TY  - JOUR
A1  - Bittorf, Patrick
A1  - Bergmann, Thorsten
A1  - Merlin, Simone
A1  - Olgasi, Chistina
A1  - Pullig, Oliver
A1  - Sanzenbacher, Ralf
A1  - Zierau, Martin
A1  - Walles, Heike
A1  - Follenzi, Antonia
A1  - Braspenning, Joris
T1  - Regulatory-Compliant Validation of a Highly Sensitive qPCR for Biodistribution Assessment of Hemophilia A Patient Cells
JF  - Molecular Therapy - Methods & Clinical Development
N2  - The investigation of the biodistribution profile of a cell-based medicinal product is a pivotal prerequisite to allow a factual benefit-risk assessment within the non-clinical to clinical translation in product development. Here, a qPCR-based method to determine the amount of human DNA in mouse DNA was validated according to the guidelines of the European Medicines Agency and the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. Furthermore, a preclinical worst-case scenario study was performed in which this method was applied to investigate the biodistribution of 2 x 10\(^6\) intravenously administered, genetically modified, blood outgrowth endothelial cells from hemophilia A patients after 24 h and 7 days. The validation of the qPCR method demonstrated high accuracy, precision, and linearity for the concentration interval of 1:1 x 10\(^3\) to 1:1 x 10\(^6\) human to mouse DNA. The application of this method in the biodistribution study resulted in the detection of human genomes in four out of the eight investigated organs after 24 h. After 7 days, no human DNA was detected in the eight organs analyzed. This biodistribution study provides mandatory data on the toxicokinetic safety profile of an actual candidate cell-based medicinal product. The extensive evaluation of the required validation parameters confirms the applicability of the qPCR method for non-clinical biodistribution studies.
KW  - outgrowth endothelial cells
KW  - real time PCR
KW  - in vivo
KW  - gene therapy
KW  - factor-VIII
KW  - murine
KW  - quantification
KW  - establishment
KW  - phenotype
KW  - xenotransplantation
Y1  - 2020
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230284
VL  - 18
ER  - 
TY  - JOUR
A1  - Farag, Heba Gamal
A1  - Froehler, Sebastian
A1  - Oexle, Konrad
A1  - Ravindran, Ethiraj
A1  - Schindler, Detlev
A1  - Staab, Timo
A1  - Huebner, Angela
A1  - Kraemer, Nadine
A1  - Chen, Wei
A1  - Kaindl, Angela M.
T1  - Abnormal centrosome and spindle morphology in a patient with autosomal recessive primary microcephaly type 2 due to compound heterozygous WDR62 gene mutation
JF  - Orphanet Journal of Rare Diseases
N2  - Background: Autosomal recessive primary microcephaly (MCPH) is a rare neurodevelopmental disease with severe microcephaly at birth due to a pronounced reduction in brain volume and intellectual disability. Biallelic mutations in the WD repeat-containing protein 62 gene WDR62 are the genetic cause of MCPH2. However, the exact underlying pathomechanism of MCPH2 remains to be clarified. 
Methods/results: We characterized the clinical, radiological, and cellular features that add to the human MCPH2 phenotype. Exome sequencing followed by Sanger sequencing in a German family with two affected daughters with primary microcephaly revealed in the index patient the compound heterozygous mutations c. 1313G>A (p.R438H) / c.2864-2867delACAG (p.D955Afs*112) of WDR62, the second of which is novel. Radiological examination displayed small frontal lobes, corpus callosum hypoplasia, simplified hippocampal gyration, and cerebellar hypoplasia. We investigated the cellular phenotype in patient-derived lymphoblastoid cells and compared it with that of healthy female controls. WDR62 expression in the patient's immortalized lymphocytes was deranged, and mitotic spindle defects as well as abnormal centrosomal protein localization were apparent. 
Conclusion: We propose that a disruption of centrosome integrity and/or spindle organization may play an important role in the development of microcephaly in MCPH2.
KW  - cell division
KW  - intellectual disability
KW  - missense mutations
KW  - protein
KW  - malformations
KW  - establishment
KW  - cytokinesis
KW  - genome
KW  - midbody
KW  - database
KW  - maintenance
KW  - families
KW  - microcephaly
KW  - WDR62 mutation
Y1  - 2013
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-123505
SN  - 1750-1172
VL  - 8
IS  - 178
ER  -