Gene therapy for hemophilia A: production of therapeutic levels of human factor VIII in vivo in mice.

Abstract

Continuous delivery of factor VIII (FVIII) protein in hemophiliacs by gene therapy will represent a major clinical advance over the current practice of infrequent administration of purified FVIII. Conceptually, retroviral vectors that can permanently insert the FVIII gene into the DNA of the host cell appear the most suitable vehicles for this specific purpose. However, most retroviral vector systems have shown a poor performance in the production of FVIII from primary cells in vitro and in vivo. Here we report the retroviral-mediated gene delivery of a B-domain-deleted human FVIII by using the MFG vector system. This vector permitted efficient transduction of the majority of the primary cells in culture without the use of a selectable marker. High levels of FVIII were produced by various transduced primary cells in vitro. Upon transplantation of primary fibroblasts into mice, therapeutic levels of FVIII in the circulation were obtained for >1 weelk The capacity of primary cells to deliver the FVIII into the circulation was strongly dependent on the site of implantation. These results represent a major step forward in development of gene therapy for treating hemophilia A.

Analysis of factor VIII mediated suppression of lentiviral vector titres

“…14 As the number of vector copies per cell was not given, and as the cell type (Huh7) and promoter (an additional 100 bp of 5 0 sequence present) were different, it is not possible to compare these data directly; however, this value is within the range we observed at higher multiplicities of infection (data not shown). The FVIII expression levels reported by Dwarki et al 8 using the MFG retroviral vector system, and also Kootstra et al 13 who used an HIV-1 vector containing an internal CAG promoter, were considerably more (at least two orders of magnitude) than we observed. It is not clear why such high-level FVIII production was achieved using these two vectors in comparison to most other reports; in both cases robust promoters were used, a 5 0 intron was present and, unlike some retroviral vectors, they did not contain a selectable marker.…”

“…Such vectors frequently displayed rearrangements or deletions suggesting selection against the presence of an intact FVIII gene in retroviral producer cells. 6,7 The generation of vectors with improved titres appears to be related to the presence of an intron 7,8 which is postulated to counteract transcriptional repression leading to increased mRNA accumulation. 9 Sustained levels of FVIII were not detected in patients administered with a retroviral construct 10 and therefore more recently studies have focused on the use of lentiviral vectors which are capable of transducing a wide variety of terminally differentiated cell types including hepatocytes.…”

Use of blood outgrowth endothelial cells for gene therapy for hemophilia A

“…The fact that human FVIII levels in murine plasma increased over time suggests that BOECs expanded more than 100-fold in vivo after their administration. To arrive at this, we used the conservative assumptions that the half-life of human FVIII in our mice was the same as previously reported for human FVIII in immunodeficient mice (1 hour) 25 and that all administered BOECs remained viable. The latter assumption is almost certainly incorrect, which means that the degree of expansion is even greater.…”

Microbubble ultrasound improves the efficiency of gene transduction in skeletal muscle in vivo with reduced tissue damage

“…Skeletal muscle possesses several qualities, such as stability, longevity and susceptibility to transfection, which make it an ideal target tissue for gene therapy, particularly for systemic delivery of proteins as therapeutic reagents such as clotting factors for haemophilia. 1 Successful gene therapy requires delivery systems which are safe, easy to apply and give efficient transgene expression. Viral delivery systems, in particular, have been shown to be efficient for gene transduction in muscle.…”