Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Aug;30(7-8):649-658.
doi: 10.1038/s41434-023-00399-w. Epub 2023 Apr 7.

Combinatorial gene therapy for epilepsy: Gene sequence positioning and AAV serotype influence expression and inhibitory effect on seizures

Esbjörn Melin  1 , My Andersson  2 , Casper R Gøtzsche  3   4 , Jenny Wickham  2 , Yuzhe Huang  4 , Julia Alicja Szczygiel  4 , Arnie Boender  2 , Søren H Christiansen  4 , Lars Pinborg  5 , David P D Woldbye #  4 , Merab Kokaia #  2
Affiliations

Combinatorial gene therapy for epilepsy: Gene sequence positioning and AAV serotype influence expression and inhibitory effect on seizures

Esbjörn Melin et al. Gene Ther. 2023 Aug.

Abstract

Gene therapy with AAV vectors carrying genes for neuropeptide Y and its receptor Y2 has been shown to inhibit seizures in multiple animal models of epilepsy. It is however unknown how the AAV serotype or the sequence order of these two transgenes in the expression cassette affects the actual parenchymal gene expression levels and the seizure-suppressant efficacy. To address these questions, we compared three viral vector serotypes (AAV1, AAV2 and AAV8) and two transgene sequence orders (NPY-IRES-Y2 and Y2-IRES-NPY) in a rat model of acutely induced seizures. Wistar male rats were injected bilaterally with viral vectors and 3 weeks later acute seizures were induced by a subcutaneous injection of kainate. The latency until 1st motor seizure, time spent in motor seizure and latency to status epilepticus were measured to evaluate the seizure-suppressing efficacy of these vectors compared to an empty cassette control vector. Based on the results, the effect of the AAV1-NPY-IRES-Y2 vector was further investigated by in vitro electrophysiology, and its ability to achieve transgene overexpression in resected human hippocampal tissue was evaluated. The AAV1-NPY-IRES-Y2 proved to be better to any other serotype or gene sequence considering both transgene expression and ability to suppress induced seizures in rats. The vector also demonstrated transgene-induced decrease of glutamate release from excitatory neuron terminals and significantly increased both NPY and Y2 expression in resected human hippocampal tissue from patients with drug-resistant temporal lobe epilepsy. These results validate the feasibility of NPY/Y2 receptor gene therapy as a therapeutic opportunity in focal epilepsies.

PubMed Disclaimer

Conflict of interest statement

This study was sponsored by the spin-off company CombiGene AB. MK and DPDW are co-founders, shareowners, and consultants of CombiGene AB. Esbjörn Melin is employed and has shares in CombiGene AB.

