Improved Methods of Retroviral Vector Transduction and Production for Gene Therapy

Abstract

To facilitate clinical applications of retroviral-mediated human gene transfer, retroviral vectors must be of high titer and free of detectable replication-competent retroviruses. The purpose of this study was to optimize methods of retroviral vector production and transduction. Studies were conducted using 22 retroviral vector producer cell lines. Inactivation of retroviral vectors was greater at 37 degrees C than at 32 degrees C. A 5- to 15-fold increase of vectors was produced at 32 degrees C compared to 37 degrees C; the vector increase at 34 degrees C was intermediate. For example, PA317/G1Na.40 grew to a titer of 1.8 x 10(7) cfu/ml at 32 degrees C, compared to 5.0 x 10(5) cfu/ml at 37 degrees C. The production of retroviral vectors was scalable achieving similar results in flasks, roller bottles, or a CellCube Bioreactor. Retroviral vectors were concentrated 15-24 times with vector recovery ranging from 91 to 96% in a Pellicon tangential flow filtration system. Retroviral supernatants were successfully lyophilized. The combination of glucose or sorbitol with gelatin resulted in recovery rates of 64-83%. In studies on transduction by retroviral vectors, centrifugation of vector supernatants onto target cells significantly increased transduction efficiency as measured by vector titration for G418 resistance, fluorescence-activated cell sorting (FACS), and polymerase chain reaction (PCR) analyses. The combination of the above methods has significantly increased the growth and transduction by this vector system.

Cell Culture Processes for the Production of Viral Vectors for Gene Therapy Purposes

“…A 5-to 15-fold increase of vectors was produced at 32°C compared to 37°C; the vector increase at 34°C was intermediate. The production of retroviral vectors was scalable achieving similar results in flasks, roller bottles, or a CellCube Bioreactor (Kotani et al 1994). This group suggested that the increased titer could be due to (i) reduced metabolic activity/growth of the cells, causing an increase in vector production; or (ii) increased stability of virus particles at lower temperatures.…”

“…In addition to packed bed and fluidized bed bioreactors, fixed bed culture systems, such as the CellCube, have been used for retrovirus production (Kotani et al 1994;Merten et al 2001a;Wikstro¨m et al 2004). The performance of the CellCube is reported to be comparable with the Celligen packed bed reactor, with each systems producing an average of 2.1 · 10 7 and 0.55 · 10 7 IP ml À1 , respectively (Merten 2004).…”

Differences among nonhuman primates in susceptibility to bone marrow progenitor transduction with retrovirus vectors

“…We initially chose to carry out the CD34 transduction cultures at 33°C rather than the typical 37°C based on previous studies in another large animal model, 7 because of the greatly extended half-life of the packaged virus at this cooler temperature, 32 and the demonstrated ability of HSC from several species, 33,34,35 including humans, 36 to survive well at 33°C. In order to determine whether this lower temperature truly offered an advantage, we directly compared the effect of temperature on the CD34 progenitor transduction rates for humans and the four nonhuman primates species under investigation.…”

“…This result is perhaps not surprising given the increased half-life of retrovirus particles at only slightly cooler temperatures. 32 The fact that 33°C is widely used as a temperature for long-term bone marrow cultures in a variety of species further suggests that carry-ing out transduction cultures at this temperature may actually enhance the survival of HSC. [33][34][35][36] Finally, studies in swine 7 and dogs 45 indicate that it is at least possible to transduce long-term engrafting HSC at 33°C.…”

Post-mitotic, differentiated myotubes efficiently produce retroviral vector from hybrid adeno-retrovirus templates

“…32 Furthermore, a number of groups have reported increased titres when incubating retroviral producer cells at lower temperatures or by reducing serum concentration, approaches that undoubtedly slow producer cell division. [32][33][34][35][36] However, the increases in retroviral vector titres using these approaches have previously been attributed to an increase in retrovirus stability at lower temperatures and easier isolation of retrovirus from medium containing lower serum concentrations.…”