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1.  Antisense Oligonucleotide-mediated Suppression of Muscle Glycogen Synthase 1 Synthesis as an Approach for Substrate Reduction Therapy of Pompe Disease 
Pompe disease is an autosomal recessive disorder caused by a deficiency of acid α-glucosidase (GAA; EC and the resultant progressive lysosomal accumulation of glycogen in skeletal and cardiac muscles. Enzyme replacement therapy using recombinant human GAA (rhGAA) has proven beneficial in addressing several aspects of the disease such as cardiomyopathy and aberrant motor function. However, residual muscle weakness, hearing loss, and the risks of arrhythmias and osteopenia persist despite enzyme therapy. Here, we evaluated the relative merits of substrate reduction therapy (by inhibiting glycogen synthesis) as a potential adjuvant strategy. A phosphorodiamidate morpholino oligonucleotide (PMO) designed to invoke exon skipping and premature stop codon usage in the transcript for muscle specific glycogen synthase (Gys1) was identified and conjugated to a cell penetrating peptide (GS-PPMO) to facilitate PMO delivery to muscle. GS-PPMO systemic administration to Pompe mice led to a dose-dependent decrease in glycogen synthase transcripts in the quadriceps, and the diaphragm but not the liver. An mRNA response in the heart was seen only at the higher dose tested. Associated with these decreases in transcript levels were correspondingly lower tissue levels of muscle specific glycogen synthase and activity. Importantly, these reductions resulted in significant decreases in the aberrant accumulation of lysosomal glycogen in the quadriceps, diaphragm, and heart of Pompe mice. Treatment was without any overt toxicity, supporting the notion that substrate reduction by GS-PPMO-mediated inhibition of muscle specific glycogen synthase represents a viable therapeutic strategy for Pompe disease after further development.
PMCID: PMC4217081  PMID: 25350581
antisense oligonucleotide; cell penetrating peptide; glycogen synthase; morpholino; nonsense-mediated decay; Pompe disease; phosphorodiamidate morpholino oligonucleotide; skeletal muscle; substrate reduction therapy
2.  Dysregulation of Multiple Facets of Glycogen Metabolism in a Murine Model of Pompe Disease 
PLoS ONE  2013;8(2):e56181.
Pompe disease, also known as glycogen storage disease (GSD) type II, is caused by deficiency of lysosomal acid α-glucosidase (GAA). The resulting glycogen accumulation causes a spectrum of disease severity ranging from a rapidly progressive course that is typically fatal by 1 to 2 years of age to a slower progressive course that causes significant morbidity and early mortality in children and adults. The aim of this study is to better understand the biochemical consequences of glycogen accumulation in the Pompe mouse. We evaluated glycogen metabolism in heart, triceps, quadriceps, and liver from wild type and several strains of GAA−/− mice. Unexpectedly, we observed that lysosomal glycogen storage correlated with a robust increase in factors that normally promote glycogen biosynthesis. The GAA−/− mouse strains were found to have elevated glycogen synthase (GS), glycogenin, hexokinase, and glucose-6-phosphate (G-6-P, the allosteric activator of GS). Treating GAA−/− mice with recombinant human GAA (rhGAA) led to a dramatic reduction in the levels of glycogen, GS, glycogenin, and G-6-P. Lysosomal glycogen storage also correlated with a dysregulation of phosphorylase, which normally breaks down cytoplasmic glycogen. Analysis of phosphorylase activity confirmed a previous report that, although phosphorylase protein levels are identical in muscle lysates from wild type and GAA−/− mice, phosphorylase activity is suppressed in the GAA−/− mice in the absence of AMP. This reduction in phosphorylase activity likely exacerbates lysosomal glycogen accumulation. If the dysregulation in glycogen metabolism observed in the mouse model of Pompe disease also occurs in Pompe patients, it may contribute to the observed broad spectrum of disease severity.
PMCID: PMC3572993  PMID: 23457523
3.  DURATION-1: Exenatide Once Weekly Produces Sustained Glycemic Control and Weight Loss Over 52 Weeks 
Diabetes Care  2010;33(6):1255-1261.
In the Diabetes Therapy Utilization: Researching Changes in A1C, Weight and Other Factors Through Intervention with Exenatide Once Weekly (DURATION-1) study, the safety and efficacy of 30 weeks of treatment with the glucagon-like peptide-1 receptor agonist exenatide once weekly (exenatide QW; 2 mg) was compared with exenatide BID in 295 patients with type 2 diabetes. We now report the safety and efficacy of exenatide QW in 1) patients who continued treatment for an additional 22 weeks (52 weeks total) and 2) patients who switched from exenatide BID to exenatide QW after 30 weeks.
