Repair of endogenous expression of dystrophin by exon skipping using anti-sense oligonucleotides (AOs) is the most promising treatment strategy for Duchenne muscular dystrophy (DMD), and is the only therapy showing clear functional improvement in the dog model (Yokota et al. 2009). There is increasing interest in a similar approach for other neuromuscular disorders, including spinal muscular atrophy, amyotrophic lateral sclerosis, and myotonic dystrophy (Singh et al. 2011, Wheeler et al. 2007). We have shown that the uncharged morpholino backbone chemistry have the broadest therapeutic window (see Overview), where weekly intravenous dosing of 960 mg/kg in mice, or 320 mg/kg in monkeys shows accumulation of AO in the kidneys with mild signs of toxicity. The doses required to sustain expression of dystrophin and alleviate symptoms in humans are likely to be quite high (20 to 100 mg/kg/wk). At present the consequences of AO accumulation on kidneys, the optimal doses and schedule required to sustain efficacy while keeping the AO kidney load to a minimum are not known. The goal of this project is to define the effect of high dose morpholino drug on kidneys both at the cellular and molecular levels, and establish sensitive and specific urinary biomarkers to help monitor treatment and dosage scheduling for Project 1.
In Aim 1 we will focus on the direct effects of high doses of morpholino AO on the kidney. We will use differential stable isotope labeling by amino acid in mouse (SILAM) strategy to systemically define protein alterations in the kidneys and urines of AO treated versus untreated mdx mice. We have already generated SILAM mouse and successfully used it to define alteration in proteome in diaphragm and liver of mdx mouse versus wild type mouse. We will use a similar strategy to first examine the proteome of the entire kidney organ and define the affected networks and then refine the analyses by targeting the affected area and cellular organelles. We will also perform comprehensive proteome profiling or urine samples to define reliable biomarkers associated with AO kidney load. With this experiment we will determine the extent of kidney damage at the molecular level and define intervention targets for Project 1.
In Aim 2 we will further refine the analysis by focusing on the affected areas in the kidneys using histoproteome profiling on tissue sections prepared with the help of Core B (Drs. Robin A. Felder and Pedro A. Jose). Our center is a leading expert in these technologies and we have a well standardized proteomic approaches, often using differentia! labeling with stable isotopes and/or label free strategy to achieve such experiments (An et al. 2010, Reeves et al. 2009, An et al. 2008, Mintz et al. 2003). Finally in Aim 3 we will assay candidate biomarkers using human urine samples collected from AO treated versus non-treated Duchenne patients (Project 1), pending an investigators'meeting at the start of Year 3. The primary outcome of this project will be a palette of urinary biomarkers able to monitor AO accumulation in the kidneys that can inform treatment schedule and patient-specific therapeutic index (Project 1). Our laboratory has extensive experience in proteome profiling of tissue and bodily fluids. For biomarkers discovery see our previous studies using cerebrospinal fluid samples (Vanderver et al. 2005 &2008, Rajagopal et al. 2011) as well as urine samples of HIV patients suffering from renal injuries (Soler-Garcia et al. 2009). Urine is a fairly accessible bodily fluid and we have all the necessary techniques and tools to achieve Project 2.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Specialized Center--Cooperative Agreements (U54)
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Special Emphasis Panel (ZHD1-DSR-A)
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Children's Research Institute
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Yu, Qing; Morales, Melissa; Li, Ning et al. (2018) Skeletal, cardiac, and respiratory muscle function and histopathology in the P448Lneo- mouse model of FKRP-deficient muscular dystrophy. Skelet Muscle 8:13
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Novak, James S; Hogarth, Marshall W; Boehler, Jessica F et al. (2017) Myoblasts and macrophages are required for therapeutic morpholino antisense oligonucleotide delivery to dystrophic muscle. Nat Commun 8:941
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