Visualizing Muscle Sialic Acid Expression in the GNED207VTgGne-/- Cmah-/- Model of GNE Myopathy: A Comparison of Dietary and Gene Therapy Approaches

2021 ◽  
pp. 1-19
Author(s):  
Kelly E. Crowe ◽  
Deborah A. Zygmunt ◽  
Kristin Heller ◽  
Louise Rodino-Klapac ◽  
Satoru Noguchi ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Single Dose ◽  
Sialic Acid ◽  
Tibialis Anterior Muscle ◽  
High Dose ◽  
Loss Of Function ◽  
Gne Myopathy ◽  
Lectin Staining ◽  
Dose Oral ◽  
Gne Gene

Background: GNE myopathy (GNEM) is a rare, adult-onset, inclusion body myopathy that results from partial loss of function mutations in the GNE gene. GNE encodes UDP-GlcNAc epimerase/Mannose-6 kinase, a protein with two enzymatic activities that comprise the committed step in biosynthesis of sialic acid (SA), an essential glycan that appears on the terminal positions of many extracellular oligosaccharide chains. These GNE mutations can cause a reduction of SA in many tissues, although pathology is restricted to skeletal muscles through a poorly understood mechanism. Objective: Despite recent advances in the field, it remains unclear which therapeutic avenue is most promising for the restoration of SA level in skeletal muscle affected by GNEM. Our objective was to assess dietary and gene therapy strategies for GNEM in Cmah-deficient GNED207VTgGne-/- mice, a model that allows for the visualization of orally delivered N-glycolylneuraminic acid (Neu5Gc), one of the two predominant SA forms in muscle. Methods: Methods included in situ physiology studies of the tibialis anterior muscle, studies of ambulation and limb grip strength, and muscle staining using MAA, SNA, and anti-Neu5Gc antibody, along with qPCR, qRT-PCR, western blot, and HPLC studies to assess virally introduced DNA, GNE gene expression, GNE protein expression, and SA expression. Results: We found that a diet enriched in Neu5Gc-containing glycoproteins had no impact on Neu5Gc immunostaining in muscles of GNEM model mice. Delivery of a single high dose oral Neu5Gc therapy, however, did increase Neu5Gc immunostaining, though to levels below those found in wild type mice. Delivery of a single dose of GNE gene therapy using a recombinant Adeno Associated Virus (rAAV) vector with a liver-specific or a muscle-specific promoter both caused increased muscle Neu5Gc immunostaining that exceeded that seen with single dose monosaccharide therapy. Conclusions: Our findings indicate that dietary loading of Neu5Gc-containing glycoproteins is not effective in increasing muscle Neu5Gc expression, while single dose oral Neu5Gc monosaccharide or GNE gene therapy are. Neu5Gc immunostaining, however, showed greater changes than did lectin staining or HPLC analysis. Taken together, these results suggest that Neu5Gc immunostaining may be more sensitive technique to follow SA expression than other more commonly used methods and that liver expression of GNE may contribute overall muscle SA content.

A phase I study of busulfan consolidation/intensification therapy for patients with high risk acute myeloid leukemia (AML)

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 6580-6580
Author(s):  
M. M. Gharibo ◽  
D. Schaar ◽  
A. Rubin ◽  
S. Grospe ◽  
L. Dudek ◽  
...  
Keyword(s):  
Single Dose ◽  
High Risk ◽  
Phase I ◽  
Dose Escalation ◽  
Infectious Complications ◽  
High Dose ◽  
Dose Escalation Study ◽  
Gm Csf ◽  
Dose Oral ◽  
Grade 3

