Abstract
Widespread genetic modification of cells in the central nervous system (CNS) with a viral vector has become possible and increasingly more efficient. We previously applied an AAV9 vector with the cytomegalovirus/chicken beta-actin (CBA) hybrid promoter and achieved wide-scale CNS transduction in neonatal and adult rats. However, this method transduces a variety of tissues in addition to the CNS. Thus we studied intravenous AAV9 gene transfer with a synapsin promoter to better target the neurons. We noted in systematic comparisons that the synapsin promoter drives lower level expression than does the CBA promoter. The engineered adeno-associated virus (AAV)-PHP.B serotype was compared with AAV9, and AAV-PHP.B did enhance the efficiency of expression. Combining the synapsin promoter with AAV-PHP.B could therefore be advantageous in terms of combining two refinements of targeting and efficiency. Wide-scale expression was used to model a disease with widespread pathology. Vectors encoding the amyotrophic lateral sclerosis (ALS)-related protein transactive response DNA-binding protein, 43 kDa (TDP-43) with the synapsin promoter and AAV-PHP.B were used for efficient CNS-targeted TDP-43 expression. Intracerebroventricular injections were also explored to limit TDP-43 expression to the CNS. The neuron-selective promoter and the AAV-PHP.B enhanced gene transfer and ALS disease modeling in adult rats.
Highlights
Adeno-associated virus (AAV) is one of the most widely used systems for gene transfer to the central nervous system (CNS), for example in optogenetics, cre-lox targeting, and CRISPR gene editing
We studied synapsin promoter-TDP-43 vectors and intracerebroventricular injections in order to address whether the motor paralysis that we see using the chicken beta-actin (CBA) promoter is due to TDP-43 expression in the CNS
The difference in brightness between the two fluorophores had no bearing on the results since the tissues were immuno-labeled with a green fluorescent protein (GFP) antibody that recognizes both proteins. cDNA for human wild-type TDP-43 was incorporated into the synapsin promoter cassette in place of yellow fluorescent protein (YFP)
Summary
Adeno-associated virus (AAV) is one of the most widely used systems for gene transfer to the central nervous system (CNS), for example in optogenetics, cre-lox targeting, and CRISPR gene editing. Many of these governing parameters of AAV gene transfer have been extensively studied with focal, intraparenchymal injections. Few of these comparative studies have been made for wide-scale CNS transduction where neurons are transduced throughout the nervous system. This study attempted to improve upon previous work with AAV9 and the hybrid cytomegalovirus/chicken beta-actin (CBA, known as the CAG promoter) promoter in terms of neuronal targeting and gene transfer efficiency. If possible, these refinements could be applied to disease modeling and gene therapy
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