ABSTRACT
The successful prophylactic treatment of hemophilia A by frequent infusions of plasma concentrates or recombinant factor VIII (hFVIII) indicates that gene therapy may be a potential alternative for the treatment of the disease. For efficient delivery and long-term expression of the hFVIII gene, a novel minimal adenovirus (mini-Ad) vector, MiniAdFVIII, has been developed. The vector is devoid of all viral genes and carries the full-length hFVIII cDNA under the control of the human 12.5-kb albumin promoter. The MiniAdFVIII vector was propagated with the assistance of an ancillary vector in 293 cells and was purified by CsCl banding. Sustained expression of hFVIII at physiologic levels (100-800 ng/mL) was achieved in mice after a single intravenous injection of MiniAdFVIII. The expressed hFVIII had a structure identical to that of recombinant hFVIII, as determined by Western blot analysis. The functionality of the protein was confirmed by the restoration of blood coagulation capacity in MiniAdFVIII-treated hemophilic mice, as determined by tail clipping observations. Although antivector or antihuman FVIII antibodies at various levels were detected, long-term expression of the transgene was observed in the mice that did not generate antibodies against the transgene product. The vector DNA persisted in the liver tissues of the mice with long-term expression. No significant histopathologic findings or toxicities were observed to be associated with the vector in the MiniAdFVIII-treated C57BL/6 mice. These results support the further development of MiniAdFVIII for clinical trials toward the treatment of hemophilia A.
Subject(s)
Adenoviridae/genetics , Factor VIII/genetics , Genetic Therapy , Genetic Vectors/genetics , Hemophilia A/therapy , Albumins/genetics , Animals , Antibodies, Heterophile/biosynthesis , DNA, Complementary/genetics , Factor VIII/biosynthesis , Factor VIII/immunology , Gene Expression , Genes, Synthetic , Genetic Vectors/pharmacokinetics , Hemophilia A/genetics , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Promoter Regions, Genetic , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Safety , Tissue Distribution , Tumor Cells, CulturedABSTRACT
To achieve efficient delivery and sustained expression of the human factor VIII cDNA in vivo, a minimal-adenoviral (mini-Ad) vector system was developed. The system is composed of a mini-Ad vector with essential cis-elements (less than 1 kb) of the viral genome, an E1-deleted ancillary Ad with packaging attenuation, and an E1-complementing production cell line. Based on this system, MiniAdFVIII was generated to deliver a 27 kb expression cassette consisting of a full-length human factor VIII cDNA flanked by human albumin promoter and genomic sequences. The MiniAdFVIII vector mediated expression of functional human factor VIII in HepG2 and 293 cells. A single-dose intravenous injection of 10(11) viral particles in hemophilic mice of MiniAdFVIII produced a sustained high-level expression of human factor VIII (at 100-800 ng/ml up to 369 days) which corrected the FVIII-deficient phenotype. Safety studies of MiniAdFVIII showed that there were no significant toxic effects in mice and dogs after single intravessel doses of up to 3 x 10(11) and 6 x 10(12) viral particles, respectively. Studies for developing the MiniAdFVIII vector with a site-specific integration mechanism and progress in the development of a human factor VIII-tolerized mouse model for pre-clinical studies of MiniAdFVIII are reported. Further pre-clinical studies and product development of MiniAdFVIII for clinical trials are also discussed.
Subject(s)
Adenoviridae/genetics , Factor VIII/genetics , Gene Transfer Techniques , Genetic Therapy , Genetic Vectors , Hemophilia A/therapy , Animals , Disease Models, Animal , Dogs , Humans , MiceABSTRACT
Theca cells have been shown to secrete transforming growth factor beta (TGF beta), but little is known regarding the regulation of thecal TGF beta secretion. To investigate the regulation of thecal TGF beta secretion and activation, rat theca-interstitial cells (TIC) isolated by Percoll gradient centrifugation were cultured with LH (0.01-10 ng/ml), androstenedione, androsterone, testosterone, or 5 alpha-dihydrotestosterone (DHT) (all 1 x 10(-9)-1 x 10(-5) M), estradiol (E2), estrone (both 1 x 10(-11)-1 x 10(-7) M), or IGF-I (30 ng/ml). Active TGF beta and total TGF beta in the conditioned medium were measured by bioassay. TIC spontaneously produced TGF beta, of which approximately 45% was in the active form. LH inhibited total TGF beta secretion (45% at 0.1 ng/ml of LH) and active TGF beta concentrations (40% at 0.3 ng/ml of LH). IGF-I inhibited active but not total TGF beta. Addition of LH did not cause any additional change in active or total TGF beta. Neither androstenedione, androsterone, testosterone, nor DHT had any effect on either active or total TGF beta secretion in the presence or absence of LH. In contrast, E2 inhibited both active (57%) and total (37%) TGF beta secretion. In the presence of LH, no additional effect of E2 was observed. ICI 182,780, a pure estrogen antagonist, reversed the E2 inhibition, suggesting that the E2 effect is mediated by estrogen receptors. We next investigated the role of E2 on TGF beta production by granulosa cells (GC).(ABSTRACT TRUNCATED AT 250 WORDS)
