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  - Riedl, Katharina A.
A1  - Kampf, Thomas
A1  - Herold, Volker
A1  - Behr, Volker C.
A1  - Bauer, Wolfgang R.
T1  - Wall shear stress analysis using 17.6 Tesla MRI: A longitudinal study in ApoE\(^{-/-}\)mice with histological analysis
JF  - PLoS One
N2  - This longitudinal study was performed to evaluate the feasibility of detecting the interaction between wall shear stress (WSS) and plaque development. 20 ApoE\(^{-/-}\)mice were separated in 12 mice with Western Diet and 8 mice with Chow Diet. Magnetic resonance (MR) scans at 17.6 Tesla and histological analysis were performed after one week, eight and twelve weeks. Allin vivoMR measurements were acquired using a flow sensitive phase contrast method for determining vectorial flow. Histological sections were stained with Hematoxylin and Eosin, Elastica van Gieson and CD68 staining. Data analysis was performed using Ensight and a Matlab-based "Flow Tool". The body weight of ApoE\(^{-/-}\)mice increased significantly over 12 weeks. WSS values increased in the Western Diet group over the time period; in contrast, in the Chow Diet group the values decreased from the first to the second measurement point. Western Diet mice showed small plaque formations with elastin fragmentations after 8 weeks and big plaque formations after 12 weeks; Chow Diet mice showed a few elastin fragmentations after 8 weeks and small plaque formations after 12 weeks. Favored by high-fat diet, plaque formation results in higher values of WSS. With wall shear stress being a known predictor for atherosclerotic plaque development, ultra highfield MRI can serve as a tool for studying the causes and beginnings of atherosclerosis.
KW  - phase-contrast MRI
KW  - flow patterns
KW  - blood flow
KW  - apolipoprotein-E
KW  - atheriosclerosis
KW  - mouse
KW  - mice
KW  - quantification
KW  - association
KW  - lesions
Y1  - 2020
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229318
VL  - 15
IS  - 8
ER  -