Generation of Retroviral Packaging and Producer Cell Lines for Large-Scale Vector Production and Clinical Application: Improved Safety and High Titer

Abstract

For many applications, human clinical therapies using retroviral vectors still require many technological improvements in key areas of vector design and production. These improvements include higher unprocessed manufacturing titers, complement-resistant vectors, and minimized potential to generate replication-competent retrovirus (RCR). To address these issues, we have developed a panel of human packaging cell lines (PCLs) with reduced homology between retroviral vector and packaging components. These reduced-homology PCLs allowed for the use of a novel high multiplicity of transduction ("high m.o. t.") method to introduce multiple copies of provector within vector-producing cell lines (VPCLs), resulting in high-titer vector without the generation of RCR. In a distinct approach to increase vector yields, we integrated manufacturing parameters into screening strategies and clone selection for large-scale vector production. Collectively, these improvements have resulted in the development of diverse VPCLs with unprocessed titers exceeding 2 x 10(7) CFU/ml. Using this technology, human Factor VIII VPCLs yielding titers as high as 2 x 10(8) CFU/ml unprocessed supernatant were generated. These cell lines produce complement-resistant vector particles (N. J. DePolo et al., J. Virol. 73: 6708-6714, 1999) and provide the basis for an ongoing Factor VIII gene therapy clinical trial.

Translating the combination of gene therapy and tissue engineering for treating recessive dystrophic epidermolysis bullosa

“…Compared to the LAMB3 cDNA (3.6 kb) (Mavilio et al, 2006), the large size of the COL7A1 cDNA (8.9 kb) has limited the generation of high-titer retroviral vectors from stable retrovirus-producer cells (Chen et al, 2002;Goto et al, 2006;Siprashvili et al, 2010). The use of high-titer retroviral packaging cell lines (293Vec cells), constructed in our laboratory, could circumvent the titer issue.…”

Direct evidence for the absence of intercellular trafficking of VP22 fused to GFP or to the herpes simplex virus thymidine kinase

“…1,11 We could assume that it should be possible to increase the efficiency of gene transfer by using the latest generation of packaging cell lines that produce high-titer recombinant retroviruses. 12,13 Another means to render the TK/GCV strategy more potent would be to increase the BE in order to kill more untransduced cancer cells. This goal can be achieved by combining the GCV treatment with other therapeutic molecules that would act in a synergistic or additive manner.…”

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

“…Thus, the use of human cell lines for the establishment of packaging cells is a step towards increased biological safety, because they lack endogenous murine retroviruses (Rigg et al 1996;Pensiero et al 1996;Forestell et al 1997). In fact, viral supernatants or producer cells derived from human cells have never given a positive test result for RCRs in small-or medium-scale assays (Sheridan et al 2000). A further advantage of using human based packaging cell lines is the fact that the glycosylation of the env protein is of a human type.…”