Publications by authors named "Guy L Odom"

23 Publications

  • Page 1 of 1

Micro-dystrophin gene therapy prevents heart failure in an improved Duchenne muscular dystrophy cardiomyopathy mouse model.

JCI Insight 2021 Apr 8;6(7). Epub 2021 Apr 8.

Department of Physiology & Cell Biology and Davis Heart and Lung Research Institute, College of Medicine, The Ohio State University, Columbus, Ohio, USA.

Gene replacement for Duchenne muscular dystrophy (DMD) with micro-dystrophins has entered clinical trials, but efficacy in preventing heart failure is unknown. Although most patients with DMD die from heart failure, cardiomyopathy is undetectable until the teens, so efficacy from trials in young boys will be unknown for a decade. Available DMD animal models were sufficient to demonstrate micro-dystrophin efficacy on earlier onset skeletal muscle pathology underlying loss of ambulation and respiratory insufficiency in patients. However, no mouse models progressed into heart failure, and dog models showed highly variable progression insufficient to evaluate efficacy of micro-dystrophin or other therapies on DMD heart failure. To overcome this barrier, we have generated the first DMD mouse model to our knowledge that reproducibly progresses into heart failure. This model shows cardiac inflammation and fibrosis occur prior to reduced function. Fibrosis does not continue to accumulate, but inflammation persists after function declines. We used this model to test micro-dystrophin gene therapy efficacy on heart failure prevention for the first time. Micro-dystrophin prevented declines in cardiac function and prohibited onset of inflammation and fibrosis. This model will allow identification of committed pathogenic steps to heart failure and testing of genetic and nongenetic therapies to optimize cardiac care for patients with DMD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/jci.insight.146511DOI Listing
April 2021

Microutrophin expression in dystrophic mice displays myofiber type differences in therapeutic effects.

PLoS Genet 2020 11 11;16(11):e1009179. Epub 2020 Nov 11.

Department of Neurology, University of Washington, Seattle, Washington, United States of America.

Gene therapy approaches for DMD using recombinant adeno-associated viral (rAAV) vectors to deliver miniaturized (or micro) dystrophin genes to striated muscles have shown significant progress. However, concerns remain about the potential for immune responses against dystrophin in some patients. Utrophin, a developmental paralogue of dystrophin, may provide a viable treatment option. Here we examine the functional capacity of an rAAV-mediated microutrophin (μUtrn) therapy in the mdx4cv mouse model of DMD. We found that rAAV-μUtrn led to improvement in dystrophic histopathology & mostly restored the architecture of the neuromuscular and myotendinous junctions. Physiological studies of tibialis anterior muscles indicated peak force maintenance, with partial improvement of specific force. A fundamental question for μUtrn therapeutics is not only can it replace critical functions of dystrophin, but whether full-length utrophin impacts the therapeutic efficacy of the smaller, highly expressed μUtrn. As such, we found that μUtrn significantly reduced the spacing of the costameric lattice relative to full-length utrophin. Further, immunostaining suggested the improvement in dystrophic pathophysiology was largely influenced by favored correction of fast 2b fibers. However, unlike μUtrn, μdystrophin (μDys) expression did not show this fiber type preference. Interestingly, μUtrn was better able to protect 2a and 2d fibers in mdx:utrn-/- mice than in mdx4cv mice where the endogenous full-length utrophin was most prevalent. Altogether, these data are consistent with the role of steric hindrance between full-length utrophin & μUtrn within the sarcolemma. Understanding the stoichiometry of this effect may be important for predicting clinical efficacy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1009179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7682874PMC
November 2020

Gene Therapy Rescues Cardiac Dysfunction in Duchenne Muscular Dystrophy Mice by Elevating Cardiomyocyte Deoxy-Adenosine Triphosphate.

JACC Basic Transl Sci 2019 Nov 2;4(7):778-791. Epub 2019 Oct 2.

Department of Neurology, University of Washington, Seattle, Washington.

Mutations in the gene encoding for dystrophin leads to structural and functional deterioration of cardiomyocytes and is a hallmark of cardiomyopathy in Duchenne muscular dystrophy (DMD) patients. Administration of recombinant adeno-associated viral vectors delivering microdystrophin or ribonucleotide reductase (RNR), under muscle-specific regulatory control, rescues both baseline and high workload-challenged hearts in an aged, DMD mouse model. However, only RNR treatments improved both systolic and diastolic function under those conditions. Cardiac-specific recombinant adeno-associated viral treatment of RNR holds therapeutic promise for improvement of cardiomyopathy in DMD patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jacbts.2019.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978556PMC
November 2019

Syntrophin binds directly to multiple spectrin-like repeats in dystrophin and mediates binding of nNOS to repeats 16-17.

