Moloney murine leukemia virus-derived retroviral vectors decay intracellularly with a half-life in the range of 5.5 to 7.5 hours

Abstract

Replication-incompetent recombinant retroviruses are currently used for gene delivery. The limited efficiency of gene transfer using these vectors hampers implementation of gene therapy. Successful integration of Moloney murine leukemia virus (MMuLV)-derived retroviral vectors into the host cell DNA requires cell division. The time difference between virus entry and cell division is variable and prolonged in slowly dividing cells. Therefore, the rate of intracellular decay of internalized vectors between the time of entry into the target cell and cell division may limit the probability of successful integration following viral entry. We present two methods that measure the intracellular stability of MMuLV-derived retroviral vectors in NIH 3T3 cells. The first is based on a temporary interruption of cell cycle progression by using cell detachment. This method provides an estimate, but not a direct measurement, of the half-life. The results show that the MMuLV intracellular half-life is on the order of but shorter than the total cell cycle time. The second method allows the direct measurement of the intracellular half-life by using two cell cycle-specific labels: 5-bromodeoxyuridine, a thymidine analog that labels cells in S-phase; and the viral vector that labels cells in mitosis. By varying the time between the administration of the two labels, the intracellular half-life is measured to be in the range of 5.5 to 7.5 h. Such a short intracellular half-life may restrict the efficiency of gene transfer by retroviral vectors, particularly in slowly dividing target cells.

Relationship between cell proliferation, cell‐cycle phase, and retroviral vector production in FLYRD18 human packaging cells

“…Figure 3B shows that the cell-cycle distribution did not change significantly for the first 8 hours after inoculation. During the first 4 hours, a small increase in the percentage of S-phase and a small decrease in the percentage of G 1 -phase cells was observed, i.e., the cells did not arrest and accumulate in G 1 for the first 6 hours of culture, as observed by Andreadis et al (1997). Here, we show no such perturbation of cell-cycle progression in the FLYRD18/LNC-hB7 cell line as a result of cell attachment.…”

“…Therefore, to measure virus titer produced in a particular cell-cycle phase, samples from separated cell fractions need to be taken within this time period. However, a number of groups have reported temporary inhibition of cell-cycle progression (up to 6 h) following the detachment of some cell types from the culture substratum (Andreadis et al, 1997;Campisi and Medrano, 1983). As mentioned above, if cell-cycle progression is essential for virus production, the cells need to be cycling immediately after inoculation to compare virus levels produced during the first few hours.…”

“…A wealth of literature exists concerning the determination of target cell cycle effects on infection by retroviral particles (Andreadis et al, 1988;Fritsch and Temin, 1977;Harel et al, 1981;Sladek and Jacobberger, 1998;Springett et al, 1989). Studies have indicated that target cells must be undergoing mitosis to allow successful infection by most retroviruses (Andreadis et al, 1997;Lewis and Emerman, 1994;Miller et al, 1990;Roe et al, 1993), excluding len-tiviruses (Lewis et al, 1994). However, the effect of cell cycle of the packaging cells used for retroviral vector production has received no attention to date.…”

Mathematical model of the rate‐limiting steps for retrovirus‐mediated gene transfer into mammalian cells

“…However, we cannot rule out the possibility that the intracellular degradation rates in actively replicating cells might be different than in stationary cells. Andreadis et al (1997) developed a method to quantify the intracellular stability of retroviral vectors in actively replicating cells without synchronization and estimated the intracellular half-life of MoMLV vectors inside NIH-3T3 cells to be 6.5 h. This method may be useful to better quantify the intracellular half-life of our retroviral vectors, since the serum starvation experiment indicated that intracellular degradation was non-negligible.…”

“…A sensitivity analysis of the model was performed by artificially raising by 10% each of the parameter values shown in Table I while keeping all of the other parameter values constant. Two different intracellular degradation rates were investigated: k di ¼ 0.0012 h À1 (Table I) (Andreadis et al, 1997) where intracellular degradation is increased, such as by the presence of host cell restriction factors. The results are shown in Figure 8.…”