Neuropediatrics 2024; 55(S 01): S1-S25
DOI: 10.1055/s-0044-1791911
Neurometabolische und Neurogenetische Erkrankungen

Characterization of Two rtTA/Ngn2-Transduced Pluripotent Stem Cell Lines from an Individual with SSADH Deficiency

S. Hofmann
1   Department I, Division of Pediatric Neurology and Metabolic Medicine, Heidelberg University Medical Faculty, Center for Pediatric and Adolescent Medicine, Heidelberg, Germany
,
M. Xi
1   Department I, Division of Pediatric Neurology and Metabolic Medicine, Heidelberg University Medical Faculty, Center for Pediatric and Adolescent Medicine, Heidelberg, Germany
,
V. Backendorf
1   Department I, Division of Pediatric Neurology and Metabolic Medicine, Heidelberg University Medical Faculty, Center for Pediatric and Adolescent Medicine, Heidelberg, Germany
,
Y. L. Behrens
2   Department of Human Genetics, Hannover Medical School (MHH), Hannover, Germany
,
T. Reinkens
2   Department of Human Genetics, Hannover Medical School (MHH), Hannover, Germany
,
M. Meyer
2   Department of Human Genetics, Hannover Medical School (MHH), Hannover, Germany
,
G. F. Hoffmann
1   Department I, Division of Pediatric Neurology and Metabolic Medicine, Heidelberg University Medical Faculty, Center for Pediatric and Adolescent Medicine, Heidelberg, Germany
,
T. Opladen
1   Department I, Division of Pediatric Neurology and Metabolic Medicine, Heidelberg University Medical Faculty, Center for Pediatric and Adolescent Medicine, Heidelberg, Germany
,
S. Jung-Klawitter
1   Department I, Division of Pediatric Neurology and Metabolic Medicine, Heidelberg University Medical Faculty, Center for Pediatric and Adolescent Medicine, Heidelberg, Germany
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Background/Purpose: Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare autosomal recessive disorder caused by mutations in the ALDH5A1 gene. The resulting enzyme defect causes a block in the degradation of γ-aminobutyric acid (GABA) and thus pathologic accumulation of GABA as well as toxic by-products. Clinically this presents in various neurologic disorders such as delayed development. Since current treatment options are limited and symptom related, new therapies are needed. Here we present two inducible rTA/Ngn-2 transduced pluripotent stem cell (iPSC) lines generated from a patient suffering from SSADHD and intended to be used for drug screening in differentiated neurons.

Methods: An SSADHD iPSC line from a patient carrying the pathogenic variants c.278C>T/c.802A>G has been transduced with the rtTA- and Ngn2-transgenes to allow for directed neuronal differentiation upon doxycycline treatment. Two clones have been manually picked and further characterized. Pluripotency was assessed by RT-PCR, IF, and FACS. The capability to differentiate into all three germ layers was shown by embryoid body (EBs) formation with spontaneous in vitro differentiation as well as targeted differentiation using the StemMACS Trilineage Differentiation Kit (Miltenyi Biotec) and IF. The ALDH5A1 variants were validated by PCR and Sanger sequencing, karyotype analysis was performed using R-banding. The ability to induce neuronal differentiation by supplementing doxycycline was assessed.

Results: The presence of pluripotency markers and differentiation potential was shown in both clones. Normal diploid karyotype as well as the presence of the variants c.278C>T and c.802A>G were verified. Functionality of the doxycycline inducible Ngn2 expression system was proven. Readouts for high throughput drug screening have been tested including TMRM and neuronal branching for suitability.

Conclusion: Both rtTA/Ngn2-inducible SSADHD iPSC lines have been fully characterized and can now be used in context of a drug repurposing approach.



Publication History

Article published online:
08 October 2024

Georg Thieme Verlag KG
Stuttgart · New York