Hum Mol Genet 2018 09;27(17):2978-2985

Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195-7290, USA.

Mutation of the gene encoding dystrophin leads to Duchenne and Becker muscular dystrophy (DMD and BMD). Currently, dystrophin is thought to function primarily as a structural protein, connecting the muscle cell actin cytoskeleton to the extra-cellular matrix. In addition to this structural role, dystrophin also plays an important role as a scaffold that organizes an array of signaling proteins including sodium, potassium, and calcium channels, kinases, and nitric oxide synthase (nNOS). Many of these signaling proteins are linked to dystrophin via syntrophin, an adapter protein that is known to bind directly to two sites in the carboxyl terminal region of dystrophin. A search of the dystrophin sequence revealed three additional potential syntrophin binding sites (SBSs) within the spectrin-like repeat (SLR) region of dystrophin. Binding assays revealed that the site at SLR 17 bound specifically to the α isoform of syntrophin while the site at SLR 22 bound specifically to the β-syntrophins. The SLR 17 α-SBS contained the core sequence known to be required for nNOS-dystrophin interaction. In vitro and in vivo assays indicate that α-syntrophin facilitates the nNOS-dystrophin interaction at this site rather than nNOS binding directly to dystrophin as previously reported. The identification of multiple SBSs within the SLR region of dystrophin demonstrates that this region functions as a signaling scaffold. The signaling role of the SLR region of dystrophin will need to be considered for effective gene replacement or exon skipping based DMD/BMD therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/ddy197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6097012PMC
September 2018

Translation of Cardiac Myosin Activation with 2-deoxy-ATP to Treat Heart Failure via an Experimental Ribonucleotide Reductase-Based Gene Therapy.

JACC Basic Transl Sci 2016 Dec;1(7):666-679

Department of Bioengineering, University of Washington, Seattle, Washington, USA.

Despite recent advances, chronic heart failure remains a significant and growing unmet medical need, reaching epidemic proportions carrying substantial morbidity, mortality, and costs. A safe and convenient therapeutic agent that produces sustained inotropic effects could ameliorate symptoms, and improve functional capacity and quality of life. We discovered small amounts of 2-deoxy-ATP (dATP) activate cardiac myosin leading to enhanced contractility in normal and failing heart muscle. Cardiac myosin activation triggers faster myosin crossbridge cycling with greater force generation during each contraction. We describe the rationale and results of a translational medicine effort to increase dATP levels using a gene therapy strategy that upregulates ribonucleotide reductase, the rate-limiting enzyme for dATP synthesis, selectively in cardiomyocytes. In small and large animal models of heart failure, a single dose of this gene therapy has led to sustained inotropic effects with no toxicity or safety concerns identified to-date. Further animal studies are being conducted with the goal of testing this agent in patients with heart failure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jacbts.2016.07.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5444879PMC
December 2016

Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy.

Nat Commun 2017 02 14;8:14454. Epub 2017 Feb 14.

Department of Neurology, University of Washington, Seattle, Washington 98195-7720, USA.

Gene replacement therapies utilizing adeno-associated viral (AAV) vectors hold great promise for treating Duchenne muscular dystrophy (DMD). A related approach uses AAV vectors to edit specific regions of the DMD gene using CRISPR/Cas9. Here we develop multiple approaches for editing the mutation in dystrophic mdx mice using single and dual AAV vector delivery of a muscle-specific Cas9 cassette together with single-guide RNA cassettes and, in one approach, a dystrophin homology region to fully correct the mutation. Muscle-restricted Cas9 expression enables direct editing of the mutation, multi-exon deletion or complete gene correction via homologous recombination in myogenic cells. Treated muscles express dystrophin in up to 70% of the myogenic area and increased force generation following intramuscular delivery. Furthermore, systemic administration of the vectors results in widespread expression of dystrophin in both skeletal and cardiac muscles. Our results demonstrate that AAV-mediated muscle-specific gene editing has significant potential for therapy of neuromuscular disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms14454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5316861PMC
February 2017

Validation of ultrasonography for non-invasive assessment of diaphragm function in muscular dystrophy.