Figures

Fig. 1
Fig. 1. Outcomes from the seizure assessment after bilateral overexpression of the transgenes.
AAV1: A latency to 1st motor seizure, B time spent in motor seizures, C latency to status epilepticus (SE). The AAV1-NPY/Y2 vector is highlighted (magenta); AAV2: D latency to 1st motor seizure, E time spent in motor seizures, F latency to SE; AAV8: G latency to 1st motor seizure, H time spent in motor seizures, I latency to SE. Data is shown as mean ± SEM. (n = 7–12 in each group). *P < 0.05 versus same serotype control (EMPTY). One-way ANOVA followed by Bonferroni’s multiple comparison post-hoc tests.
Fig. 2
Fig. 2. NPY expression levels after bilateral viral vector administration as measured by means of grey value in images from immunohistochemistry.
Data is shown as mean ± SEM. The data was analyzed with a one-way ANOVA followed by Bonferroni’s multiple comparison test. ***P < 0.001.
Fig. 3
Fig. 3. Y2 receptor functional binding intensity in the hippocampus after bilateral viral vector administration.
Data is shown as mean ± SEM. Data was analysed with a one-way ANOVA followed by Bonferroni’s multiple comparison test. ***P < 0.001.
Fig. 4
Fig. 4. Train stimulation in slices from AAV1-NPY/Y2 treated animals decreases glutamate release in neighbouring synapses through NPY-Y2 receptor activation.
A Experimental setup, showing placement of stimulation (S1 and S2) and recording electrodes (Rec.) in the stratum radiatum, CA1 area of the acute hippocampal slice (left) and schematic depiction of the stimulation protocol (right). The effect of transgene NPY expression on excitatory neurotransmission was also examined by applying repetitive train stimulation (TS) with 100 Hz, 10 stimulations, in one group of presynaptic fibers/pathway (S1), preceded and followed at 500 ms interval by PP-stimulation (20 Hz) in a neighbouring, convergent but independent presynaptic pathway (S2). We compared the amplitude of the fEPSPS evoked by paired-pulse stimulation (PP) before (PP1) and after train stimulation (PP2, blue B). Example sweeps showing fEPSP evoked by S2 before (black sweep) and after TS-stimulation (blue sweep), showing a reduction in the first fEPSP and concomitant increase in PPR after TS-stimulation. Right) This reduction is smaller in the AAV1-NPY/Y2 treated animals after application of the Y2-receptor antagonist BIIE0246. C Bar graph showing average depression of fEPSP after TS-stimulation in AAV1-NPY/Y2 (n = 5), AAV1-Y2/NPY (n = 7), and AAV1-EMPTY (n = 7). The TS-induced depression of fEPSP is reduced after application of BIIE0246 in the AAV1-NPY/Y2 treatment-group (n = 5), but not in AAV1-Y2/NPY (n = 7) or AAV1-EMPTY (n = 7). AAV1-NPY/Y2 (left bars), AAV1-Y2/NPY (middle bars), and AAV1-EMPTY (right bars). D Examples of immunohistochemical staining for NPY after electrophysiology. Scalebar 100 μm, **P < 0.01, ***P < 0.001, Student’s paired t-test. Data is shown as mean ± SEM.
Fig. 5
Fig. 5. Increased NPY-immunoreactivity in AAV1-NPY/Y2 treated organotypic hippocampal slices from TLE-patients.
Example images of immunohistochemical stainings of NPY in AAV1-EMPTY (left) and AAV1-NPY/Y2-treated (right) organotypic slices from one patient operated for intractable TLE. Scale bar 50 μm, iml (inner molecular layer), gcl (granule cell layer), hil (hilus), NPY (neuropeptide Y, red), HO (Hoescht stain, blue).
Fig. 6
Fig. 6. mRNA levels for NPY and Y2 are increased in AAV1-NPY/Y2-treated organotypic hippocampal slices from three TLE-patients.
A NPY mRNA. B Y2 mRNA. C Y1. D Y5 mRNA levels. Data are shown as individual qPCR Ct-values normalized to GAPDH and means ± SEM. Low Ct-values (denoted as “-”) correspond to higher levels of mRNA.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Duncan JS, Sander JW, Sisodiya SM, Walker MC. Adult epilepsy. Lancet. 2006;367:1087–100. doi: 10.1016/S0140-6736(06)68477-8. - DOI - PubMed
    1. Fiest KM, Sauro KM, Wiebe S, Patten SB, Kwon C-S, Dykeman J, et al. Prevalence and incidence of epilepsy: A systematic review and meta-analysis of international studies. Neurology. 2017;88:296–303. doi: 10.1212/WNL.0000000000003509. - DOI - PMC - PubMed
    1. Pugliatti M, Beghi E, Forsgren L, Ekman M, Sobocki P. Estimating the cost of epilepsy in Europe: A review with economic modeling. Epilepsia. 2007;48:2224–33. doi: 10.1111/j.1528-1167.2007.01251.x. - DOI - PubMed
    1. Picot MC, Baldy-Moulinier M, Daurès JP, Dujols P, Crespel A. The prevalence of epilepsy and pharmacoresistant epilepsy in adults: A population-based study in a Western European country. Epilepsia. 2008;49:1230–8. doi: 10.1111/j.1528-1167.2008.01579.x. - DOI - PubMed
    1. Yilmazer-Hanke DM, Wolf HK, Schramm J, Elger CE, Wiestler OD, Blümcke I. Subregional pathology of the amygdala complex and entorhinal region in surgical specimens from patients with pharmacoresistant temporal lobe epilepsy. J Neuropathol Exp Neurol. 2000;59:907–20. doi: 10.1093/jnen/59.10.907. - DOI - PubMed