Mammalian C-type retroviruses are inactivated by human serum, following triggering of the classical complement cascade. This may have inhibited transmission to humans of C-type oncoviruses from other mammals. Indeed, the retroviruses human immunodeficiency virus and human T-cell leukaemia virus are resistant to human complement. Antibody-independent activation of human C1q, the first component of the classical pathway, by retroviral envelope proteins has been described. However, retroviruses produced from human cells are resistant to inactivation by human complement and human serum is known to contain antibodies directed against carbohydrates on retroviral envelopes. Gal(alpha 1-3)Gal terminal carbohydrates are expressed by most mammals but are absent in humans, which lack a functional (alpha 1-3)galactosyltransferase gene. Here, we demonstrate that anti-Gal(alpha 1-3)Gal antibodies in human serum inactivate retroviruses produced from animal cells. Expression of porcine (alpha 1-3)galactosyltransferase in human cells renders the cells and the retroviruses they produce sensitive to human serum.
The relative efficiency of transduction of gene therapy target cells was measured for retroviruses bearing the envelopes of amphotropic murine leukemia virus (MLV-A), xenotropic murine leukemia virus (MLV-X), gibbon ape leukemia virus (GALV), feline leukemia virus subgroup B (FeLV-B), and the feline endogenous virus RD114. These viruses use various cell-surface receptors. Activated peripheral blood lymphocytes (PBL) and primary melanoma cultures were infected relatively poorly by MLV-X pseudotypes. RD114 pseudotypes infected PBL relatively well, whereas bone marrow progenitor cells were efficiently infected by all viruses. Helper-free virus bearing the envelopes of MLV-A, RD114, or GALV was similarly tested. All infected melanoma or bone marrow progenitor cells efficiently, whereas MLV-A was relatively inefficient for infection of PBL. The general utility of RD114 pseudotyped virus for gene delivery coupled with its resistance to inactivation by human serum makes this envelope the most suitable choice for in vivo gene therapy.
Cationic liposomes enhanced the rate of transduction of target cells with retroviral vectors. The greatest effect was seen with the formulation DC-Chol/DOPE, which gave a 20-fold increase in initial transduction rate. This allowed an efficiency of transduction after brief exposure of target cells to virus plus liposome that could be achieved only after extensive exposure to virus alone. Enhancement with DC-Chol/DOPE was optimal when stable virion-liposome complexes were preformed. The transduction rate for complexed virus, as for virus used alone or with the polycation Polybrene, showed first-order dependence on virus concentration. Cationic liposomes, but not Polybrene, were able to mediate envelope-independent transduction, but optimal efficiency required envelope-receptor interaction. When virus complexed with DC-Chol/DOPE was used to transduce human mesothelioma xenografts, transduction was enhanced four- to fivefold compared to that for virus alone. Since the efficacy of gene therapy is dependent on the number of cells modified, which is in turn dependent upon the balance between transduction and biological clearance of the vector, the ability of cationic liposomes to form stable complexes with retroviral vectors and enhance their rate of infection is likely to be important for in vivo application.
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