J Physiol 2016 12 13;594(24):7215-7227. Epub 2016 Oct 13.

Department of Physiology and Biophysics, University of Washington, Seattle, WA, 98195, USA.

Key Points: Duchenne muscular dystrophy (DMD) is a severe, degenerative muscle disease that is commonly studied using the mdx mouse. The mdx diaphragm muscle closely mimics the pathophysiological changes in DMD muscles. mdx diaphragm force is commonly assessed ex vivo, precluding time course studies. Here we used ultrasonography to evaluate time-dependent changes in diaphragm function in vivo, by measuring diaphragm movement amplitude. In mdx mice, diaphragm amplitude decreased with age and values were much lower than for wild-type mice. Importantly, diaphragm amplitude strongly correlated with ex vivo specific force values. Micro-dystrophin administration increased mdx diaphragm amplitude by 26% after 4 weeks. Diaphragm amplitude correlated positively with ex vivo force values and negatively with diaphragm fibrosis, a major cause of DMD muscle weakness. These studies validate diaphragm ultrasonography as a reliable technique for assessing time-dependent changes in mdx diaphragm function in vivo. This technique will be valuable for testing potential therapies for DMD.

Abstract: Duchenne muscular dystrophy (DMD) is a severe, degenerative muscle disease caused by dystrophin mutations. The mdx mouse is a widely used animal model of DMD. The mdx diaphragm muscle most closely recapitulates key features of DMD muscles, including progressive fibrosis and considerable force loss. Diaphragm function in mdx mice is commonly evaluated by specific force measurements ex vivo. While useful, this method only measures force from a small muscle sample at one time point. Therefore, accurate assessment of diaphragm function in vivo would provide an important advance to study the time course of functional decline and treatment benefits. Here, we evaluated an ultrasonography technique for measuring time-dependent changes of diaphragm function in mdx mice. Diaphragm movement amplitude values for mdx mice were considerably lower than those for wild-type, decreased from 8 to 18 months of age, and correlated strongly with ex vivo specific force. We then investigated the time course of diaphragm amplitude changes following administration of an adeno-associated viral vector expressing Flag-micro-dystrophin (AAV-μDys) to young adult mdx mice. Diaphragm amplitude peaked 4 weeks after AAV-μDys administration, and was 26% greater than control mdx mice at this time. This value decreased slightly to 21% above mdx controls after 12 weeks of treatment. Importantly, diaphragm amplitude again correlated strongly with ex vivo specific force. Also, diaphragm amplitude and specific force negatively correlated with fibrosis levels in the muscle. Together, our results validate diaphragm ultrasonography as a reliable technique for assessing time-dependent changes in dystrophic diaphragm function in vivo, and for evaluating potential therapies for DMD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1113/JP272707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5157096PMC
December 2016

Progress and prospects of gene therapy clinical trials for the muscular dystrophies.

Hum Mol Genet 2016 Apr 8;25(R1):R9-17. Epub 2015 Oct 8.

Department of Neurology and

Clinical trials represent a critical avenue for new treatment development, where early phases (I, I/II) are designed to test safety and effectiveness of new therapeutics or diagnostic indicators. A number of recent advances have spurred renewed optimism toward initiating clinical trials and developing refined therapies for the muscular dystrophies (MD's) and other myogenic disorders. MD's encompass a heterogeneous group of degenerative disorders often characterized by progressive muscle weakness and fragility. Many of these diseases result from mutations in genes encoding proteins of the dystrophin-glycoprotein complex (DGC). The most common and severe form among children is Duchenne muscular dystrophy, caused by mutations in the dystrophin gene, with an average life expectancy around 25 years of age. Another group of MD's referred to as the limb-girdle muscular dystrophies (LGMDs) can affect boys or girls, with different types caused by mutations in different genes. Mutation of the α-sarcoglycan gene, also a DGC component, causes LGMD2D and represents the most common form of LGMD. Early preclinical and clinical trial findings support the feasibility of gene therapy via recombinant adeno-associated viral vectors as a viable treatment approach for many MDs. In this mini-review, we present an overview of recent progress in clinical gene therapy trials of the MD's and touch upon promising preclinical advances.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/ddv420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4802376PMC
April 2016

AAV6-mediated Cardiac-specific Overexpression of Ribonucleotide Reductase Enhances Myocardial Contractility.

