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Improved gene delivery to adult mouse spinal cord through the use of engineered hybrid adeno-associated viral serotypes.

ABSTRACT: Adeno-associated viral (AAV) vectors are often used in gene therapy for neurological disorders because of its safety profile and promising results in clinical trials. One challenge to AAV gene therapy is effective transduction of large numbers of the appropriate cell type, which can be overcome by modulating the viral capsid through DNA shuffling. Our previous study demonstrates that Rec2, among a family of novel engineered hybrid capsid serotypes (Rec1~4) transduces adipose tissue with far superior efficiency than naturally occurring AAV serotypes. Here we assessed the transduction of adult spinal cord at two different doses of AAV vectors expressing green fluorescent protein (2 × 109 or 4 × 108 viral particles) via intraparenchymal injection at the thoracic vertebral level T9. In comparison with an equal dose of the currently preferable AAV9 serotype, Rec3 serotype transduced a broader region of the spinal cord up to ~1.5?cm longitudinally and displayed higher transgene expression and increased maximal transduction rates of astrocytes at either dose and neurons at the lower dose. These novel engineered hybrid vectors could provide powerful tools at lower production costs to manipulate gene expression in the spinal cord for mechanistic studies or provide potent vehicles for gene therapy delivery, such as neurotrophins, to the spinal cord.

SUBMITTER: Siu JJ

PROVIDER: S-EPMC5472488 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Improved gene delivery to adult mouse spinal cord through the use of engineered hybrid adeno-associated viral serotypes.

Siu J J JJ Queen N J NJ Huang W W Yin F Q FQ Liu X X Wang C C McTigue D M DM Cao L L

Gene therapy 20170425 6

Adeno-associated viral (AAV) vectors are often used in gene therapy for neurological disorders because of its safety profile and promising results in clinical trials. One challenge to AAV gene therapy is effective transduction of large numbers of the appropriate cell type, which can be overcome by modulating the viral capsid through DNA shuffling. Our previous study demonstrates that Rec2, among a family of novel engineered hybrid capsid serotypes (Rec1~4) transduces adipose tissue with far supe ...[more]

PMID: 28440798

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Project description:RATIONALE:Somatic overexpression in mice using an adeno-associated virus (AAV) as gene transfer vectors has become a valuable tool to analyze the roles of specific genes in cardiac diseases. The lack of atrial-specific AAV vector has been a major obstacle for studies into the pathogenesis of atrial diseases. Moreover, gene therapy studies for atrial fibrillation would benefit from atrial-specific vectors. Atrial natriuretic factor (ANF) promoter drives gene expression specifically in atrial cardiomyocytes. OBJECTIVE:To establish the platform of atrial specific in vivo gene delivery by AAV-ANF. METHODS AND RESULTS:We constructed AAV vectors based on serotype 9 (AAV9) that are driven by the atrial-specific ANF promoter. Hearts from mice injected with AAV9-ANF-GFP (green fluorescent protein) exhibited strong and atrial-specific GFP expression without notable GFP in ventricular tissue. In contrast, similar vectors containing a cardiac troponin T promoter (AAV9-TNT4-GFP) showed GFP expression in all 4 chambers of the heart, while AAV9 with an enhanced chicken ?-actin promoter (AAV-enCB-GFP) caused ubiquitous GFP expression. Next, we used Rosa26mT/mG (membrane-targeted tandem dimer Tomato/membrane-targeted GFP), a double-fluorescent Cre reporter mouse that expresses membrane-targeted tandem dimer Tomato before Cre-mediated excision, and membrane-targeted GFP after excision. AAV9-ANF-Cre led to highly efficient LoxP recombination in membrane-targeted tandem dimer Tomato/membrane-targeted green fluorescent protein mice with high specificity for the atria. We measured the frequency of transduced cardiomyocytes in atria by detecting Cre-dependent GFP expression from the Rosa26mT/mG allele. AAV9 dose was positively correlated with the number of GFP-positive atrial cardiomyocytes. Finally, we assessed whether the AAV9-ANF-Cre vector could be used to induce atrial-specific gene knockdown in proof-of-principle experiments using conditional JPH2 (junctophilin-2) knockdown mice. Four weeks after AAV9-ANF-Cre injection, a strong reduction in atrial expression of JPH2 protein was observed. Furthermore, there was evidence for abnormal Ca2+ handling in atrial myocytes isolated from mice with atrial-restricted JPH2 deficiency. CONCLUSIONS:AAV9-ANF vectors produce efficient, dose-dependent, and atrial-specific gene expression following a single-dose systemic delivery in mice. This vector is a novel reagent for both mechanistic and gene therapy studies on atrial diseases.

Dataset Information

Improved gene delivery to adult mouse spinal cord through the use of engineered hybrid adeno-associated viral serotypes.

Publications

Improved gene delivery to adult mouse spinal cord through the use of engineered hybrid adeno-associated viral serotypes.

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