Of mice and…more mice: The search for an effective treatment for muscular dystrophy using gene therapy [abstract]

Faculty Mentor: Dongsheng Duan, Molecular Biology and ImmunologyAbstract only availableGene therapy represents a promising future treatment for Duchenne muscular dystrophy (DMD), the most severe and debilitating form of muscular dystrophy. Mutations in the dystrophin gene are the cause of DMD. Since the dystrophin gene is too large to be packaged in gene therapy vehicles, truncated forms of the gene must be used when administering gene therapy. Previous studies have shown that some of the truncated dystrophin genes capable of ameliorating certain skeletal muscle problems associated with DMD. However, it is not clear whether these truncated genes can improve overall performance. In this experiment, we hypothesize than an optimized mini-dystrophin gene can improve cardiovascular endurance if it is expressed in the heart of the mdx mouse, a mouse model for DMD. A treadmill test was then performed on these transgenic mice and similarly aged controls. Using a Columbus Instruments Treadmill with a shock grid at the base, the mice were trained with increasing intensity for five days, and then tested for overall cardiovascular endurance on the sixth day. Groups of mice were either assigned to an uphill (+7o) or a downhill (-15o) treadmill grade. After training, the mice were placed on a treadmill moving at a medium pace as a warm up for 20 minutes, then the speed was gradually increased until the mouse was exhausted. When a mouse sits on the shock grid for greater than five seconds and fails to reenter the treadmill with gentle prodding, the mouse is defined as exhausted. The transgenic mice consistently ran further, and for a longer period of time than the control mdx mice, which have no dystrophin expression. In the uphill test the transgenic mice ran an average of 603+/-134.73 meters (n=11), while the mdx mice ran an average of 484.25+/-89.9 meters (n=4) and the BL10 ran an average of 686.13+/-77.1 meters (n=8). In the downhill test, the transgenic mice ran an average of 657.78+/-173.76 meters (n=9), with mdx and BL10 still to be tested. This data suggests that heart specific expression of the minigene may greatly improve cardiovascular health in dystrophic mice. In addition to the mini gene, a micro-dystrophin gene has also been proposed for DMD gene therapy. To further evaluate the therapeutic efficacy of the microgene in skeletal muscle, we performed a second study.  In this study, we used a Columbus Instruments Grip Strength Meter to measure the overall force development from limb muscle in the transgenic mice and similarly aged con

Novel mini/micro-dystrophin genes restore nNOS to the sarcolemma and improve the therapeutic outcome for Duchenne Muscular Dystrophy [abstract]

Duchenne muscular dystrophy (DMD) is a muscle disease caused by mutations of the dystrophin gene. Gene replacement therapy represents a very promising approach to cure this disease. The dystrophin gene is one of the largest genes in the genome and it exceeds the carrying capacity of adenoassociated viral vector (AAV) and lentiviral vector, the most powerful gene delivery vehicles for muscle. The truncated mini/micro-dystrophin genes have been developed to overcome this hurdle. Despite improvement of muscle function and the dystrophic phenotype by these mini/micro-dystrophin genes, none of them can restore sarcolemmal neuronal nitric oxide synthase (nNOS). Sarcolemmal nNOS plays a crucial role in maintaining blood perfusion during muscle contraction. In DMD patients, sarcolemmal nNOS is lost. Consequently, it leads to functional ischemia and muscle damage. To improve the therapeutic efficacy of the truncated dystrophin genes, one has to develop the new synthetic dystrophin genes with the ability to restore sarcolemmal nNOS. The motif for nNOS sarcolemmal localization was identified and incorporated into new mini/microdystrophin genes. The effect of sarcolemmal nNOS restoration on the muscle functions, blood perfusion in contracting muscle and exercise performance was evaluated. Dystrophin spectrin-like repeats 16 and 17 (R16/17) are required for sarcolemmal nNOS localization. The synthetic mini/micro-dystrophin genes carrying R16/17 significantly improved muscle function, blood perfusion and exercise capacity. Our newly developed R16/17 mini/micro-dystrophin genes provide functions close to that of the full-length dystrophin gene. They represent excellent candidate genes for DMD gene therapy