Mol Ther 2016 Feb 21;24(2):240-250. Epub 2015 Sep 21.

Department of Bioengineering, University of Washington, Seattle, Washington, USA; Center for Cardiovascular Biology, Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington, USA. Electronic address:

Impaired systolic function, resulting from acute injury or congenital defects, leads to cardiac complications and heart failure. Current therapies slow disease progression but do not rescue cardiac function. We previously reported that elevating the cellular 2 deoxy-ATP (dATP) pool in transgenic mice via increased expression of ribonucleotide reductase (RNR), the enzyme that catalyzes deoxy-nucleotide production, increases myosin-actin interaction and enhances cardiac muscle contractility. For the current studies, we initially injected wild-type mice retro-orbitally with a mixture of adeno-associated virus serotype-6 (rAAV6) containing a miniaturized cardiac-specific regulatory cassette (cTnT(455)) composed of enhancer and promotor portions of the human cardiac troponin T gene (TNNT2) ligated to rat cDNAs encoding either the Rrm1 or Rrm2 subunit. Subsequent studies optimized the system by creating a tandem human RRM1-RRM2 cDNA with a P2A self-cleaving peptide site between the subunits. Both rat and human Rrm1/Rrm2 cDNAs resulted in RNR enzyme overexpression exclusively in the heart and led to a significant elevation of left ventricular (LV) function in normal mice and infarcted rats, measured by echocardiography or isolated heart perfusions, without adverse cardiac remodeling. Our study suggests that increasing RNR levels via rAAV-mediated cardiac-specific expression provide a novel gene therapy approach to potentially enhance cardiac systolic function in animal models and patients with heart failure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/mt.2015.176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4817808PMC
February 2016

Recombinant adeno-associated viral (rAAV) vectors mediate efficient gene transduction in cultured neonatal and adult microglia.

J Neurochem 2016 Jan 20;136 Suppl 1:49-62. Epub 2015 Mar 20.

Department of Neurology, University of Washington, Seattle, Washington, USA.

Microglia are a specialized population of myeloid cells that mediate CNS innate immune responses. Efforts to identify the cellular and molecular mechanisms that regulate microglia behaviors have been hampered by the lack of effective tools for manipulating gene expression. Cultured microglia are refractory to most chemical and electrical transfection methods, yielding little or no gene delivery and causing toxicity and/or inflammatory activation. Recombinant adeno-associated viral (rAAVs) vectors are non-enveloped, single-stranded DNA vectors commonly used to transduce many primary cell types and tissues. In this study, we evaluated the feasibility and efficiency of utilizing rAAV serotype 2 (rAAV2) to modulate gene expression in cultured microglia. rAAV2 yields high transduction and causes minimal toxicity or inflammatory response in both neonatal and adult microglia. To demonstrate that rAAV transduction can induce functional protein expression, we used rAAV2 expressing Cre recombinase to successfully excise a LoxP-flanked miR155 gene in cultured microglia. We further evaluated rAAV serotypes 5, 6, 8, and 9, and observed that all efficiently transduced cultured microglia to varying degrees of success and caused little or no alteration in inflammatory gene expression. These results provide strong encouragement for the application of rAAV-mediated gene expression in microglia for mechanistic and therapeutic purposes. Neonatal microglia are functionally distinct from adult microglia, although the majority of in vitro studies utilize rodent neonatal microglia cultures because of difficulties of culturing adult cells. In addition, cultured microglia are refractory to most methods for modifying gene expression. Here, we developed a novel protocol for culturing adult microglia and evaluated the feasibility and efficiency of utilizing Recombinant Adeno-Associated Virus (rAAV) to modulate gene expression in cultured microglia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jnc.13081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4547919PMC
January 2016

Dystrobrevin increases dystrophin's binding to the dystrophin-glycoprotein complex and provides protection during cardiac stress.

J Mol Cell Cardiol 2014 Nov 24;76:106-15. Epub 2014 Aug 24.

