muscle damage in DMD patients and mouse models. Little is known about the contribution of membrane-associated nNOS to blood flow regulation in dystrophin-deficient DMD dogs. We tested the hypothesis that the loss of sarcolemmal nNOS abolishes protective sympatholysis in contracting muscle of affected dogs. Haemodynamic responses to noradrenaline in the brachial artery were evaluated at rest and during contraction in the absence and presence of NOS inhibitors. We found sympatholysis was significantly compromised in DMD dogs, as well as in normal dogs treated with a selective nNOS inhibitor, suggesting that the absence of sarcolemmal nNOS underlies defective sympatholysis in the canine DMD model. Surprisingly, inhibition of all NOS isoforms did not completely abolish sympatholysis in normal dogs, suggesting sympatholysis in canine muscle also involves NO-independent mechanism(s). Our study established a foundation for using the dog model to test therapies aimed at restoring nNOS homeostasis in DMD.

Dual adeno-associated virus (AAV) technology was developed in 2000 to double the packaging capacity of the AAV vector. The proof of principle has been demonstrated in various mouse models. Yet, pivotal evidence is lacking in large animal models of human diseases. Here we report expression of a 7-kb canine ΔH2–R15 mini-dystrophin gene using a pair of dual AAV vectors in the canine model of Duchenne muscular dystrophy (DMD). The ΔH2–R15 minigene is by far the most potent synthetic dystrophin gene engineered for DMD gene therapy. We packaged minigene dual vectors in Y731F tyrosine-modified AAV-9 and delivered to the extensor carpi ulnaris muscle of a 12-month-old affected dog at the dose of 2 × 10(13) viral genome particles/vector/muscle. Widespread mini-dystrophin expression was observed 2 months after gene transfer. The missing dystrophin-associated glycoprotein complex was restored. Treatment also reduced muscle degeneration and fibrosis and improved myofiber size distribution. Importantly, dual AAV therapy greatly protected the muscle from eccentric contraction-induced force loss. Our data provide the first clear evidence that dual AAV therapy can be translated to a diseased large mammal. Further development of dual AAV technology may lead to effective therapies for DMD and many other diseases in human patients.

Micro-dystrophins are highly promising candidates for treating Duchenne muscular dystrophy, a lethal muscle disease caused by dystrophin deficiency. Here, we report robust disease rescue in the severe DBA/2J-mdx model with a neuronal nitric oxide synthase (nNOS)-binding micro-dystrophin vector. 2 × 1013 vector genome particles/mouse of the vector were delivered intravenously to 10-week-old mice and were evaluated at 6 months of age. Saturated micro-dystrophin expression was detected in all skeletal muscles and the heart and restored the dystrophin-associated glycoprotein complex and nNOS. In skeletal muscle, therapy substantially reduced fibrosis and calcification and significantly attenuated inflammation. Centronucleation was significantly decreased in the tibialis anterior (TA) and extensor digitorum longus (EDL) muscles but not in the quadriceps. Muscle function was normalized in the TA and significantly improved in the EDL muscle. Heart histology and function were also evaluated. Consistent with the literature, DBA/2J-mdx mice showed myocardial calcification and fibrosis and cardiac hemodynamics was compromised. Surprisingly, similar myocardial pathology and hemodynamic defects were detected in control DBA/2J mice. As a result, interpretation of the cardiac data proved difficult due to the confounding phenotype in control DBA/2J mice. Our results support further development of this microgene vector for clinical translation. Further, DBA/2J-mdx mice are not good models for Duchenne cardiomyopathy.

Background Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic X-linked recessive muscle diseases caused by mutations in the DMD gene, with DMD being the more severe form. We have recently shown that increased plasma low-density lipoprotein–associated cholesterol causes severe muscle wasting in the mdx mouse, a mild DMD model, which suggested that plasma lipids may play a critical role in DMD. We have also observed that loss of dystrophin in mice causes unexpected elevations in plasma lipoprotein levels. Objective The objectives of the study were to determine whether patients with DMD and BMD also present with clinically relevant plasma lipoprotein abnormalities and to mitigate the presence of confounders (medications and lifestyle) by analyzing the plasma from patients with DMD/BMD and unmedicated dogs with DMD, the most relevant model of DMD. Methods Levels of low-density lipoprotein–associated cholesterol, high-density lipoprotein cholesterol, and triglycerides were analyzed in patients with DMD and BMD and female carriers. Samples from unmedicated, ambulatory dogs with DMD, unaffected carriers, and normal controls were also analyzed. Results We report that 97% and 64% of all pediatric patients with DMD (33 of 36) and BMD (6 of 11) are dyslipidemic, along with an unusually high incidence in adult patients with BMD. All dogs with DMD showed plasma lipid abnormalities that progressively worsened with age. Most strikingly, unaffected carrier dogs also showed plasma lipid abnormalities similar to affected dogs with DMD. Dyslipidemia is likely not secondary to liver damage as unaffected carriers showed no plasma aminotransferase elevation. Conclusions The high incidence of plasma lipid abnormalities in dystrophin-deficient plasma may depict a new type of genetic dyslipidemia. Abnormal lipid levels in dystrophinopathic samples in the absence of muscle damage suggest a primary state of dyslipidemia. Whether dyslipidemia plays a causal role in patients with DMD warrants further investigation, which could lead to new diagnostic and therapeutic options.

