Amphotropic retrovirus transduction is inhibited by high doses of particle‐associated envelope proteins

Abstract

Using a panel of amphotropic murine leukemia virus packaging cell lines that differed only in their levels of envelope protein (gp70) expression, we examined the relationship between transduction and the number of envelope proteins per virus. We generated virus stocks that contained different levels of virus-associated envelope proteins, purified them from gp70 that was not associated with the viruses, quantified their titers, and measured the efficiency with which they transduced NIH 3T3, TE671, and HeLa cells. As expected, titers increased monotonically with viral envelope protein number. Titers are measured using highly dilute virus, however, and are often not predictive of gene transfer when high doses of virus are used, as is done in gene therapy protocols. Interestingly, when we used high doses of virus, we observed significantly different trends: gene transfer increased, reached a maximum, and then declined sharply as the number of envelope proteins per virus increased. The highest levels of gene transfer occurred when cells were transduced with a moderate dose of virus that contained low levels of envelope protein. Our results indicate that transduction is inhibited when viruses that contain large numbers of envelope proteins are used. This is most likely because each virus, when it binds to a cell, delivers a large payload of envelope proteins that occupy or inactivate multiple virus receptors, reducing or eliminating the susceptibility of the cell to being transduced by additional viruses. The implications of our findings for the design of improved retroviral vectors for human gene therapy are discussed.

Removal of envelope protein‐free retroviral vectors by anion‐exchange chromatography to improve product quality

“…Consequently, retroviral supernatants have a large proportion of noninfectious vectors, the ratio of total to IPs usually being above 100:1 [7,8]. The presence of noninfectious vectors at such high levels may result in transduction efficiency inhibition, due to interference with viral receptors [9], and also constitute an extra bio-burden that might produce some immunological response in the patients [7, 10 -12]. Thus, the removal of noninfectious vectors is desirable to increase the quality of the final RV product.…”

Improved Coinfection with Amphotropic Pseudotyped Retroviral Vectors

“…A convenient and efficient method for concentrating amphotropic retroviral vectors was introduced recently; this comprises flocculation of the vector with high concentrations of polymers such as Polybrene followed by a brief centrifugation [ 10 , 11 ]. This process also separates active viral particles from inactive particles that can compete for receptors on target cells [ 12 ]. Polymers, usually Polybrene, are also used in standard infection protocols to increase attachment of viral vectors to target cells [ 13 ].…”