Department of Integrative Biology and Physiology, University of Minnesota, Minneapolis, MN, USA. Electronic address:

Duchenne muscular dystrophy is a fatal progressive disease of both cardiac and skeletal muscle resulting from the mutations in the DMD gene and loss of the protein dystrophin. Alpha-dystrobrevin (α-DB) tightly associates with dystrophin but the significance of this interaction within cardiac myocytes is poorly understood. In the current study, the functional role of α-DB in cardiomyocytes and its implications for dystrophin function are examined. Cardiac stress testing demonstrated significant heart disease in α-DB null (adbn(-/-)) mice, which displayed mortality and lesion sizes that were equivalent to those seen in dystrophin-deficient mdx mice. Despite normal expression and subcellular localization of dystrophin in the adbn(-/-) heart, there is a significant decrease in the strength of dystrophin's interaction with the membrane-bound dystrophin-associated glycoprotein complex (DGC). A similar weakening of the dystrophin-membrane interface was observed in mice lacking the sarcoglycan complex. Cardiomyocytes from adbn(-/-) mice were smaller and responded less to adrenergic receptor induced hypertrophy. The basal decrease in size could not be attributed to aberrant Akt activation. In addition, the organization of the microtubule network was significantly altered in adbn(-/-) cardiac myocytes, while the total expression of tubulin was unchanged in adbn(-/-) hearts. These studies demonstrate that α-DB is a multifunctional protein that increases dystrophin's binding to the dystrophin-glycoprotein complex, and is critical for the full functionality of dystrophin.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.yjmcc.2014.08.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4271192PMC
November 2014

Phosphorylation within the cysteine-rich region of dystrophin enhances its association with β-dystroglycan and identifies a potential novel therapeutic target for skeletal muscle wasting.

Hum Mol Genet 2014 Dec 31;23(25):6697-711. Epub 2014 Jul 31.

Department of Neurology, University of Washington School of Medicine, Seattle, WA 98195-7720, USA Department of Biochemistry, University of Washington School of Medicine, Seattle, WA 98195-7350, USA and Program in Molecular and Cellular Biology, University of Washington School of Medicine, Seattle, WA 98195-7275, USA

Mutations in dystrophin lead to Duchenne muscular dystrophy, which is among the most common human genetic disorders. Dystrophin nucleates assembly of the dystrophin-glycoprotein complex (DGC), and a defective DGC disrupts an essential link between the intracellular cytoskeleton and the basal lamina, leading to progressive muscle wasting. In vitro studies have suggested that dystrophin phosphorylation may affect interactions with actin or syntrophin, yet whether this occurs in vivo or affects protein function remains unknown. Utilizing nanoflow liquid chromatography mass spectrometry, we identified 18 phosphorylated residues within endogenous dystrophin. Mutagenesis revealed that phosphorylation at S3059 enhances the dystrophin-dystroglycan interaction and 3D modeling utilizing the Rosetta software program provided a structural model for how phosphorylation enhances this interaction. These findings demonstrate that phosphorylation is a key mechanism regulating the interaction between dystrophin and the DGC and reveal that posttranslational modification of a single amino acid directly modulates the function of dystrophin.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/ddu388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4245040PMC
December 2014

Muscle structure influences utrophin expression in mdx mice.

PLoS Genet 2014 Jun 12;10(6):e1004431. Epub 2014 Jun 12.

Department of Neurology, University of Washington, Seattle, Washington, United States of America.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by mutations in the dystrophin gene. To examine the influence of muscle structure on the pathogenesis of DMD we generated mdx4cv:desmin double knockout (dko) mice. The dko male mice died of apparent cardiorespiratory failure at a median age of 76 days compared to 609 days for the desmin-/- mice. An ∼ 2.5 fold increase in utrophin expression in the dko skeletal muscles prevented necrosis in ∼ 91% of 1a, 2a and 2d/x fiber-types. In contrast, utrophin expression was reduced in the extrasynaptic sarcolemma of the dko fast 2b fibers leading to increased membrane fragility and dystrophic pathology. Despite lacking extrasynaptic utrophin, the dko fast 2b fibers were less dystrophic than the mdx4cv fast 2b fibers suggesting utrophin-independent mechanisms were also contributing to the reduced dystrophic pathology. We found no overt change in the regenerative capacity of muscle stem cells when comparing the wild-type, desmin-/-, mdx4cv and dko gastrocnemius muscles injured with notexin. Utrophin could form costameric striations with α-sarcomeric actin in the dko to maintain the integrity of the membrane, but the lack of restoration of the NODS (nNOS, α-dystrobrevin 1 and 2, α1-syntrophin) complex and desmin coincided with profound changes to the sarcomere alignment in the diaphragm, deposition of collagen between the myofibers, and impaired diaphragm function. We conclude that the dko mice may provide new insights into the structural mechanisms that influence endogenous utrophin expression that are pertinent for developing a therapy for DMD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1004431DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4055409PMC
June 2014

Extracorporeal delivery of rAAV with metabolic exchange and oxygenation.

