Establishment of complement-resistant retroviral vector by homologous restriction factor 20 gene

Abstract

Homologous restriction factor 20 (HRF20, CD59) is one of cessfully selected by complement-dependent selection, the complement regulatory factors. In this study, the which showed significant expression of the HRF20 antigen. complement-resistant retroviral vector, which possessesIn addition, these cells, transduced with tissue plasminthe HRF20 gene as a selection gene, was constructed and ogen activator (tPA) cDNA using complement-resistant examined. The virus-producing cell, transduced with retroviral vector, expressed tPA antigen after complementcomplement-resistant retroviral vector, was established dependent selection. These findings suggest that compafter complement-dependent selection. NIH3T3 and PK15 lement-resistant retroviral vector can be used for the cells transduced with this virus-producing cell were sucdouble transduction of HRF20, as well as other genes.Keywords: complement-dependent selection; gene therapy; retroviral vectorThe technique of gene transfer has been developed over the past decade and enables gene therapy to be applied to patients suffering from inherent metabolic diseases and malignancies. 1,2 The use of retroviral vectors is the most efficient method of gene therapy and their cDNA usually consists of both a therapeutic and a selection gene such as the neomycin-resistance gene or hygromycin-resistance gene. [3][4][5] Using these selection drugs, the retroviral vector-producing cells are selected 10 to 14 days after transduction. Recent investigation has demonstrated the effect of in vivo gene transfer with retroviral vector-producing cells on the treatment of experimental and clinical brain tumors using the herpes simplex virus thymidine kinase (HSVtk) gene, the mechanism of which has been termed the bystander effect. [6][7][8] However, it is difficult to introduce retroviral vector-producing cells in vivo for human gene therapy because of the immunological reaction to vector-producing cells by human complement. It has been reported that the homologous restriction factor 20 (HRF20, CD59) gene, one of the regulators of the complement activation (RCA) molecule, can be used for rapid selection of transduced cells. 9 Due to the transduction of the RCA molecule, xenogeneic cells are resistant to human complement attack. [10][11][12] To insert the HRF20 gene, as well as other therapeutic genes into vector-producing cells, the authors established a novel complement-resistant retroviral vector, which possesses the HRF20 gene as a selection gene.Complement-resistant retroviral vector (LRHL) was constructed by replacing the neomycin resistance gene (Neo R ) of the LRNL retroviral vector with the HRF20 (Figure 1). To produce virus-producing cells, LRHL retroviral vector was transduced into ⌿2 ecotropic packaging cells, and then the supernatant was poured into PA317 amphotropic packaging cells. After these cells were selected using complement-dependent selection, six colonies were grown. The expression of the HRF20 antigen on these cells was shown to be strongly positive, the mean channel shift ...

Feasibility of Double-Expression Retroviral Vector Using Complement Regulatory Factor Gene

“…This fragment was ligated into the EcoRI site of pSP72 vector (Promega Corp., Madison, WI). HRF20 cDNA with HindIII/ClaI sites, digested from this vector, was ligated into LR(N)L vector, the NeoR cDNA of which was removed by partial digestion using both HindIII and ClaI [9]. Then DAF cDNA and tissue plasminogen activator (tPA) cDNA were ligated to LRHL vector (LDAFRHL and LtPARHL) using a DNA ligation kit (Takara Biologicals) with SalI linker and BamHI linker, respectively [10,11].…”

Complement and anti-α-galactosyl natural antibody-mediated inactivation of murine retrovirus occurs in adult serum but not in umbilical cord serum

“…The problem of retrovirus vector and or packaging cell line inactivation by human serum has been approached in various ways. These include development of complement-resistant vectors, [10][11][12] serum, 14 animal serum, or even in serum-free medium. 15 Most of these newer vectors and packaging cell lines are not yet well characterized for human use.…”