Bone Marrow Stromal Cells as Targets for Gene Therapy of Hemophilia A

Abstract

Attempts to develop an ex vivo gene therapy strategy for hemophilia A, using either primary T cells or bone marrow (BM) stem/progenitor cells have been unsuccessful, due to the inability of these cell types to express coagulation factor VIII (FVIII). As an alternative, we evaluated the potential of BM-derived stromal cells which can be readily obtained and expanded in vitro. Human and murine BM stromal cells were transduced with an intron-based Moloney murine leukemia virus (MoMLV) retroviral vector expressing a B-domain-deleted human factor VIII cDNA (designated as MFG-FVIIIdeltaB). Transduction efficiencies were increased 10- to 15-fold by phosphate depletion and centrifugation, which obviated the need for selective enrichment of the transduced BM stromal cells. This resulted in high FVIII expression levels in transduced human (180 +/- 4 ng FVIII/10[6] cells per 24 hr) and mouse (900 +/- 130 ng FVIII/10[6] cells per 24 hr) BM stromal cells. Pseudotyping of the MFG-FVIIIdeltaB retroviral vectors with the gibbon ape leukemia virus envelope (GALV-env) resulted in significantly higher transduction efficiencies (100 +/- 20%) and FVIII expression levels (390 +/- 10 ng FVIII/10[6] cells per 24 hr) in transduced human BM stromal cells than with standard amphotropic vectors. This difference in transduction efficiency correlated with the higher titer of the GALV-env pseudotyped viral vectors and with the higher GALV receptor (GLVR-1) versus amphotropic receptor (GLVR-2) mRNA expression levels in human BM stromal cells. These findings demonstrate the potential of BM stromal cells for gene therapy in general and hemophilia A in particular.

Potential of mesenchymal stem cells in gene therapy approaches for inherited and acquired diseases

“…In a related study, transgene expression from murine stem cell virus-based vectors in vivo lasted for up to 12 weeks in human MSCs, adhered to ceramic cubes and implanted into severe combined immunodeficient (SCID) mice [15]. However, transduction of MSCs with MoMLV and murine stem cell virus-based vectors were shown to be inefficient, as they required drug selection to enrich transduced cells [13,15], multiple rounds of transduction for several days [14,16,17], or highly concentrated vector stocks [18]. In addition, efficient transduction of human MSCs by amphotropic MoMLV and murine stem cell virus pseudotypes was found to be limited by the expression levels of the amphotropic viral receptor.…”

“…The amphotropic receptor is a phosphate transporter whose expression is increased in the absence of phosphate. Chuah et al [16] used a phosphate starvation procedure to increase transduction of MSCs by amphotropic MoMLV-based vectors.…”

“…The proteins included the Escherichia coli β-galactosidase [15,40], GFP [14,18,19] and red fluorescent protein (DsRed) [19], as well as many therapeutic proteins, including coagulation factors VIII [12,16,17] and IX [41][42][43], IL-3 [15,44,45] and IL-7 [46], human growth hormone [41], human erythropoietin (hEPO) [47] and murine erythropoietin (mEPO) [48], arylsulfatase A [49,50], tyrosine hydroxylase GTP cyclohydrolase I [13,51], α-L-iduronidase [52], β-hexosaminidase A [53] and bone morphogenetic protein (BMP) [54]. It remains to be determined how MSCs perform relative to other mammalian expression systems, such as Chinese hamster ovary cells, in terms of transgene expression levels.…”

Transfer and Stable Transgene Expression of a Mammalian Artificial Chromosome into Bone Marrow‐Derived Human Mesenchymal Stem Cells

“…This list includes the replacement of genes to correct acquired or inherited disorders of bone, muscle, or cartilage by transfer of genes encoding bone morphogenic proteins -2, -4, -9 [21,[32][33][34][35][36][37][38][39][40], the transfer of genes encoding the blood coagulation factors VIII and IX for the treatment of hemophilia [41][42][43][44][45][46][47], human growth hormone [46,48], insulin-like growth factor 1 [49], and interleukin-3 [50]. This list also includes: erythropoietin [51], α-l-iduronidase for treatment of mucopolysaccharidosis type I [52], proα2 collagen (I) for treatment of osteogenesis imperfecta [53], sox9 [54] to enhance chondrogenesis, and interferon to treat tumors [55].…”

Hematopoietic stem cell gene therapy leads to marked visceral organ improvements and a delayed onset of neurological abnormalities in the acid sphingomyelinase deficient mouse model of Niemann–Pick disease

“…These data are consistent with those of other investigators who have shown long-term and high-level expression of transgenes in mice or rats transplanted with MFG-transduced hematopoietic cells. [22][23][24] Concordant with the high level enzymatic expression, an almost complete correction of the reticuloendothelial system pathology, which was observed only when high-level engraftment (ie Ͼ90%) was achieved by BMT, 18 was seen in gene therapy-treated animals engrafted 30% or less.…”