Durable engraftment of genetically modified FVIII‐secreting autologous bone marrow stromal cells in the intramedullary microenvironment

Abstract

Genetically modified FVIII‐expressing autologous bone marrow‐derived mesenchymal stromal cells (BMSCs) could cure haemophilia A. However, culture‐expanded BMSCs engraft poorly in extramedullary sites. Here, we compared the intramedullary cavity, skeletal muscle, subcutaneous tissue and systemic circulation as tissue microenvironments that could support durable engraftment of FVIII‐secreting BMSC in vivo. A zinc finger nuclease integrated human FVIII transgene into PPP1R12C (intron 1) of culture‐expanded primary canine BMSCs. FVIII‐secretory capacity of implanted BMSCs in each dog was expressed as an individualized therapy index (number of viable BMSCs implanted × FVIII activity secreted/million BMSCs/24 hours). Plasma samples before and after implantation were assayed for transgenic FVIII protein using an anti‐human FVIII antibody having negligible cross‐reactivity with canine FVIII. Plasma transgenic FVIII persisted for at least 48 weeks after implantation in the intramedullary cavity. Transgenic FVIII protein levels were low after intramuscular implantation and undetectable after both intravenous infusion and subcutaneous implantation. All plasma samples were negative for anti‐human FVIII antibodies. Plasma concentrations and durability of transgenic FVIII secretion showed no correlation with the therapy index. Thus, the implantation site microenvironment is crucial. The intramedullary microenvironment, but not extramedullary tissues, supported durable engraftment of genetically modified autologous FVIII‐secreting BMSCs.

Genome-edited adult stem cells: Next-generation advanced therapy medicinal products

“…In this setting, GE has been used to insert hFIX and hFVIII into the AAVS1 locus of MSCs through homologous recombination (HR) in order to treat hemophilia A and B. 82,83 GE-MSCS are also considered a potential alternative treatment for neurodegenerative diseases such as Parkinson's disease (PD). Using this approach, MSCs have been engineered to secrete soluble receptors of advanced glycation end products from AAVS1 loci.…”

In Vitro Conditioning of Adipose-Derived Mesenchymal Stem Cells by the Endothelial Microenvironment: Modeling Cell Responsiveness towards Non-Genetic Correction of Haemophilia A

“…Although recently receiving promising results in phase I/II trials [5,6], adenoassociated virus (AAV) gene therapy is not exempt from important concerns, including capsid-specific T-cell responses, which reduce transgene expression and cause hepatotoxicity. Another consideration to be made is in regards to the fact that viral vector production on a clinical scale is costly and variable [7]. Furthermore, the clinical development of AAV-FVIII still appears to be more complicated in comparison with AAV-factor IX (FIX) due to (1) the difficulty of efficient AAV vector packaging for large FVIII cDNA, and (2) the uncertain choice of liver target cell [8].…”

“…Considering that, HA cell therapy is based on the hypothesis that the transplantation of exogenous cells capable of releasing FVIII could become a functional cure for coagulation deficiency. A variety of genetically modified cells has been tested so far as delivery vehicles for FVIII, including hematopoietic stem/progenitor cells [13][14][15], bone marrow-derived mesenchymal stem cells [7,16,17], endothelial cells [18], human placental cells [19], and adipose stromal cells [20]. Despite promising pre-clinical results, the development of effective cell therapy approaches for HA treatment may benefit from providing insights into non-genetically modified cell populations able to produce FVIII and endowed with high engraftment potential.…”