Adenosine kinase, glutamine synthetase and EAAT2 as gene therapy targets for temporal lobe epilepsy

Abstract

Astrocytes are an attractive cell target for gene therapy but the validation of new therapeutic candidates is needed. We determined whether adeno-associated viral (AAV) vector-mediated overexpression of glutamine synthetase (GS) or excitatory amino acid transporter 2 (EAAT2), or expression of microRNA targeting adenosine kinase (miR-ADK) in hippocampal astrocytes in the rat brain could modulate susceptibility to kainate-induced seizures and neuronal cell loss. Transgene expression was found predominantly in astrocytes following direct injection of glial-targeting AAV9 vectors by 3 weeks post-injection. ADK expression in miR-ADK vector-injected rats was reduced by 94–96% and was associated with a ~50% reduction in the duration of kainate-induced seizures and greater protection of dentate hilar neurons but not CA3 neurons compared to miR-control vector-injected rats. In contrast, infusion of AAV-GS and EAAT2 vectors did not afford any protection against seizures or neuronal damage as the level of transcriptional activity of the glial-fibrillary acidic promoter was too low to drive any significant increase in transgenic GS or EAAT2 relative to the high endogenous levels of these proteins. Our findings support ADK as a prime therapeutic target for gene therapy of temporal lobe epilepsy and suggest alternative approaches including the use of stronger glial promoters are needed to increase transgenic GS and EAAT2 expression to levels that may be required to affect seizure induction and propagation.

Transcriptional activity of novel ALDH1L1 promoters in the rat brain following AAV vector-mediated gene transfer

“…In comparison to the detection of the transgene in astrocytes in hippocampal sections from a rat that had been injected with an AAV9-GFAP-dYFP 10 (Figure 5a), we did not observe any eGFP-expressing cells with an astrocytic morphology in the ALDH1L1-eGFP or Syn-eGFP injected brains (confirmed with double-immunofluorescent labeling; data not shown), consistent with our observations in the substantia nigra. Unexpectedly, there was a wide variability in eGFP expression levels between animals allocated to both immunohistochemistry and Western blot analysis within each treatment group.…”

“…To characterize their tropism in the rat SNpc, titer-matched AAV9 vectors (2 × 10 9 genomes) were unilaterally injected into the rat SNpc ( n = 3 rats per vector), and cellular transduction patterns of transgene expression were analyzed at 3 weeks by immunohistochemical analysis. An additional subgroup of rats injected with an AAV9 vector expressing a yellow fluorescent protein reporter gene (YFP) under the control of a 2.2 kb GFAP promoter (AAV9-GFAP-YFP) that targets astrocytes as well as neurons in the rat hippocampus 10 was included to enable comparisons between the cellular transduction patterns mediated by the different promoters.…”

Recombinant Adeno-Associated Virus Serotype 6 (rAAV6) Potently and Preferentially Transduces Rat Astrocytes In vitro and In vivo

“…To date, astrocyte-specific gene transduction was reported with “canonical” rAAV5, rAAV8, and rAAV9 driving gene expression under control of the astrocytic Gfa2 promoter (see for example Young et al, 2014; Furman et al, 2016). However, astrocytic tropism of rAAV5, rAAV8, and rAAV9 may be strongly influenced by packaging or other factors.…”

Destination Brain: the Past, Present, and Future of Therapeutic Gene Delivery

“…Therapeutically, AAV9 has been used to deliver human erythropoietin (hEPO) (Yang et al, 2013), EAAT2, glutamine synthase (GS), miRNA against adenosine kinase (Young et al, 2014), and β-galactoside (Weismann et al, 2015). In an experimental PD model, a single intrastriatal dose of AAV9-hEPO was preceded by an additional intrastriatal or intramuscular injection to evaluate effects on immunogenicity and transduction efficiency.…”

“…Though intrastriatal or intramuscular might not be preferred routes of administration, these findings highlight the importance of assessing dosing route and frequency. AAV9-EAAT2 and -GS delivery to rat hippocampal astrocytes did not alter kainate-induced seizures, while AAV9-miRNA against adenosine kinase reduced seizure duration suggesting a possible therapeutic usage (Young et al, 2014). Intravascular injection of AAV9-β-galactoside led to CNS and peripheral organ transduction.…”