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Neuraminidase-mediated desialylation augments AAV9-mediated gene expression in skeletal muscle.

ABSTRACT: BACKGROUND:Following systemic delivery, AAV9-mediated gene expression is significantly increased in ischemic versus non-ischemic muscle, suggesting that AAV9 is an attractive vector for treating peripheral arterial disease. Potential mechanisms underlying ischemia-augmented expression include: (i) increased vascular permeability and (ii) "unmasking" of endogenous AAV9 receptors. In the present study, we aimed to reconstitute the ischemic induction of AAV9 in vivo, using local injection of histamine (to increase vascular permeability) and neuraminidase (to desialylate cell surface glycans). METHODS:Bioassays were performed to optimize the effects of histamine and neuraminidase after intramuscular injection. Histamine and/or neuraminidase were then injected intramuscularly shortly before intravenous injection of an AAV9 vector expressing luciferase. Luciferase expression was serially assessed with bioluminescence imaging. At the end of the study, tissues were harvested for assays of luciferase activity and AAV9 genome copy number aiming to assess AAV-mediated gene expression and transduction, respectively. RESULTS:Intramuscular injection of either neuraminidase or neuraminidase plus histamine significantly increased both transduction and gene expression, whereas histamine alone had little effect. Pre-injection with neuraminidase increased AAV9-mediated gene delivery by four- to nine-fold and luciferase activity by 60-100-fold. Luciferase activity in neuraminidase-injected muscle was > 100-fold higher than in any off-target tissue (including heart, liver and brain). CONCLUSIONS:The ischemic induction of AAV9-mediated gene expression in muscle can largely be reconstituted by pre-injecting neuraminidase intranmuscularly. This strategy may prove useful in future human gene therapy protocols as a quick and efficient means to selectively target systemically injected AAV9 to localized regions of muscle, thus decreasing the potential for adverse effects in off-target tissues.

SUBMITTER: Zhu H

PROVIDER: S-EPMC6209320 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Neuraminidase-mediated desialylation augments AAV9-mediated gene expression in skeletal muscle.

Zhu Hongling H Wang Tao T John Lye Robert R French Brent A BA Annex Brian H BH

The journal of gene medicine 20180904 9

<h4>Background</h4>Following systemic delivery, AAV9-mediated gene expression is significantly increased in ischemic versus non-ischemic muscle, suggesting that AAV9 is an attractive vector for treating peripheral arterial disease. Potential mechanisms underlying ischemia-augmented expression include: (i) increased vascular permeability and (ii) "unmasking" of endogenous AAV9 receptors. In the present study, we aimed to reconstitute the ischemic induction of AAV9 in vivo, using local injection o ...[more]

PMID: 30101537

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Project description:BACKGROUND:We propose micro single-photon emission computed tomography/computed tomography imaging of the hNIS (human sodium/iodide symporter) to noninvasively quantify adeno-associated virus 9 (AAV9)-mediated gene expression in a murine model of peripheral artery disease. METHODS:AAV9-hNIS (2×1011 viral genome particles) was injected into nonischemic or ischemic gastrocnemius muscles of C57Bl/6J mice following unilateral hindlimb ischemia ± the ?-sialidase NA (neuraminidase). Control nonischemic limbs were injected with phosphate buffered saline or remained noninjected. Twelve mice underwent micro single-photon emission computed tomography/computed tomography imaging after serial injection of pertechnetate (99mTcO4-), a NIS substrate, up to 28 days after AAV9-hNIS injection. Twenty four animals were euthanized at selected times over 1 month for ex vivo validation. Forty-two animals were imaged with 99mTcO4- ± the selective NIS inhibitor perchlorate on day 10, to ascertain specificity of radiotracer uptake. Tissue was harvested for ex vivo validation. A modified version of the U-Net deep learning algorithm was used for image quantification. RESULTS:As quantitated by standardized uptake value, there was a gradual temporal increase in 99mTcO4- uptake in muscles treated with AAV9-hNIS. Hindlimb ischemia, NA, and hindlimb ischemia plus NA increased the magnitude of 99mTcO4- uptake by 4- to 5-fold compared with nonischemic muscle treated with only AAV9-hNIS. Perchlorate treatment significantly reduced 99mTcO4- uptake in AAV9-hNIS-treated muscles, demonstrating uptake specificity. The imaging results correlated well with ex vivo well counting (r2=0.9375; P<0.0001) and immunoblot analysis of NIS protein (r2=0.65; P<0.0001). CONCLUSIONS:Micro single-photon emission computed tomography/computed tomography imaging of hNIS-mediated 99mTcO4- uptake allows for accurate in vivo quantification of AAV9-driven gene expression, which increases under ischemic conditions or neuraminidase desialylation in skeletal muscle.