CRISPR editing of muscle stem cells (MuSCs) with adeno-associated virus serotype-9 (AAV9) holds promise for sustained gene repair therapy for muscular dystrophies. However, conflicting evidence exists on whether AAV9 transduces MuSCs. To rigorously address this question, we used a muscle graft model. The grafted muscle underwent complete necrosis before regenerating from its MuSCs. We injected AAV9.Cre into Ai14 mice. These mice express tdTomato upon Cre-mediated removal of a floxed stop codon. About 28%–47% and 24%–89% of Pax7(+) MuSCs expressed tdTomato in pre-grafts and regenerated grafts (p > 0.05), respectively, suggesting AAV9 efficiently transduced MuSCs, and AAV9-edited MuSCs renewed successfully. Robust MuSC transduction was further confirmed by delivering AAV9.Cre to Pax7-ZsGreen-Ai14 mice in which Pax7(+) MuSCs are genetically labeled by ZsGreen. Next, we co-injected AAV9.Cas9 and AAV9.gRNA to dystrophic mdx mice to repair the mutated dystrophin gene. CRISPR-treated and untreated muscles were grafted to immune-deficient, dystrophin-null NSG.mdx4cv mice. Grafts regenerated from CRISPR-treated muscle contained the edited genome and yielded 2.7-fold more dystrophin(+) cells (p = 0.015). Importantly, increased dystrophin expression was not due to enhanced formation of revertant fibers or de novo transduction by residual CRISPR vectors in the graft. We conclude that AAV9 effectively transduces MuSCs. AAV9 CRISPR editing of MuSCs may provide enduring therapy.

Many diseases affect multiple tissues and/or organ systems, or affect tissues that are broadly distributed. For these diseases, an effective gene therapy will require systemic delivery of the therapeutic vector to all affected locations. Adeno-associated virus (AAV) has been used as a gene therapy vector for decades in preclinical studies and human trials. These studies have shown outstanding safety and efficacy of the AAV vector for gene therapy. Recent studies have revealed yet another unique feature of the AAV vector. Specifically, AAV can lead to bodywide gene transfer following a single intravascular injection. Here we describe the protocols for effective systemic delivery of AAV in both neonatal and adult mice and dogs. We also share lessons we learned from systemic gene therapy in the murine and canine models of Duchenne muscular dystrophy.

Background and objective Gait analysis is valuable for studying neuromuscular and skeletal diseases. Wearable motion sensors or inertial measurement units (IMUs) have become common for human gait analysis. Canines are important large animal models for translational research of human diseases. Our objective is to develop a method for accurate and reliable determination of the timing of each stride in dogs using a wearable IMU. Methods We built a wireless IMU sensor using off-the-shelf components. We also developed a MATLAB algorithm for data acquisition and stride timing determination. Stride parameters from 1,259 steps of three adult mixed breed dogs were determined across a range of six height-normalized speeds using the IMU system. The IMU results were validated by frame-by-frame manual counting of high-speed video recordings. Results Comparing IMU derived results with video revealed that the mean error ± standard deviation for stride, stance, and swing duration was 0.001 ± 0.025, -0.001 ± 0.030, and 0.001 ± 0.019 s respectively. A mean error ± standard deviation of 0.000 ± 0.020 and -0.008 ± 0.027 s was obtained for determining toe-off and toe-touch events respectively. Only one step was missed by the algorithm in the video dataset of 1,259 steps. Conclusion We have developed and validated an IMU method for automatic canine gait analysis. Our method can be used for studying neuromuscular diseases in veterinary clinics and in translational research.