Improving the hybrid adeno-associated viral vectors with the exonic splicing enhancer

Abstract only availableAdeno-associated virus (AAV) is a rising champion for gene therapy due to its powerful and safe gene delivery properties. Yet the small packaging capacity of AAV limits its application for large therapeutic genes. The hybrid dual vectors show promise in overcoming this limitation (Ghosh 2008). In this approach, the gene of interest is split into 5' and 3' parts. Each part is packaged in a separate AAV virion. The 5' vector carries a splice donor, and the 3' vector a splice acceptor. Both also share an overlapping sequence. Recombination occurs simultaneously under two pathways, either through homologous recombination mediated by the overlapping sequence or head-to-tail ITR-mediated recombination. The recombined genome is spliced to generate mature mRNA. In an effort to further improve the hybrid vectors, we replaced the alkaline phosphatase (AP) overlapping sequence in a previously published LacZ hybrid vector set with an exonic splicing enhancer (ESE) sequence. The ESE sequences are known to increase splicing efficiency (Skordis, 2003). We hypothesized that the ESE sequence should increase splicing efficiency in the hybrid vectors, hence, lead to greater gene expression. To test this hypothesis, we transfected 293 cells with a series of different LacZ constructs. We then measured LacZ protein expression by cytochemical staining and enzymatic assay. Green fluorescent protein (GFP) containing plasmid was co-transfected as an internal control for transfection efficiency. The ESE-intron LacZ, unmodified intact LacZ, intron LacZ and AP-intron LacZ yielded β-galactosidase (LacZ) activity of 475 ± 36, 361 ± 18, 283 ± 9 and 225 ± 35 units/µg protein, respectively. Next, we split the ESE-intron LacZ gene into two separate constructs, which can be used to make recombinant AAV. Encouragingly, co-transfection of the split LacZ ESE-intron constructs resulted in 5.61 ± 0.76 units/µg protein β-galactosidase activity while the previously described split LacZ AP-intron and split LacZ intron constructs resulted in 3.93 ± 0.36 and 3.41 ± 0.36 units/µg protein β-galactosidase activity, respectively. Our results suggest that the ESE-intron vector is operating at par with the intact gene and may provide better gene delivery than the current generation of hybrid vectors. Future study with the corresponding recombinant AAV viruses may further reveal the utility of ESE containing hybrid vectors.Summer Research Internship in Cell & Molecular Biolog

SERCA2a gene transfer improves electrocardiographic performance in aged mdx mice

<p>Abstract</p> <p>Background</p> <p>Cardiomyocyte calcium overloading has been implicated in the pathogenesis of Duchenne muscular dystrophy (DMD) heart disease. The cardiac isoform of sarcoplasmic reticulum calcium ATPase (SERCA2a) plays a major role in removing cytosolic calcium during heart muscle relaxation. Here, we tested the hypothesis that SERCA2a over-expression may mitigate electrocardiography (ECG) abnormalities in old female mdx mice, a murine model of DMD cardiomyopathy.</p> <p>Methods</p> <p>1 × 10¹²viral genome particles/mouse of adeno-associated virus serotype-9 (AAV-9) SERCA2a vector was delivered to 12-m-old female mdx mice (N = 5) via a single bolus tail vein injection. AAV transduction and the ECG profile were examined eight months later.</p> <p>Results</p> <p>The vector genome was detected in the hearts of all AAV-injected mdx mice. Immunofluorescence staining and western blot confirmed SERCA2a over-expression in the mdx heart. Untreated mdx mice showed characteristic tachycardia, PR interval reduction and QT interval prolongation. AAV-9 SERCA2a treatment corrected these ECG abnormalities.</p> <p>Conclusions</p> <p>Our results suggest that AAV SERCA2a therapy may hold great promise in treating dystrophin-deficient heart disease.</p

Gene therapy for Duchenne muscular dystrophy heart disease requires treating both heart and skeletal muscle