Project description:BackgroundGene transfer by electroporation (DNA electrotransfer) to muscle results in high level long term transgenic expression, showing great promise for treatment of e.g. protein deficiency syndromes. However little is known about the effects of DNA electrotransfer on muscle fibres. We have therefore investigated transcriptional changes through gene expression profile analyses, morphological changes by histological analysis, and physiological changes by force generation measurements. DNA electrotransfer was obtained using a combination of a short high voltage pulse (HV, 1000 V/cm, 100 mus) followed by a long low voltage pulse (LV, 100 V/cm, 400 ms); a pulse combination optimised for efficient and safe gene transfer. Muscles were transfected with green fluorescent protein (GFP) and excised at 4 hours, 48 hours or 3 weeks after treatment.ResultsDifferentially expressed genes were investigated by microarray analysis, and descriptive statistics were performed to evaluate the effects of 1) electroporation, 2) DNA injection, and 3) time after treatment. The biological significance of the results was assessed by gene annotation and supervised cluster analysis.Generally, electroporation caused down-regulation of structural proteins e.g. sarcospan and catalytic enzymes. Injection of DNA induced down-regulation of intracellular transport proteins e.g. sentrin. The effects on muscle fibres were transient as the expression profiles 3 weeks after treatment were closely related with the control muscles. Most interestingly, no changes in the expression of proteins involved in inflammatory responses or muscle regeneration was detected, indicating limited muscle damage and regeneration. Histological analysis revealed structural changes with loss of cell integrity and striation pattern in some fibres after DNA+HV+LV treatment, while HV+LV pulses alone showed preservation of cell integrity. No difference in the force generation capacity was observed in the muscles 2 weeks after DNA electrotransfer.ConclusionThe small and transient changes found in the gene expression profiles are of great importance, as this demonstrates that DNA electrotransfer is safe with minor effects on the muscle host cells. These findings are essential for introducing the DNA electrotransfer to muscle for clinical use. Indeed the HV+LV pulse combination used has been optimised to ensure highly efficient and safe DNA electrotransfer.

Project description:BackgroundIn livestock populations the genetic contribution to muscling is intensively monitored in the progeny of industry sires and used as a tool in selective breeding programs. The genes and pathways conferring this genetic merit are largely undefined. Genetic variation within a population has potential, amongst other mechanisms, to alter gene expression via cis- or trans-acting mechanisms in a manner that impacts the functional activities of specific pathways that contribute to muscling traits. By integrating sire-based genetic merit information for a muscling trait with progeny-based gene expression data we directly tested the hypothesis that there is genetic structure in the gene expression program in ovine skeletal muscle.ResultsThe genetic performance of six sires for a well defined muscling trait, longissimus lumborum muscle depth, was measured using extensive progeny testing and expressed as an Estimated Breeding Value by comparison with contemporary sires. Microarray gene expression data were obtained for longissimus lumborum samples taken from forty progeny of the six sires (4-8 progeny/sire). Initial unsupervised hierarchical clustering analysis revealed strong genetic architecture to the gene expression data, which also discriminated the sire-based Estimated Breeding Value for the trait. An integrated systems biology approach was then used to identify the major functional pathways contributing to the genetics of enhanced muscling by using both Estimated Breeding Value weighted gene co-expression network analysis and a differential gene co-expression network analysis. The modules of genes revealed by these analyses were enriched for a number of functional terms summarised as muscle sarcomere organisation and development, protein catabolism (proteosome), RNA processing, mitochondrial function and transcriptional regulation.ConclusionsThis study has revealed strong genetic structure in the gene expression program within ovine longissimus lumborum muscle. The balance between muscle protein synthesis, at the levels of both transcription and translation control, and protein catabolism mediated by regulated proteolysis is likely to be the primary determinant of the genetic merit for the muscling trait in this sheep population. There is also evidence that high genetic merit for muscling is associated with a fibre type shift toward fast glycolytic fibres. This study provides insight into mechanisms, presumably subject to strong artificial selection, that underpin enhanced muscling in sheep populations.

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Neuraminidase-mediated desialylation augments AAV9-mediated gene expression in skeletal muscle.

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Neuraminidase-mediated desialylation augments AAV9-mediated gene expression in skeletal muscle.

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