466. Automated Generation of Genetically Modified Human CD34+ Cells in a Functionally Closed System

Abstract

Hereditary genetic disorders have been shown to be effectively treated by re-introduction of the wild-type gene product via viral modification of autologous CD34+ progenitor cells. However, current manufacturing processes are performed manually or at best by using semi-automated procedures. A standardized production of cellular therapeutic agents and their genetic modification is a major requirement to move cellular and gene therapies from their current translational setting into routine clinical use. A closed and highly automated manufacturing procedure would lead to large improvements in product performance, product safety and operator safety. We have recently developed a functionally closed and fully automated cell processing device, the CliniMACS Prodigy®, which enables complex cellular products to be manufactured.In this functionally closed system, the following steps have been automated: concentration and washing of blood products, magnetic labeling and enrichment of CD34+ cells, cultivation and pre-activation of the enriched CD34+ cells, transduction with lentiviral vectors and resuspension of the final cell product in buffer for infusion.Automated cell preparation, cell labeling and magnetic CD34+ cell separation was achieved from both mobilized and non-mobilized apheresis samples as well as bone marrow aspirates. CD34+ cells were enriched to purities exceeding 90%. Small scale experiments (10e5-10e7 purified CD34+ cells) performed with a newly developed automated transduction process, incorporating a single transduction cycle, resulted in high transduction efficiencies (up to 90%). A scale-up of the procedure was performed using larger numbers of cells (>10e7) enriched from mobilized apheresis material. CD34+ cells were transduced with a clinical-grade lentiviral vector using a two hit protocol already described in a clinical setting and used as standard for a control experiment run in parallel (Scaramuzza et al. 2013, Mol. Ther). Equivalent gene marking efficiencies were obtained. Moreover, gene modified cells maintained full engraftment potential as demonstrated by biodistribution studies in NSG mice.These preliminary data demonstrate that automated cell processing and genetic manipulation of haematopoetic progenitor cells can be efficiently performed in a closed system. Work in progress aims to further optimize this procedure and improve the efficiency of CD34+ cell transduction to levels superior to those currently achieved by manual protocols.