A Dual Therapy for SLC6A1 Variant-Mediated Developmental Epileptic Encephalopathy: Gene Restoration Plus 4-phenylbutyrate
Abstract number :
1.438
Submission category :
1. Basic Mechanisms / 1D. Mechanisms of Therapeutic Interventions
Year :
2022
Submission ID :
2232850
Source :
www.aesnet.org
Presentation date :
12/3/2022 12:00:00 PM
Published date :
Nov 22, 2022, 05:27 AM
Authors :
Carson Flamm, BA – Department of Neurology, Vanderbilt Brain Institute, Vanderbilt University Medical Center; Wangzhen Shen, MD – Department of Neurology, Vanderbilt Brain Institute, Vanderbilt University Medical Center, Nashville; Karishma Randhave, BS – Department of Neurology, Vanderbilt Brain Institute, Vanderbilt University Medical Center, Nashville; Muhammad Khan, PhD – Department of Neurology, Vanderbilt Brain Institute, Vanderbilt University Medical Center, Nashville; Jing-Qiong Kang, MD, PhD – Department of Neurology, Vanderbilt Brain Institute, Vanderbilt University Medical Center, Nashville
This is a Late-Breaking abstract.
Rationale: We have previously identified that a partial or a complete loss of GABA uptake function is a major pathophysiology for SLC6A1 mutation mediated neurodevelopmental disorders. We have identified that 4-phenylbutyrate (PBA) can partially rescue disease phenotype in the patient mutation bearing knockin mouse models. The disorders are likely life-long because the existence of the mutation. The high dosage and potential side effect call for alternative treatment strategies such as gene therapy. We thus decide to thoroughly compare these two mutations in cell and mouse models. We hypothesize that combined treatment with PBA and gene therapy may be the most effective, as it could reduce toxicity resulting from high doses of PBA and prevent gene therapy overcorrection.
Methods: In cell models, HEK293T cells or human induced pluripotent stem cells (iPSC) differentiated astrocytes were transfected with the wildtype SLC6A1 or the mutant SLC6A1(A288V) or SLC6A1(S295L) cDNAs. The lives are fluxed and evaluated for GABA uptake with 3H radiolabeling GABA uptake assay. Total protein from cell lysates were subjected to SDS-PAGE and immunoblotted for GAT-1 and GFP. We tested gene therapy in the knockin mouse models Slc6a1+/A288V and Slc6a1+/S295L. We first generated the transgenic mouse HSlc6a1tg with a conditional overexpression of humanized Slc6a1 BAC transgene via PiggyBAC mediated integration, global overexpression of which can be activated via breeding with HprtCre mice. In breeding mice overexpressing hSlc6a1 with Slc6a1+/A288V and Slc6a1+/S295L mice, we aim to determine whether hSlc6a1 overexpression can compensate for A288V or S295L mutation-mediated loss of GABA uptake function. We additionally aim to test if hSlc6a1 overexpression in conjunction with PBA treatment can restore GABA uptake and reduce the necessary PBA dosage or the gene dosage of the human Slc6a1.
Results: In cell models, the mutant GAT-1(A288V) had ~30% of remaining GABA uptake of the wildtype while The GAT1(S295L) had less than 3% of remaining GABA uptake compared with the wildtype. Both the GAT-1(A288V) and the GAT-1(S295L) proteins were retained inside endoplasmic reticulum with no or reduced cell surface expression. PBA increased GABA uptake and cell surface expression of GAT-1 for both mutations. A combinatorial use of PBA and wildtype Slc6a1 achieved more complete restoration of GABA uptake with a lower concentration of PBA and a lower amount of wildtype allele gene dosage than PBA or gene therapy alone.
Conclusions: We concluded that a dual therapy with gene therapy in combination with PBA could reduce the side effect from PBA and reduce the dosage required for gene therapy. Because the dosage of PBA is easily modifiable, this mode of treatment will help prevent overcorrection from gene therapy.
Funding: The work was supported by research grants from SLC6A1 Connect and NIH R01 NS121718 to KJQ.
Basic Mechanisms