Sci Rep 2013 ;3:1538

Department of Medicine, Division of Nephrology, University of Washington, Seattle, WA 98195-7720, USA.

Over the past decade much progress has been made towards the treatment of disease with recombinant adeno-associated viral vectors, ranging from cancer to muscular dystrophies, and autoimmune diseases to cystic fibrosis. Given inherent challenges of vector delivery we developed a system incorporating commercially available dialysis equipment. This concept was evaluated in vitro utilizing rAAV expressing the reporter gene human placental alkaline phosphatase. A number of pre-circulating conditions were assessed. Vector recovery was evaluated by quantitative vector genome analysis and cellular transduction assays. A dialysis circulation time course was established, and results were recorded across varied conditions ranging from approximately 2 to 90% retention of viable vector. This approach is unique in that it focuses on efficient localized, isolated and continual delivery of vector to target tissues, provides for the preservation of tissue integrity with dialysis for metabolic exchange and allows for the transfer of oxygen through a secondary membrane post-dialysis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep01538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3607836PMC
September 2013

Gene replacement therapies for duchenne muscular dystrophy using adeno-associated viral vectors.

Curr Gene Ther 2012 Jun;12(3):139-51

Department of Neurology, University of Washington, Seattle, WA 98195-7720, USA.

The muscular dystrophies collectively represent a major health challenge, as few significant treatment options currently exist for any of these disorders. Recent years have witnessed a proliferation of novel approaches to therapy, spanning increased testing of existing and new pharmaceuticals, DNA delivery (both anti-sense oligonucleotides and plasmid DNA), gene therapies and stem cell technologies. While none of these has reached the point of being used in clinical practice, all show promise for being able to impact different types of muscular dystrophies. Our group has focused on developing direct gene replacement strategies to treat recessively inherited forms of muscular dystrophy, particularly Duchenne and Becker muscular dystrophy (DMD/BMD). Both forms of dystrophy are caused by mutations in the dystrophin gene and all cases can in theory be treated by gene replacement using synthetic forms of the dystrophin gene. The major challenges for success of this approach are the development of a suitable gene delivery shuttle, generating a suitable gene expression cassette able to be carried by such a shuttle, and achieving safe and effective delivery without elicitation of a destructive immune response. This review summarizes the current state of the art in terms of using adeno-associated viral vectors to deliver synthetic dystrophin genes for the purpose of developing gene therapy for DMD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4242712PMC
http://dx.doi.org/10.2174/156652312800840603DOI Listing
June 2012

Animal models of muscular dystrophy.

Prog Mol Biol Transl Sci 2012 ;105:83-111

Division of Medical Genetics, Department of Neurology, University of Washington, Seattle, Washington, USA.

The muscular dystrophies (MDs) represent a diverse collection of inherited human disorders, which affect to varying degrees skeletal, cardiac, and sometimes smooth muscle (Emery, 2002). To date, more than 50 different genes have been implicated as causing one or more types of MD (Bansal et al., 2003). In many cases, invaluable insights into disease mechanisms, structure and function of gene products, and approaches for therapeutic interventions have benefited from the study of animal models of the different MDs (Arnett et al., 2009). The large number of genes that are associated with MD and the tremendous number of animal models that have been developed preclude a complete discussion of each in the context of this review. However, we summarize here a number of the more commonly used models together with a mixture of different types of gene and MD, which serves to give a general overview of the value of animal models of MD for research and therapeutic development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/B978-0-12-394596-9.00004-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4872622PMC
May 2012

Gene therapy of mdx mice with large truncated dystrophins generated by recombination using rAAV6.

Mol Ther 2011 Jan 21;19(1):36-45. Epub 2010 Sep 21.

Department of Neurology, University of Washington School of Medicine, Seattle, Washington 98195-7720, USA.

