ASGCT 23rd Annual Meeting, virtual format, May 12-15, 2020https://annualmeeting.asgct.org/am20/ You can grasp the size of the event by the 590 pages of Abstracts.
Abstracts (Sernova: P581/590; #1359):
ASGCT Abstracts; SVA #1359 1359. Combined Gene and Cell Therapy for the Treatment of Hemophilia A within an Implantable Therapeutic Device Alessia Cucci1 , Cristina Olgasi1 , Simone Merlin1 , Chiara Borsotti1 , Thorsten Bergmann2 , Patrick Bittorf2 , Kelcey Patterson3 , Delfina M. Mazzuca3 , Alexandra Stolzing4 , Martin Zierau5 ,
Philip M. Toleikis3 , Fabrizio Benedicenti6 , Andrea Calabria6 , Eugenio Montini6 , Joris Braspenning2 , Antonia Follenzi1 1 Department of Health Sciences, Universit del Piemonte Orientale, Novara, Italy,2 Department of Tissue Engineering and Regenerative Medicine, University Hospital Wrzburg, Wrzburg, Germany,
3 Sernova Corp, London, ON, Canada,4 Centre for Biological Engineering School of Mechanical, Electrical and Manufacturing Engineering, Loughborough University, Loughborough, United Kingdom,5 IMS Integrierte Management Systeme e. K, Heppenheim, Germany,6 TIGET Vector Integration Core, San Raffaele Telethon Institute for Gene Therapy, SR-Tiget, Milan, Italy
Hemophilia A (HA) is a rare bleeding disorder caused by absence or dysfunction of FVIII protein. New regenerative medicine approaches to treat haemophilia A require insights into cell compartments capable of producing FVIII. We and others previously demonstrated that FVIII is produced specifically in endothelial cells. The aim of our work is to develop a novel ex vivo cell-based therapy using a medical device (Cell Pouch™, Sernova Corp.) leading to an improvement in patient quality of life. We isolated blood outgrowth endothelial cells (BOECs) from healthy and patients’ blood. BOECs express the endothelial markers (e.g. CD31, KDR, Tie-2, VEC, vWF) both at RNA and protein levels and were able to form tubules network when cultured in matrigel. HA BOECs were transduced with a lentiviral vector (LV) carrying the B domain deleted form of FVIII under the Vascular Endothelial Cadherin promoter (LV-VEC.hFVIII) and were characterized for endothelial phenotype and for the number of integrated LV copies/ cell (~3). We observed that FVIII was expressed by 80% of LV-VEC. hFVIII transduced cells and was efficiently secreted in the supernatant. ELISA assay showed that LV-VEC.hFVIII BOECs secreted higher amount of FVIII (24 ng/ml) compared to not transduced (4.5 ng/ml). Ten million LV-VEC.hFVIII-BOECs were transplanted intraperitoneally in association with cytodex® 3 microcarrier beads in NOD/SCID γ-null HA mice (NSG-HA, n=6). BOECs survived and secreted FVIII at therapeutic levels (12%) up to 18 weeks and ameliorate the bleeding phenotype of HA mice. Subsequently, a large-scale expansion of BOECs was performed prior transplantation in Cell Pouch™. The large scale expanded cells were investigated for the presence of chromosomal aberration, senescence markers and tumorigenic potential showing a safe profile suggesting that BOECs can be expanded without negative consequences. Finally, LV-transduced HA BOECs were transplanted into an implanted prevascularized, scalable medical device (Cell Pouch™) under the skin, optimized for sustained secretion of therapeutic FVIII in the NSG-HA mice, showing BOECs engraftment up to 12 weeks. Moreover, genomic integration profile of LV transduced BOECs was analyzed at different expansion passages or procedure timepoints. The molecular analysis showed the classic integration pattern of LVs, with a marked tendency to integrate within transcription units and no preference for promoter or regulatory elements. Therefore, no enrichment for oncogenes was observed, as well as expansion of clones with integration in common integration sites or biases towards gene classes related to cancer genes, but it is necessary to produce a longer time points for safety endpoints. Furthermore, a transcriptomic approach by RNA-seq analysis has been used to assess the comprehensive changes in mRNA expression in healthy, HA and LV-VEC.hFVIII transduced BOECs and data are in process to be analyzed. Overall, these results pave the way for future clinical testing in HA patients by transplantation of GMP produced autologous gene corrected BOECs within implanted device.