Muscle-specific, liver-detargeted adeno-associated virus gene therapy rescues Pompe phenotype in adult and neonate Gaa-/- mice.

Pompe disease (PD) is a neuromuscular disorder caused by acid α-glucosidase (GAA) deficiency. Reduced GAA activity leads to pathological glycogen accumulation in cardiac and skeletal muscles responsible for severe heart impairment, respiratory defects, and muscle weakness. Enzyme replacement therapy with recombinant human GAA (rhGAA) is the standard-of-care treatment for PD, however, its efficacy is limited due to poor uptake in muscle and the development of an immune response. Multiple clinical trials are ongoing in PD with adeno-associated virus (AAV) vectors based on liver- and muscle-targeting. Current gene therapy approaches are limited by liver proliferation, poor muscle targeting, and the potential immune response to the hGAA transgene. To generate a treatment tailored to infantile-onset PD, we took advantage of a novel AAV capsid able to increase skeletal muscle targeting compared to AAV9 while reducing liver overload. When combined with a liver-muscle tandem promoter (LiMP), and despite the extensive liver-detargeting, this vector had a limited immune response to the hGAA transgene. This combination of capsid and promoter with improved muscle expression and specificity allowed for glycogen clearance in cardiac and skeletal muscles of Gaa-/- adult mice. In neonate Gaa-/- , complete rescue of glycogen content and muscle strength was observed 6 months after AAV vector injection. Our work highlights the importance of residual liver expression to control the immune response toward a potentially immunogenic transgene expressed in muscle. In conclusion, the demonstration of the efficacy of a muscle-specific AAV capsid-promoter combination for the full rescue of PD manifestation in both neonate and adult Gaa-/- provides a potential therapeutic avenue for the infantile-onset form of this devastating disease.

Impact of conservation measures on demography and genetic variability of livestock breeds.

Conservation of animal genetic resources requires regular monitoring and interventions to maintain population size and manage genetic variability. This study uses genealogical information to evaluate the impact of conservation measures in Europe, using (i) data from the Domestic Animal Diversity Information System (DAD-IS) and (ii) a posteriori assessment of the impact of various conservation measures on the genetic variability of 17 at-risk breeds with a wide range of interventions. Analysis of data from DAD-IS showed that 68% of national breed populations reported to receive financial support showed increasing demographic trends, v. 51% for those that did not. The majority of the 17 at-risk breeds have increased their numbers of registered animals over the last 20 years, but the changes in genetic variability per breed have not always matched the trend in population size. These differences in trends observed in the different metrics might be explained by the tensions between interventions to maintain genetic variability, and development initiatives which lead to intensification of selection.

Systematic Review and Meta-Analysis of Diagnostic Accuracy of Serum Refractometry and Brix Refractometry for the Diagnosis of Inadequate Transfer of Passive Immunity in Calves.

BACKGROUND: Transfer of passive immunity in calves can be assessed by direct measurement of immunoglobulin G (IgG) by methods such as radial immunodiffusion (RID) or turbidimetric immunoassay (TIA). IgG can also be measured indirectly by methods such as serum refractometry (REF) or Brix refractometry (BRIX). OBJECTIVES: To determine the accuracy of REF and BRIX for assessment of inadequate transfer of passive immunity (ITPI) in calves. DESIGN: Systematic review and meta-analysis of diagnostic accuracy studies. METHODS: Databases (PubMed and CAB Abstract, Searchable Proceedings of Animal Science) and Google Scholar were searched for relevant studies. Studies were eligible if the accuracy (sensitivity and specificity) of REF or BRIX was determined using direct measurement of IgG by RID or turbidimetry as the reference standard. The study population included calves <14 days old that were fed with natural colostrum (colostrum replacement products were excluded). Quality assessment was performed by the QUADAS-2 tool. Hierarchical models were used for meta-analysis. RESULTS: From 1,291 references identified, 13 studies of 3,788 calves were included. Of these, 11 studies evaluated REF and 5 studies evaluated BRIX. The median (range) prevalence of ITPI (defined as calves with IgG <10 g/L by RID or TIA) was 21% (1.3-56%). Risk of bias and applicability concerns were generally low or unclear. For REF, summary estimates were obtained for 2 different cutoffs: 5.2 g/dL (6 studies) and 5.5 g/dL (5 studies). For the 5.2 g/dL cutoff, the summary sensitivity (95% CI) and specificity (95% CI) were 76.1% (63.8-85.2%) and 89.3% (82.3-93.7%), and 88.2% (80.2-93.3%) and 77.9% (74.5-81.0%) for the 5.5 g/dL cutoff. Due to the low number of studies using the same cutoffs, summary estimates could not be obtained for BRIX. CONCLUSIONS AND CLINICAL IMPORTANCE: Despite their widespread use on dairy farms, evidence about the optimal strategy for using refractometry, including the optimal cutoff, are sparse (especially for BRIX). When using REF to rule out ITPI in herds, the 5.5 g/dL cutoff may be used whereas for ruling in ITPI, the 5.2 g/dL cutoff may be used.