The National Center for Advancing Translational Sciences (NCATS) at the National Institutes of Health (NIH) was established in December 2011. NCATS mission is to catalyze the generation of innovative methods and technologies that will enhance the development, testing, and implementation of interventions that improve human health across a wide range of human diseases and conditions. Notably, the “innovative methods and technologies” that are NCATS focus include gene therapy, and the “wide range of human diseases” includes rare diseases, which are a special area of focus for NCATS, and for which gene therapy may have a particularly large impact. In contrast to other institutes and centers at NIH, NCATS does not focus on individual diseases or organ systems, but on what is common among them, and on the common roadblocks and limitations in the translational science process. NCATS defines translation as the process of turning observations in the laboratory, clinic, and community into interventions that improve the health of individuals and the public—from diagnostics and therapeutics to medical procedures and behavioral changes. We define “translational science” as the field of investigation that seeks to understand the scientific and operational principles underlying each step of the translational process. We study translation on a system-wide level, focusing on creative solutions to both scientific and operational problems. A hallmark of NCATS approach to translation is collaboration and team science. The enormous range of disciplines—from target qualification through intervention development to demonstration of clinical efficacy to implementation in the community—means that successful translation requires multidisciplinary teams including individuals from diverse scientific backgrounds, as well as patients and patient advocates, working together. Nowhere is the importance of collaboration and team science seen more clearly than in rare diseases. As we will highlight below, patients and patient advocacy groups (PAGs) are an essential part of collaborative teams, and in many cases are driving rare disease intervention development, including gene therapy. One of the biggest hurdles facing therapeutics development in rare diseases is the sheer number of individual conditions, commonly estimated at around 6000. Well over 90% of these rare conditions have no FDA-approved treatment, and at the current rate of new drug development, hundreds of years will pass before all rare diseases are treatable with a therapy shown to be safe and effective. To address this major challenge, NCATS is particularly interested in “platform”-type approaches that can be readily adapted to multiple diseases. One example is high-throughput screening of all clinically approved drugs1 to find those that can be “repurposed” expeditiously to treat other diseases. Another strategy is to take advantage of expanding knowledge of disease biology, and focus drug development on shared underlying molecular etiologies rather than clinical phenotype.2 Gene therapy approaches, including viral vector-mediated ex vivo or in vivo gene transfer, genome editing, and other nucleic acid therapeutics, are inherent platforms, and of obvious relevance for the treatment of the more than 4000 known rare monogenic disorders. In this editorial, we first consider NCATS support for gene therapy across its different programs, including NCATS new initiatives to establish gene therapy-based platforms for rare diseases. We then highlight some areas and aspects of gene therapy we see as particularly promising and how they relate to NCATS mission and priorities.

Duchenne muscular dystrophy (DMD) is a lethal X-linked childhood muscle wasting disease caused by mutations in the dystrophin gene. Nanobiotechnology-based therapies (such as synthetic nanoparticles and naturally existing viral and nonviral nanoparticles) hold great promise to replace and repair the mutated dystrophin gene and significantly change the disease course. While a majority of DMD nanotherapies are still in early preclinical development, several [such as adeno-associated virus (AAV)-mediated systemic micro-dystrophin gene therapy] are advancing for phase I clinical trials. Recent regulatory approval of Ataluren (a nonsense mutation read-through chemical) in Europe and Exondys51 (an exon-skipping antisense oligonucleotide drug) in the United States shall offer critical insight in how to move DMD nanotherapy to human patients. Progress in novel, optimized nano-delivery systems may further improve emerging molecular therapeutic modalities for DMD. Despite these progresses, DMD nanotherapy faces a number of unique challenges. Specifically, the dystrophin gene is one of the largest genes in the genome while nanoparticles have an inherent size limitation per definition. Furthermore, muscle is the largest tissue in the body and accounts for 40% of the body mass. How to achieve efficient bodywide muscle targeting in human patients with nanomedication remains a significant translational hurdle. New creative approaches in the design of the miniature micro-dystrophin gene, engineering of muscle-specific synthetic AAV capsids, and novel nanoparticle-mediated exon-skipping are likely to result in major breakthroughs in DMD therapy. WIREs Nanomed Nanobiotechnol 2018, 10:e1472. doi: 10.1002/wnan.1472 This article is categorized under: Biology-Inspired Nanomaterials > Protein and Virus-Based Structures Therapeutic Approaches and Drug Discovery > Emerging Technologies.

The portfolio of the National Center for Advancing Translational Sciences (NCATS) rare-diseases therapeutic development program comprises 28 research projects initiated at the preclinical stage. Historical data reveal substantially lower costs and higher success rates but longer preclinical timelines for the NCATS projects relative to the industry averages for early-stage translational medical research and development (R&D) typically cited in literature. Here, we evaluate the potential risks and rewards of investing in a portfolio of rare-disease therapeutics. Using a “megafund” financing structure, NCATS data, and valuation estimates from a panel of industry experts, we simulate a hypothetical megafund in which senior and junior debt yielded 5 and 8%, respectively. The simulated expected return to equity was 14.7%, corresponding to a modified internal rate of return of 21.6%. These returns and the likelihood of private-sector funding can be enhanced through third-party funding guarantees from philanthropies, patient advocacy groups, and government agencies., MIT Laboratory for Financial Engineering, National Institutes of Health (U.S.). Intramural Research Program, National Institutes of Health (U.S.). National Center for Advancing Translational Sciences