We wished to determine if primary canine marrow mononuclear cells (MNC) would support the expression of human FVIII and thereby serve as a potential target for gene therapy of hemophilia A. To this end, we created HIV-1 vectors that express B-domain deleted human FVIII (BDFVIII) using the viral LTR promoter or constitutively active cellular promoters: the mouse phosphoglycerate kinase (PGK) or elongation factor 1α (EF1α) promoters. The vector that expressed BDFVIII from the LTR also encoded a functional single-exon Tat protein [
Srinivasakumar and Schuening (2000) J. Virol. 74:6659–6668
]. These promoters have been previously shown, by us and others, to express transgenes at high levels in hematopoietic cells [Srinivasakumar et. al., (2002). J. Virol. 76:7334–7342
]. Separate vector stocks were prepared for each of the above vectors by transient transfection of 293T cells [Srinivasakumar (2002) Methods Mol. Med. 69:275–302
] and their titers were determined by real-time quantitative PCR of genomic DNA isolated from vector transduced 293T cells (Table 1). Each vector stock was then used for infection of primary canine marrow MNC by three spin-infections over 2 days on CH296-coated 6-well plates. The medium was replaced at the end of the transduction procedure. Conditioned medium was harvested 3-days after transduction and assayed for functional FVIII activity using the Coamatic Factor VIII kit (Chromogenix, Monza, Italy). The results, shown in Table 1, indicate that vector-derived FVIII could be detected in the supernatants of canine marrow MNCs transduced with all HIV-1 vectors. Highest levels of functional FVIII were obtained with the Tat-encoding bicistronic HIV-1 vector. These results demonstrate that canine marrow MNC support expression and secretion of functional human FVIII. Further experiments are warranted to determine if the levels of FVIII observed in vitro will translate into therapeutic benefit in vivo in canine models of hemophilia A.Table 1.
Expression of human BDFVIII in canine marrow mononuclear cells transduced by HIV-1 vectors.