In this randomized, multicenter, comparator-controlled, open-label trial, 258 patients entered the 22-week open-ended assessment phase (n = 128 QW-only; n = 130 BID→QW). A1C, fasting plasma glucose (FPG), body weight, blood pressure, fasting lipids, safety, and tolerability were assessed.
Patients continuing exenatide QW maintained A1C improvements through 52 weeks (least squares mean −2.0% [95% CI −2.1 to −1.8%]). Patients switching from exenatide BID to exenatide QW achieved further A1C improvements; both groups exhibited the same A1C reduction and mean A1C (6.6%) at week 52. At week 52, 71 and 54% of all patients achieved A1C <7.0% and ≤6.5%, respectively. In both treatment arms, FPG was reduced by >40 mg/dl, and body weight was reduced by >4 kg after 52 weeks. Nausea occurred less frequently in this assessment period and was predominantly mild. No major hypoglycemia was observed.
Exenatide QW elicited sustained improvements in glycemic control and body weight through 52 weeks of treatment. Patients switching to exenatide QW experienced further improvements in A1C and FPG, with sustained weight loss.
PMCID: PMC2875434  PMID: 20215461
4.  Exenatide once weekly treatment maintained improvements in glycemic control and weight loss over 2 years 
The once-weekly (QW) formulation of the glucagon-like peptide-1 receptor agonist exenatide has been demonstrated to improve A1C, fasting plasma glucose (FPG), body weight, serum lipid profiles, and blood pressure in patients with type 2 diabetes through 52 weeks of treatment. In this report, we describe the 2-year results of the open-label, open-ended extension to the DURATION-1 trial of exenatide QW for type 2 diabetes.
A 2-stage protocol was used: patients received either exenatide QW (2 mg) or exenatide twice daily for 30 weeks (5 μg for the first 4 weeks and 10 μg thereafter), followed by 1.5 years of treatment with exenatide QW (2 mg), for a total of 2 years (104 weeks) of exenatide treatment. Of the 295 (intent-to-treat [ITT]) patients who entered the trial, 73% (n = 216) completed 2 years of treatment (completer population). Baseline characteristics (mean ± SE) for these patients were: A1C, 8.2 ± 0.1%; FPG, 168.4 ± 43.0 mg/dL; body weight, 101.1 ± 18.7 kg; and diabetes duration, 7 ± 5 years.
In the completer population, significant improvements (LS mean ± SE [95% CI]) were maintained after 2 years of treatment in A1C (-1.71 ± 0.08% [-1.86 to -1.55%]), FPG (-40.1 ± 2.9 mg/dL [-45.7 to -34.5 mg/dL]), and body weight (-2.61 ± 0.52 kg [-3.64 to -1.58 kg]) compared with baseline. The percentages of patients who achieved an A1C of <7.0% and ≤6.5% at 2 years were 60% and 39%, respectively. A significant reduction in systolic blood pressure (SBP; -3.0 ± 1.0 mmHg [-4.9 to -1.1 mmHg]) was maintained through 2 years of treatment. Serum lipid profiles were also significantly improved, including triglycerides (geometric LS mean change from baseline, -15 ± 2.7% [-21% to -10%]), total cholesterol (-8.6 ± 2.8 mg/dL [-14.0 to -3.1 mg/dL]), and low-density lipoproteins (-4.5 ± 2.2 mg/dL [-8.9 to -0.01 mg/dL]). Changes in A1C, body weight, FPG, SBP, and lipids in the ITT population were similar to those seen in the completer population. Nausea (predominantly mild in intensity) was the most common adverse event, although the frequency and intensity of nausea decreased over time. No severe hypoglycemia was observed.
Exenatide QW was well tolerated during the 2-year treatment period. This study demonstrated sustained glucose control and weight loss throughout 2 years of treatment with exenatide QW.
