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....

In aiming to develop a gene therapy approach for hemophilia B, we expressed and characterized human factor IX in rat capillary endothelial cells (CECs). Moloney murine leukemia virus-derived retrovirus vectors that contain human factor IX cDNA linked to heterologous promoters and the neomycin-resistant gene were constructed and employed to prepare recombinant retroviruses. Rat CECs and NIH 3T3 cells infected with these viruses were selected with the neomycin analogue, G418 sulfate, and tested for expression of factor IX. A construct with the factor IX cDNA under direct control by long terminal repeat gave the highest level of expression (0.84 and 3.6 pg per 106 cells per day for CECs and NIH 3T3 cells, respectively) as quantitated by immunoassays as well as clotting activity assays. A single RNA transcript of 4.4 kilobases predicted by the construct and a recombinant factor IX of 68 kilodaltons identical to purified plasma factor IX were found. The recombinant human factor IX produced showed full clotting activity, demonstrating that CECs have an efficient mechanism for posttranslational modifications, including y-carboxylation, essential for its biological activity. These results, in addition to other properties of the endothelium, including large number ofcells, accessibility, and direct contact with the circulating blood, suggest that CECs can serve as an efficient drug delivery vehicle producing factor IX in a somatic gene therapy for hemophilia B.

Retroviral-mediated transfer of new genetic information into keratinocytes is a key step in epidermal gene therapy. An obstacle to the use of retroviruses for gene therapy is that although high levels of expression of the transduced gene can be maintained in tissue culture, expression is often lost when the cells are transplanted to an animal host. To examine some of the factors involved in this instability of expression, we transduced keratinocytes with a retrovirus encoding the gene for human factor IX and monitored secretion of the transduced gene. We observed continued secretion of factor IX through five passages in culture. When, however, sheets of these cells were grafted to athymic mice, factor IX expression was reduced or lost within 6 wk. We show that the reduction of factor IX expression in grafted keratinocytes did not result from a loss of grafted cells, nor was there a block to systemic delivery of a secreted endogenous product.

SummaryA mouse factor IX cDNA was isolated and characterrzed. The cDNA was 1,837 bp in length and contained the coding region as well as short 5’ and 3’ untranslated sequences. Northern blot analysis of liver RNA showed two mRNA species of 3.2 kb (major) and 2.2 kb (minor) for the mouse factor IX. An antisgnse RNA probe prepared from the mouse cDNA was employed to determine the steady state level of factor IX mRNA in mouse liver at various developmental stages. The factor IX mRNA level was very low (2–5% of the adult level) during the gestational period until day –3 (gestational day 17) followed by a rapid increase at day –2 through birth. This phase of rapid increase was followed by a gradual increase before it reached the adult level at around 20 to 24 days. At birth, the factor IX mRNA level was found to be at about 43% of that of the adult. The rnRNA levels in mouse liver agreed well with the plasma factor IX activity levels. These results indicate that reduced factor IX activity in newborns is due to the low levels of factor IX mRNA available for translation.

Recombinant adenovirus has recently become a promising gene delivery vehicle that may be used therapeutically for various medical disorders. However, in vivo expression of transgenes delivered by E1 region-deleted adenoviral vectors is transient in immunocompetent animals. It has been proposed that destruction of adenovirally transduced cells by the host immune mechanisms, particularly cytotoxic T-lymphocytes, may play a major role in limiting the duration of transgene expression in vivo. In the present study, Southern blot analysis of genomic DNA prepared from transduced liver tissues showed the persistent presence of the viral genome in both immunocompetent and immunodeficient animals, indicating the survival of the adenovirally transduced liver cells. Furthermore, active expression of the surviving factor IX transgenes was shown by the presence of recombinant human factor IX as well as specific human factor IX mRNA and protein in the transduced liver tissues. The transient appearance of human factor IX in the circulation of normal as well as partially immunodeficient mice is primarily due to the generation of mouse antihuman factor IX antibodies in these mice rather than host immune destruction of transduced cells. These results suggest that liver cells transduced with recombinant adenoviral vectors can escape from being destroyed by the host immune mechanism in normal animals. The present study thus provides a new rationale for further engineering of adenoviral vectors into a durable expression system for gene therapy of various diseases including congenital disorders such as hemophilia B.