Recombinant adeno-associated viral (rAAV) vector-mediated gene transfer represents a promising approach for many diseases. However, the applicability of rAAV vectors has long been hindered by the small (~4.8 kb) DNA packaging capacity. This limitation can hamper the packaging and delivery of critical regulatory elements and/or larger coding sequences, such as the ~14-kb dystrophin complementary DNA (cDNA) that is of interest for gene therapy of Duchenne muscular dystrophy (DMD). Here, we have demonstrated reconstitution of an expression cassette (7.3 kb) encoding a highly functional "minidystrophin" protein (ΔH2-R19, 222 kd) in vivo following intravascular co-delivery of two independent rAAV6 vectors sharing a central homologous recombinogenic region of 372 nucleotides. Similar to previously reported trans-splicing approaches, one rAAV vector provides the promoter with the ~1/2 initial portion of minidystrophin, while the second vector provides the remaining minidystrophin cDNA followed by the polyadenylation signal. Significantly, administering a modest dose [2 × 10(12) vector genomes (vg)] of the two minidystrophin-encoding rAAV vectors to dystrophic mice elicited an improvement of physiological performance indicative of prevention or amelioration of the disease state. These studies provide evidence that functional dystrophin transgenes larger than that typically carried by a single rAAV genome can be reconstituted in vivo by homologous recombination (HR) following intravascular co-delivery with rAAV6.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/mt.2010.205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3017440PMC
January 2011

Preclinical studies for gene therapy of Duchenne muscular dystrophy.

J Child Neurol 2010 Sep 24;25(9):1149-57. Epub 2010 May 24.

Department of Neurology, University of Washington School of Medicine, Seattle, Washington 98195-7720, USA.

The muscular dystrophies are a diverse group of genetic disorders without an effective treatment. Because they are caused by mutations in various genes, the most direct way to treat them involves correcting the underlying gene defect (ie, gene therapy). Such a gene therapy approach involves delivering a therapeutic gene cassette to essentially all the muscles of the body in a safe and efficacious manner. The authors describe gene delivery methods using vectors derived from adeno-associated virus that are showing great promise in preclinical studies for treatment of Duchenne muscular dystrophy. It is hoped that variations on these methods might be applicable for most, if not all, of the different types of muscular dystrophy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/0883073810371006DOI Listing
September 2010

Microutrophin delivery through rAAV6 increases lifespan and improves muscle function in dystrophic dystrophin/utrophin-deficient mice.

Mol Ther 2008 Sep 29;16(9):1539-45. Epub 2008 Jul 29.

Department of Neurology, Senator Paul D Wellstone Muscular Dystrophy Cooperative Research Center, University of Washington School of Medicine, Seattle, Washington, USA.

Duchenne muscular dystrophy (DMD), the most prevalent lethal genetic disorder in children, is caused by mutations in the 2.2-MB dystrophin gene. Absence of dystrophin and the dystrophin-glycoprotein complex (DGC) from the sarcolemma leads to severe muscle wasting and eventual respiratory and/or cardiac failure. There is presently no effective therapy for DMD. Several lines of evidence have suggested that methods to increase expression of utrophin, a dystrophin paralog, show promise as a treatment for DMD. Adeno-associated viral (AAV) vectors are a promising vehicle for gene transfer to muscle, but microutrophin transgenes small enough to be carried by AAV have not been tested for function. In this study, we intravenously administered recombinant AAV (rAAV2/6) harboring a murine codon-optimized microutrophin (DeltaR4-R21/DeltaCT) transgene to adult dystrophin(-/-)/utrophin(-/-) (mdx:utrn(-/-)) double-knockout mice. Five-month-old mice demonstrated localization of microutrophin to the sarcolemma in all the muscles tested. These muscles displayed restoration of the DGC, increased myofiber size, and a considerable improvement in physiological performance when compared with untreated mdx:utrn(-/-) mice. Overall, microutrophin delivery alleviated most of the pathophysiological abnormalities associated with muscular dystrophy in the mdx:utrn(-/-) mouse model. This approach may hold promise as a treatment option for DMD because it avoids the potential immune responses that are associated with the delivery of exogenous dystrophin.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/mt.2008.149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2643133PMC
September 2008

rAAV6-microdystrophin rescues aberrant Golgi complex organization in mdx skeletal muscles.

Traffic 2007 Oct 20;8(10):1424-39. Epub 2007 Aug 20.

Department of Physiology and Biophysics, University of Washington, Box 357290, 1959 NE Pacific Street, Seattle, WA 98195, USA.