6580 Background: Elderly AML patients have a poor prognosis. More effective well-tolerated treatments are needed to improve outcomes. Busulfan is an effective anti-leukemia drug commonly used in transplant regimens. We designed a phase I dose escalation study of single dose busulfan as consolidation/intensification therapy for elderly patients with AML. Methods: Patients in CR1 not candidates for transplantation or anticipated to benefit from high-dose cytarabine were enrolled. Cohorts were studied at 100mg/m2-120mg/m2, as a single oral dose of busulfan. The starting dose was based on a previous report (Ranson et al. Br J Haematol 79:162.1991). GM-CSF was started after busulfan until ANC> 2000/mm3. Dilantin was used for seizure prophylaxis. Results: 15 patients median age 71 (range 64–82) received treatment and are evaluable for toxicity, 14 are evaluable for response. No DLT was observed. Ongoing remission was seen in 3 patients (range 3 +- 7 + years). One patient (AML/MDS) was in remission for 52 months and died from small cell lung cancer and 1 patient was in remission for 15 months and died from cervical cancer. Two additional patients had prolonged remissions (32 and 55 months) prior to relapse. Seven patients relapsed between 4 weeks and 6 months. Treatment was well tolerated with no prolonged neutropenia. In patients who recovered without evidence of leukemia transient grade 3/4 thrombocytopenia occurred with average duration of 16 days (10–23 days) requiring 0–5 transfusions (average 2). Only 2 patients required RBC transfusion. No infectious complications or hospitalizations occurred in the absence of recurrent AML. Three patients experienced side effects from GM-CSF and received G-CSF in it’s place. Two patients experienced grade 2 fatigue and dyspnea. Conclusions: Single dose oral busulfan is an effective and well-tolerated outpatient consolidation/intensification therapy for patients with high-risk, AML. Prolonged remissions have been seen in 6 of 14 evaluable patients treated to date. Only transient bone marrow suppression requiring minimal transfusion support was seen. There were no hospitalizations or infections noted in patients without recurrent AML. The dose escalation will continue until MTD reached. No significant financial relationships to disclose.

Use of single-dose oral gabapentin to attenuate fear responses in cage-trap confined community cats: a double-blind, placebo-controlled field trial

2017 ◽  
Vol 20 (6) ◽  
pp. 535-543 ◽  
Author(s):  
Katherine E Pankratz ◽  
Kelli K Ferris ◽  
Emily H Griffith ◽  
Barbara L Sherman
Keyword(s):  
Single Dose ◽  
Controlled Study ◽  
High Dose ◽  
Inclusion Criteria ◽  
Double Blind ◽  
Double Blind Placebo ◽  
Facial Injuries ◽  
Treatment Groups ◽  
Dose Oral ◽  
Sedation Scores

Objectives This double-blind, placebo-controlled study evaluated the safety and efficacy of single-dose oral gabapentin administered for the attenuation of fear responses in cage-trap confined community cats. Methods Community cats presented in cage traps for trap–neuter–return (TNR) were recruited and screened for inclusion. Each enrolled cat was randomly assigned and administered one of three equal-volume, single-dose treatments: placebo, low-dose gabapentin (50 mg) or high-dose gabapentin (100 mg). At baseline, 1, 2, 3 and 12 h post-administration, a single, blinded observer scored each cat for signs of fear and sedation using published paradigms, calculated the respiratory rate and documented any observable facial injuries. Results Fifty-three cats met the inclusion criteria and completed the study. Cat stress score (a measure of fear) was lower in cats that received gabapentin (50 or 100 mg) than in cats that received placebo (50 mg: P = 0.027; 100 mg: P = 0.029), with the greatest reduction at 2 h post-treatment ( P = 0.0007). Respiratory rates did not differ between treatment groups. There was no difference in sedation scores between the groups ( P = 0.86) at any time point ( P = 0.09). Cat facial injuries did not vary by treatment group or over time. No adverse effects were detected specific to gabapentin administration. At 1 h, hypersalivation was observed in four cats across all treatment groups. All cats recovered from surgery and anesthesia uneventfully. Conclusions and relevance This study supports the hypothesis that 50 mg or 100 mg gabapentin (9.2–47.6 mg/kg per cat) reduces fear responses in confined community cats without measurable sedation over 3 h post-administration vs placebo. Gabapentin treatment was well tolerated in this population of cats. Further studies are recommended to investigate the use of oral gabapentin earlier in the TNR process, such as immediately after trapping or prior to transport for the prevention of confinement-related injuries.

