Work package 2: Genetic defect repair, cell selection and characterisation
Leader: Prof. Antonia Follenzi, Università del Piemonte Orientale "Amedeo Avogadro"
The main objectives of this work package are to produce high titer Lentiviral Vectors (LVs) expressing FVIII under the control of an endothelial-specific promoter to transduce the BOECs obtained in WP1.
The objectives will be fulfilled by the following tasks:
- Production of LVs, carrying the gene of interest, such as a marker gene (e.g. GFP) or the coagulation B-domain deleted FVIII (BDD) under the control of an Endothelial specific promoter (Task 1)
- BOECs LV-transduction and FVIII expression analysis to define the best LV-dose to be used in patients cells (Task 2)
- LV integrational analysis of transduced BOECs (Task 3)
- Scale up production and optimization of LV in a GMP facility for clinical translation (Task 4)
Description of the tasks
New approaches to cure haemophilia A require insights into cell compartments capable of producing FVIII. Here, in vitro transduction and efficiency assessments will be performed to define the best amount of LVs to be used for BOECs transduction in order to proceed with in vivo testing of transduced BOECs. Once the optimal transduction conditions are established, we will transfer the protocol to GMP to prepare FVIII-expressing BOECs to be transplanted in vivo within the Cell Pouch™, as described in WP5, in a clinical evaluation for patients use.
Task 1: Lentiviral vectors production.
The vector used in this study (EC.FVIII.LV) is a self-inactivating LV carrying FVIII under the control of an endothelial promoter, which will be produced with a safe third-generation packaging system.
Task 2: Lentiviral transduction of BOECs and characterisation for FVIII expression.
To analyse the specificity of LV, we will perform initial studies with VEC-GFP-LV in BOECs isolated from several patients and received by Partner 1 UKW. We will evaluate the best doses of vector, to obtain the maximal transduction frequency, average and range of transgene expression level per cell. FVIII protein-containing culture medium will be collected and measured with highly sensitive coagulation activity assays. To minimise ex vivo toxicity affecting cells, we will shorten the incubation time and examine various concentrations of LV to transduce BOECs while maintaining cell viability long term for expansion and transplantation in the Cell Pouch™ implanted in immunocompromised haemophilia A mice by Partner 5 SERC (WP5).
Task 3: LV integration analysis.
To determine whether there will be any cause for concern regarding the safety and efficacy of the LV in transduced BOECs, we will analyse the LV genomic integrations in the patients’ transduced cells.
Task 4: Scale up and optimisation for GMP level.
Once assessed all the variables necessary to obtain BOECs expressing FVIII suitable for implantation in the Sernova Cell Pouch™ (WP5), we will collaborate with Partner 2 IMS (WP6) to optimise a protocol for LV production for BOECs transduction to be performed in an approved GMP facility for therapeutic application to meet regulatory standards for safety, quality and consistency in vector production.