Muscular dystrophies are a diverse group of severe degenerative muscle diseases. Recent interest in the role of the Golgi complex (GC) in muscle disease has been piqued by findings that several dystrophies result from mutations in putative Golgi-resident glycosyltransferases. Given this new role of the Golgi in sarcolemmal stability, we hypothesized that abnormal Golgi distribution, regulation and/or function may constitute part of the pathology of other dystrophies, where the primary defect is independent of Golgi function. Thus, we investigated GC organization in the dystrophin-deficient muscles of mdx mice, a mouse model for Duchenne muscular dystrophy. We report aberrant organization of the synaptic and extrasynaptic GC in skeletal muscles of mdx mice. The GC is mislocalized and improperly concentrated at the surface and core of mdx myofibers. Golgi complex localization is disrupted after the onset of necrosis and normal redistribution is impaired during regeneration of mdx muscle fibers. Disruption of the microtubule cytoskeleton may account in part for aberrant GC localization in mdx myofibers. Golgi complex distribution is restored to wild type and microtubule cytoskeleton organization is significantly improved by recombinant adeno-associated virus 6-mediated expression of DeltaR4-R23/DeltaCT microdystrophin showing a novel mode of microdystrophin functionality. In summary, GC distribution abnormalities are a novel component of mdx skeletal muscle pathology rescued by microdystrophin expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1600-0854.2007.00622.xDOI Listing
October 2007

Viral-mediated gene therapy for the muscular dystrophies: successes, limitations and recent advances.

Biochim Biophys Acta 2007 Feb 26;1772(2):243-62. Epub 2006 Sep 26.

Department of Neurology, Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, University of Washington School of Medicine, 1959 NE Pacific Street, Seattle, WA 98195-7720, USA.

Much progress has been made over the past decade elucidating the molecular basis for a variety of muscular dystrophies (MDs). Accordingly, there are examples of mouse models of MD whose disease progression has been halted in large part with the use of viral vector technology. Even so, we must acknowledge significant limitations of present vector systems that must be overcome prior to successful treatment of humans with such approaches. This review will present a variety of viral-mediated therapeutic strategies aimed at counteracting the muscle-wasting symptoms associated with muscular dystrophy. We include viral vector systems used for muscle gene transfer, with a particular emphasis on adeno-associated virus. Findings of several encouraging studies focusing on repair of the mutant dystrophin gene are also included. Lastly, we present a discussion of muscle compensatory therapeutics being considered that include pathways involved in the up-regulation of utrophin, promotion of cellular adhesion, enhancement of muscle mass, and antagonism of the inflammatory response. Considering the complexity of the muscular dystrophies, it appears likely that a multilayered approach tailored to a patient sub-group may be warranted in order to effectively contest the progression of this devastating disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbadis.2006.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1894910PMC
February 2007

Predicting mammalian SINE subfamily activity from A-tail length.

Mol Biol Evol 2004 Nov 5;21(11):2140-8. Epub 2004 Aug 5.

Tulane Cancer Center, and Department of Epidemiology, Tulane University Health Sciences Center, New Orleans, Louisiana, USA.

Based on previous observations that newly inserted LINEs and SINEs have particularly long 3' A-tails, which shorten rapidly during evolutionary time, we have analyzed the rat and mouse genomes for evidence of recently inserted SINEs and LINEs. We find that the youngest predicted subfamilies of rodent identifier (ID) elements, a rodent-specific SINE derived from tRNA(Ala), are preferentially associated with A-tails over 50 bases in the rat genome, as predicted. Furthermore, these studies detected a subfamily of ID elements that has made over 15,000 copies that is younger than any previously reported ID subfamily. We use PCR analysis of genomic loci to demonstrate that all subfamily members tested inserted after the divergence of Rattus norvegicus from Rattus rattus. We also found evidence that the rodent B1 family of elements is much more active currently in mouse than in rat. These data provide useful estimates of recent activity from all of the mammalian retrotransposons, as well as allowing identification of the most recent insertions for use as population and speciation markers in those species. Both the current rat ID and mouse B1 elements that are active have small, specific interruptions in their 3' A-tail sequences. We suggest that these interruptions stabilize the length of the A-tails and contribute to the activity of these subfamilies. We present a model in which the dynamics of the 3' A-tail may be a central controlling factor in SINE activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/molbev/msh225DOI Listing
November 2004