scholarly journals Generation and Characterization of Skeletal Muscle Cell based Model for GNE Myopathy: Implications in Actin Dynamics

2020 ◽  
Author(s):  
Shamulailatpam Shreedarshanee Devi ◽  
Rashmi Yadav ◽  
Fluencephila Mashangva ◽  
Priyanka Chaudhary ◽  
Shweta Sharma ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cell Migration ◽  
Sialic Acid ◽  
Muscle Cell ◽  
Model System ◽  
Skeletal Muscle Cell ◽  
Actin Dynamics ◽  
Knock Out ◽  
Gne Myopathy ◽  
Gne Gene

Abstract UDP-N-acetyl glucosamine-2 epimerase/ N-acetyl mannosamine kinase (GNE) catalyzes key enzymatic reactions in the biosynthesis of sialic acid. Mutation in GNE gene causes GNE-Myopathy characterized by adult onset muscle weakness and degeneration. GNE is involved in different cellular processes like adhesion, apoptosis, ER stress and autophagy. Lack of appropriate model system limits drug and treatment options for GNE Myopathy as GNE knock out was found to be embryonically lethal. In the present study, we have generated L6 rat skeletal muscle cell-based model system for GNE Myopathy where GNE gene is knocked out at exon 3 using AAV mediated SEPT homology recombination. The cell line was heterozygous for GNE gene with one wild type and one truncated allele as confirmed by sequencing. The phenotype showed reduced GNE epimerase activity with little reduction in sialic acid content. In addition, the GNE knock out cell line revealed altered cytoskeletal organization with disrupted actin filament. The signaling cascade regulating actin dynamics such as Rho A, Cofilin, FAK and Src were altered leading to reduced cell migration in GNE heterozygous cells. Our study indicates possible role of GNE in regulating actin dynamics and cell migration of skeletal muscle cell. The skeletal muscle cell based system offers great potential in understanding pathomechanism and target identification for GNE Myopathy.

Recombinant adeno-associated virus 9-mediated expression of Kallistatin suppresses lung tumor growth in mice

2020 ◽  
Vol 20 ◽  
Author(s):  
Weihong Qu ◽  
Jianguo Zhao ◽  
Yaqing Wu ◽  
Ruian Xu ◽  
Shaowu Liu
Keyword(s):  
Lung Cancer ◽  
Gene Therapy ◽  
Tumor Growth ◽  
Lung Tumor ◽  
Control Group ◽  
High Dose ◽  
Blank Control Group ◽  
Adeno Associated Virus ◽  
Tumor Tissues ◽  
Subcutaneous Xenograft

Background:: Lung cancer remains the most common cause of cancer-related deaths in China and worldwide. Traditional surgery and chemotherapy do not offer an effective cure although gene therapy may be a promising future alter-native. Kallistatin (Kal) is an endogenous inhibitor of angiogenesis and tumorigenesis. Recombinant adeno-associated virus (rAAV) is considered the most promising vector for gene therapy of many diseases due to persistent and long-term transgen-ic expression. Objective:: The aim of this study was to investigate whether rAAV9-Kal inhibited NCI-H446 subcutaneous xenograft tumor growth in mice. Method:: The subcutaneous xenograft mode were induced by subcutaneous injection of 2×106 H446 cells into the dorsal skin of BALB/c nude mice. The mice were administered with ssrAAV9-Kal (single-stranded rAAV9) or dsrAAV9-Kal (double-stranded rAAV9）by intraperitoneal injection (I.P.). Tumor microvessel density (MVD) was examined by anti-CD34 stain-ing to evaluate tumor angiogenesis. Results:: Compared with the PBS (blank control) group, tumor growth in the high-dose ssrAAV9-Kal group was inhibited by 40% by day 49, and the MVD of tumor tissues was significantly decreased. Conclusion:: The results indicate that this therapeutic strategy is a promising approach for clinical cancer therapy and impli-cate rAAV9-Kal as a candidate for gene therapy of lung cancer.

Sign in / Sign up

Export Citation Format

Share Document