Abstract only availableDuchenne muscular dystrophy (DMD) is a lethal muscle wasting disease caused by mutations in the dystrophin gene. Affected children are wheelchair bound by the age of ten and die in their mid-twenties from respiratory and/or cardiac failure. Gene therapy represents a promising avenue for curing DMD. While significant progress has been made for treatment of skeletal muscle disease, few studies have investigated the potential of gene therapy to treat heart disease. A cure for DMD requires rescuing both skeletal and heart muscles. Gene therapy aims to deliver a functional copy of the dystrophin gene to affected muscle cells. However, the dystrophin gene is the largest gene in the body and cannot be effectively delivered with any currently available methods. This led researchers to develop abbreviated versions of the dystrophin gene. The most promising of these genes is a 7 kb mini-dystrophin gene which can completely restore skeletal muscle in the mdx mouse model of DMD. The potential of the mini-dystrophin gene for treating heart disease is uncertain. Cardiac specific mini-dystrophin gene expression improved but did not normalize heart function. To investigate whether the incomplete cardiac rescue is due to skeletal muscle disease, we generated double transgenic male mdx mice which expressed the mini-dystrophin gene in both heart and skeletal muscle. We performed comprehensive skeletal and cardiac muscle testing at 6 months of age. Restoration of skeletal muscle function was confirmed by the grip strength assay. Next, we performed an uphill treadmill assay to gauge overall cardiac performance. Double transgenic mice ran significantly farther than cardiac transgenic mice. Finally, we performed electrocardiographic (ECG) analysis to examine the function of the cardiac conduction system. ECG analysis revealed an improved heart rate for double transgenic mice when compared to heart-only transgenic mice. Taken together, these results support a role for skeletal muscle disease in modulating heart function. Furthermore, these findings highlight the importance of tailoring gene therapy approaches to treat both the heart and skeletal muscle.Life Sciences Undergraduate Research Opportunity Progra

Overexpression of BMI-1 Promotes Cell Growth and Resistance to Cisplatin Treatment in Osteosarcoma

Background: BMI-1 is a member of the polycomb group of genes (PcGs), and it has been implicated in the development and progression of several malignancies, but its role in osteosarcoma remains to be elucidated. Methodology/Principal Findings: In the present study, we found that BMI-1 was overexpressed in different types of osteosarcomas. Downregulation of BMI-1 by lentivirus mediated RNA interference (RNAi) significantly impaired cell viability and colony formation in vitro and tumorigenesis in vivo of osteosarcoma cells. BMI-1 knockdown sensitized cells to cisplatininduced apoptosis through inhibition of PI3K/AKT pathway. Moreover, BMI-1-depletion-induced phenotype could be rescued by forced expression of BMI-1 wobble mutant which is resistant to inhibition by the small interfering RNA (siRNA). Conclusions/Significance: These findings suggest a crucial role for BMI-1 in osteosarcoma pathogenesis

Learning from Easy to Complex: Adaptive Multi-curricula Learning for Neural Dialogue Generation

Current state-of-the-art neural dialogue systems are mainly data-driven and are trained on human-generated responses. However, due to the subjectivity and open-ended nature of human conversations, the complexity of training dialogues varies greatly. The noise and uneven complexity of query-response pairs impede the learning efficiency and effects of the neural dialogue generation models. What is more, so far, there are no unified dialogue complexity measurements, and the dialogue complexity embodies multiple aspects of attributes---specificity, repetitiveness, relevance, etc. Inspired by human behaviors of learning to converse, where children learn from easy dialogues to complex ones and dynamically adjust their learning progress, in this paper, we first analyze five dialogue attributes to measure the dialogue complexity in multiple perspectives on three publicly available corpora. Then, we propose an adaptive multi-curricula learning framework to schedule a committee of the organized curricula. The framework is established upon the reinforcement learning paradigm, which automatically chooses different curricula at the evolving learning process according to the learning status of the neural dialogue generation model. Extensive experiments conducted on five state-of-the-art models demonstrate its learning efficiency and effectiveness with respect to 13 automatic evaluation metrics and human judgments.Comment: Accepted to AAAI 202