Expression of human factor IX in mice after injection of genetically modified myoblasts.

Abstract

Hemophilia B is an X chromosome-linked recessive bleeding disorder. To develop a somatic gene therapy for this disease, we have examined whether mouse skeletal myoblasts can serve as efficient vehicles for systemic delivery of recombinant factor IX. When mouse myoblasts (C2C12) transduced with a Moloney murine leukemia virus-based vector containing the bacterial f-galactosidase gene were i 'ected into mouse skeletal muscles, they fused with the existing and regenerating myofibers and continued to express P-galactosidase. C2C12 myoblasts that were infected with recombinant retroviruses containing a human factor Ia cDNA secreted biologically active human factor IX into the culture medium at a rate of 2.6 zg per 10' cells per day. Myotubes derived from these cells in culture continued to express human factor IX (0.68 itg/day from myotubes derived from 10' C2C12 cells).After injection of the transduced C2C12 myoblasts into skeletal muscles of mice, the systemic level of recombinant human factor IX was found to be as high as -1 ,ug/ml of serum. These results provide the rationale for using skeletal myoblasts as an efficient gene delivery vehicle in the somatic gene therapy for hemophilia B.Factor IX is a plasma glycoprotein that plays a pivotal role in the middle phase of the blood coagulation cascade (1). It is normally synthesized in liver and secreted into the circulation. A deficiency of biologically active factor IX in circulation results in an X chromosome-linked recessive bleeding disorder, hemophilia B (Christmas disease). The current treatment for this disease by plasma protein replacement therapy is effective but complicated by serious side effects, such as possible exposure of patients to blood-borne pathogenic viruses, including hepatitis and human immunodeficiency viruses. Somatic cell gene therapy may provide an alternative safe treatment for this disorder (2). In such an approach, the normal factor IX gene is transferred into target somatic cells that can stably produce active factor IX and transport it into the circulation. The somatic cells used must be able to efficiently carry out various post-translational modifications, such as y-carboxylation, required for the biological activity of factor IX (1). Genetically modified skin fibroblasts implanted in mice as in dermis or subcutaneous implants can produce and secrete recombinant factor IX into the circulation (3, 4). This approach, however, has suffered from poor stability of expression (5) and inefficient transportation of recombinant proteins into the circulation (4).Skeletal myoblasts have several unique properties that make them attractive for use in somatic gene therapy. Proliferating myoblasts are readily isolated and cultured in large numbers (6, 7). More importantly, these cells can fuse with existing muscle fibers when injected into muscle tissues (8).In this report, we demonstrate the expression of the recombinant genes for human factor IX and f3-galactosidase by injecting genetically modified myoblasts into mouse skeletal muscles....

Direct intramuscular injection with recombinant AAV vectors results in sustained expression in a dog model of hemophilia

“…Most muscledirected approaches for factor IX expression have involved the ex vivo transduction of myoblasts, which can be re-implanted and result in secretion of factor IX into the circulation. 5,6,17 These studies have gone far to establish that both myoblasts and mature myotubes are capable of the synthesis and required post-translational modifications of factor IX protein. In addition to ex vivo approaches, secretion of circulating proteins following direct muscle injection has also been demonstrated.…”

“…The ability of myoblasts, in particular, not only to transcribe the factor IX transgene but also to perform the series of post-translational modifications required for functional activity has been established. 5,6 Genetically modified muscle cells are capable of producing a range of secreted proteins, including human growth hormone, erythropoietin, alpha-1-antitrypsin, and interleukin-10. [7][8][9][10] Recently, Xiao et al 3 demonstrated in vivo that mouse skeletal muscle, transduced with an E. coli ␤-galactosidase gene in a recombinant AAV (rAAV) vector, expressed the transgene for greater than 1.5 years in immunocompetent animals; Southern analysis of genomic DNA from transduced muscle suggested the expression persisted via high molecular weight viral genomes.…”

Treatment of severe hypercholesterolemia in apolipoprotein E-deficient mice by intramuscular injection of plasmid DNA

“…35 Retroviral vectors require dividing cells for nuclear entry, 36 thus cannot be used for efficient gene delivery to uninjured muscle. 37,38 Retroviral vectors have been used ex vivo to genetically modify dividing myoblasts, 39 followed by surgical implantation; however, the requirement for substantial tissue-culture manipulation and surgical expertise limit their ordinary use in man. Therefore, the development of a simple, safe and effective method of gene delivery to muscle resulting in long-term expression would be desirable.…”

Persistent expression of canine factor IX in hemophilia B canines

“…13,16 Skeletal muscle is capable of performing the necessary post-translational modifications required for functional FIX protein. 17 rAAV-2 targeting of skeletal muscle produces human FIX circulating levels up to 7% of normal human levels in immunodeficient mice. 18 The Chapel Hill strain hemophilia B canine is a well characterized animal model, 19,20 which has been used extensively for preclinical testing of anti-hemophilic products.…”