Trial Registration NCT00308139
PMCID: PMC3112417  PMID: 21529363
5.  Evaluation of neonatally-induced mild diabetes in rats: Maternal and fetal repercussions 
Many experimental studies have been performed to evaluate mild diabetes effects. However, results are divergent regarding glycemia and insulin measurement, fetal macrossomia, and placental weights. The aim was to investigate repercussions of neonatally-induced mild diabetes on the maternal organism and presence of congenital defects in their offspring in other mild diabetes model. On the day of birth, female offspring were distributed into two groups: Group streptozotocin (STZ): received 100 mg STZ/kg body weight, and Control Group: received vehicle in a similar time period. Maternal weights and glycemias were determined at days 0, 7, 14 and 21 of pregnancy. At day 21 of pregnancy, the rats were anesthetized and a laparotomy was performed to weigh and analyze living fetuses and placentas. The fetuses were classified as small (SPA), appropriate (APA) and large (LPA) for pregnancy age. Fetuses were also analyzed for the presence of external anomalies and processed for skeletal anomaly and ossification sites analysis. Statistical significance was considered as p < 0.05. In STZ group, there was increased glycemia at 0 and 14 days of pregnancy, lower weights throughout pregnancy, higher placental weight and index, an increased proportion of fetuses classified as SPA and LPA, and their fetuses presented with an increased frequency of abnormal sternebra, and absent cervical nuclei, which were not enough to cause the emergence of skeletal anomalies. Thus, this study shows that mild diabetes altered fetal development, characterized by intrauterine growth restriction. Further, the reached glycemia does not lead to any major congenital defects in the fetuses of streptozotocin-induced mild diabetic rats.
PMCID: PMC2904722  PMID: 20529353
6.  Repercussions of mild diabetes on pregnancy in Wistar rats and on the fetal development 
Experimental models are necessary to elucidate diabetes pathophysiological mechanisms not yet understood in humans. Objective: To evaluate the repercussions of the mild diabetes, considering two methodologies, on the pregnancy of Wistar rats and on the development of their offspring.
In the 1st induction, female offspring were distributed into two experimental groups: Group streptozotocin (STZ, n = 67): received the β-cytotoxic agent (100 mg STZ/kg body weight - sc) on the 1st day of the life; and Non-diabetic Group (ND, n = 14): received the vehicle in a similar time period. In the adult life, the animals were mated. After a positive diagnosis of pregnancy (0), female rats from group STZ presenting with lower glycemia than 120 mg/dL received more 20 mg STZ/kg (ip) at day 7 of pregnancy (2nd induction). The female rats with glycemia higher than 120 mg/dL were discarded because they reproduced results already found in the literature. In the mornings of days 0, 7, 14 and 21 of the pregnancy glycemia was determined. At day 21 of pregnancy (at term), the female rats were anesthetized and killed for maternal reproductive performance and fetal development analysis. The data were analyzed using Student-Newman-Keuls, Chi-square and Zero-inflated Poisson (ZIP) Tests (p < 0.05).
STZ rats presented increased rates of pre (STZ = 22.0%; ND = 5.1%) and post-implantation losses (STZ = 26.1%; ND = 5.7%), reduced rates of fetuses with appropriate weight for gestational age (STZ = 66%; ND = 93%) and reduced degree of development (ossification sites).
Mild diabetes led a negative impact on maternal reproductive performance and caused intrauterine growth restriction and impaired fetal development.
PMCID: PMC2874517  PMID: 20416073
7.  Significant Closure of the Human Immunodeficiency Virus Type 1 and Hepatitis C Virus Preseroconversion Detection Windows with a Transcription-Mediated-Amplification-Driven Assay 
Journal of Clinical Microbiology  2002;40(5):1761-1766.
While the present generation of serology-based assays has significantly decreased the number of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infections acquired by transfusion, the possibility of infected donations escaping detection still exists. The average seronegative viremic window duration during which immunological assays are unable to detect the virus is estimated to be between 16 and 22 days for HIV-1 and approximately 70 days for HCV. Significant reduction of detection window duration was demonstrated using a nucleic acid amplification assay, the Procleix HIV-1/HCV Assay, which utilizes transcription-mediated amplification technology to simultaneously detect HIV-1 and HCV RNAs. For 26 commercially available HIV-1 seroconversion panels tested, specimens were reactive in the HIV-1/HCV assay at the same time as or earlier than in serological assays. Overall, the HIV-1/HCV assay was able to reduce the detection window duration by an average of 14 days and 6 days compared to tests relying on recognition of HIV-1 antibody and p24 antigen, respectively. For 24 commercially available HCV seroconversion panels tested, the specimens were reactive in the HIV-1/HCV assay at an earlier blood sampling date than in serological assays, reducing the detection window duration by an average of 26 days. Similar results were obtained in testing the HIV-1 and HCV seroconversion panels in the virus-specific HIV-1- and HCV-discriminatory assays, respectively. In conclusion, the HIV-1/HCV assay and corresponding discriminatory assays significantly reduced detection window durations compared to immunoassays.
PMCID: PMC130666  PMID: 11980957

Results 1-7 (7)