Small Increases in pH Enhance Retroviral Vector Transduction Efficiency of NIH‐3T3 Cells

Abstract

Increases in pH between 7.1 and 7.7 increase the efficiency of polybrene (Pb)- and protamine sulfate (PS)-aided retroviral transduction of NIH-3T3 cells in a serum-lot-dependent manner. The increase in Pb-aided transduction efficiency at pH 7.7, relative to the value at pH 7.33, ranged from 13% to 49% for three serum lots. For a constant Moloney murine leukemia virus (MMLV) vector dilution at pH 7.33, three different serum lots resulted in absolute transduction efficiencies ranging from 29% to 53% using Pb. At the same vector dilution, PS-aided transduction was less effective on an absolute basis than Pb-aided transduction, but the benefit of elevated pH was more pronounced with PS. There was a similar enhancement with PS at elevated pH for a murine stem cell virus (MSCV) vector as for the MMLV vector. The benefit at pH 7.7 for PS-aided transduction was partially due to greater PS stability at elevated pH. Heat inactivating the serum supplement or adding protease inhibitors helped to stabilize PS. This increased the absolute transduction efficiency but decreased the relative benefit of elevated pH to a level similar to that for Pb-aided transduction. Incubating Pb with the vector at pH 7.1 for 10 min, prior to readjusting to pH 7.7 and transducing the cells, was sufficient to abrogate the beneficial effects of transduction at pH 7.7. In contrast, prior exposure of PS with vector at pH 7.1 did not affect subsequent transduction at pH 7.7. These results indicate that pH is an important variable in retroviral transduction and that the relative benefits of Pb or PS on retroviral vector transduction will vary with the pH, polymer addition method, and serum lot.

Effect of Fetal Bovine Serum on Foamy and Lentiviral Vector Production

“…/hgtb.2013 In summary, we have shown that there can be significant variability between different FBS production lots in the production of viral vectors. Our data are consistent with prior studies with lentiviral vectors (Denning et al, 2013) as well as MMLV and MSCV vectors ( Jensen et al, 2003). Our data also indicate that when serum will be used for the production of more than one type of retroviral vector, it is prudent to test the production of all vectors.…”

“…Although there has been a movement toward serum-free medium for the production of viral vectors (Falkner et al, 2006), FBS is still commonly used for the production of preclinical and clinical viral vector preparations (DiGiusto et al, 2010). The importance of macromolecules, specifically lipids such as cholesterol, to obtain high-quality vector preparations has been described for the production of MLV vectors (Rodrigues et al, 2009) and pH may also play a role ( Jensen et al, 2003). The presence of antiviral antibodies such as bovine foamy virus (BFV) antibodies may have also affected the resulting foamy viral titers.…”

“…Although it is generally accepted that the quality of serum lots varies and that testing serum lots for specific applications is beneficial, the comparison of different serum lots for the production of lentiviral and foamy viral vector preparations has not to our knowledge been previously reported. Previous studies have assessed the effect of different serum samples, including the effect of heat inactivation and polycations on transduction efficiency with lentiviral vectors (Denning et al, 2013), as well as the effect of the pH of serum samples for Moloney murine leukemia virus (MMLV) and murine stem cell virus (MSCV) vector transduction efficiency ( Jensen et al, 2003).…”

Optimization of the Transductional Efficiency of Lentiviral Vectors: Effect of Sera and Polycations

“…However, as specified in the protocol of the manufacturer of pLenti vectors (Invitrogen), polybrene may be toxic to primary cell cultures such as primary neurons and therefore should be carefully titrated [29]. Since a previously published study suggested that small variances in the pH of transduction media may significantly influence transduction efficiency [16], transduction culture mediums with pH ranging from 7.0 to 7.6 were tested. Optimal transduction of DEAE-dextran-containing cultures typically occurred at pH 7.0 – 7.2 (Figure 2D); however, the effect of pH appeared to be minor.…”

“…However, several recent studies have demonstrated that the initial step of virus binding does not involve specific envelope-receptor interactions but rather receptor-independent binding events [14, 15]. The efficiency of this initial event and, consequently, lentiviral transduction is diminished by strong electrostatic repulsion between the negatively charged cell and an approaching enveloped virus [16, 17]. Methods designed to overcome this problem include centrifugation of targets cells with virus at low speeds, co-localization of cells and virus on immobilized proteins, and employing multiple rounds of transduction [17, 18].…”