Adeno-associated virus pseudotype 5 vector improves gene transfer in arthritic joints.

The potential for gene delivery to joints, using recombinant adeno-associated virus (rAAV) vectors for the treatment of rheumatoid arthritis (RA), has received much attention. Different serotypes have different virion shell proteins and, as a consequence, vary in their tropism for diverse tissues. The aim of this study was to compare the transduction efficiency of different AAV serotypes encoding murine secreted alkaline phosphatase (mSEAP) or Escherichia coli beta-galactosidase for intraarticular gene delivery in an experimental model of arthritis. The vectors contained AAV2 terminal repeats flanking the reporter gene in an AAV1, AAV2, or AAV5 capsid, producing the pseudotypes rAAV-2/1, rAAV-2/2, and rAAV-2/5. Left knee joints of mice with collagen-induced arthritis were injected and transgene expression was analyzed by chemiluminescence or direct in situ staining of frozen sections. We show for the first time that intraarticular gene transfer with AAV- 2/5 was far more efficient than with the other serotypes tested. Transgene expression was detectable as early as 7 days after injection, reached a maximum at 21 days, and was stably expressed for at least 130 days, whereas AAV-2/1- and AAV-2/2-mediated expression levels were barely detectable. These findings provide a practical application for future local AAV-mediated gene therapy trials in RA.

Mechanism of DNA damage photosensitized by trisbipyrazyl ruthenium complex. Unusual role of Cu/Zn superoxide dismutase.

Trisbipyrazyl ruthenium(II) (Ru[bpz]3(2+)) was examined as DNA photosensitizer. Damage resulting from the photolysis of synthetic oligonucleotides has been monitored by polyacrylamide gel electrophoresis. Photoadduct formation is found on both single- and double-stranded oligonucleotides. On oligonucleotide duplex, oxidative damage occurs selectively at the 5'G of the 5'GG3' site and to a lesser extent at the 5'G of a GA sequence. These findings suggest the involvement of electron transfer and show that this mechanism is the main DNA damaging process involved in Ru(bpz)3(2+) photosensitization. In addition, photoadducts and oxidative damage are both highly affected by an increase of salt concentration in the reaction medium, stressing the importance of direct interactions between nucleic acid bases and the excited ruthenium complex for efficient electron transfer. On single-stranded oligonucleotides, all the guanines are oxidized to the same extent. In this case, oxidative damage, which is not affected by an increase of salt in the solution, has been attributed, in part, to singlet oxygen. More importantly, Cu/Zn superoxide dismutase (SOD) strongly enhances the yield of all damage, correlated to an increase of both electron transfer and singlet oxygen production. This original activity of SOD is the first example of bioactivation of a polyazaaromatic ruthenium complex.

Investigation of the positive response of ethyl acrylate in the mouse lymphoma genotoxicity assay.

To develop a better understanding of the relationship between ethyl acrylate (EA)-induced cytotoxicity and mutation frequency in the mouse lymphoma assay (MLA) we measured the effects of EA treatment to ML cells on: (1) nonprotein sulfhydryl (NPS) levels; (2) mitochondrial rhodamine 123 (Rh123) uptake; (3) the DNA elution slope (single-strand breakage) and Y intercept of fitted curves (cytotoxicity and double-strand breakage) in the alkaline elution assay; (4) the appearance of apoptosis; and (5) the pulsed-field gel electrophoretic resolution of high-molecular-weight DNA. EA reduced NPS in both a time- and concentration-dependent manner. By 30 min, > or = 20 micrograms/ml EA reduced NPS by 50% or greater. By 4 h, > or = 10 micrograms/ml markedly decreased both NPS cell content (> or = 71.5% reduction) and mitochondrial Rh123 uptake (10-50 micrograms/ml; 9-62%), the latter effect being further enhanced by washing and incubation for an additional 2 h (12-85%). EA did not induce single-strand breaks in the alkaline elution assay. Only highly cytotoxic EA concentrations (80-87% reduction in RCG at 40-50 micrograms/ml) caused both increases in the elution slope and parallel drops (Y intercept) in the elution curve in the alkaline elution assay. Conventional agarose gel electrophoretic analysis of the DNA neutral fraction of these high dose preparations showed evidence for both apoptosis (180-bp oligonucleosomal DNA laddering effect) and random smearing of DNA (necrosis). Pulsed-field gel electrophoresis of directly loaded high dose cell preparations revealed both high- and low-molecular-weight DNA double-strand breaks, but only at the highest concentrations. These observations indicated that the EA-induced mutagenic response correlated best with cellular cytotoxicity mediated by NPS depletion and mitochondrial membrane impairment.