A Novel Human Amphotropic Packaging Cell Line: High Titer, Complement Resistance, and Improved Safety

Abstract

Successful retroviral-mediated gene therapy will depend on safe, efficient packaging cell lines for vector particle production. Existing packaging lines for murine leukemia virus (MLV)-based vectors are predominantly derived from NIH/3T3 cells which carry endogenous MLV sequences that could participate in recombination to form replication-competent retrovirus (RCR). To identify cells devoid of such sequences, we screened genomic DNA from eight cell lines. DNA from the human 293 cell line did not cross-hybridize with MLV sequences, and these cells were able to secrete Gag particles after transfection. We derived a stable amphotropic packaging cell line (called ProPak-A) in 293 cells in which the Gag-Pol and Env (packaging) functions are expressed separately from a heterologous (non-MLV) promoter, to maximally reduce homology between packaging and vector sequences. ProPak-A-based producer cells are efficient, yielding higher stable titers than PA317-based producers. In addition, a vector that consistently gave rise to RCR in PA317 cells never resulted in detectable RCR in ProPak-A-based producer cultures. We have also shown that ProPak-A-packaged particles are not inactivated by human serum. Thus, the packaging cells we describe are as efficient and safer than the amphotropic packaging cells most commonly used in clinical gene therapy work and are also more appropriate for in vivo gene delivery.

Complement regulatory proteins are incorporated into lentiviral vectors and protect particles against complement inactivation

“…7,8 For instance, retroviral vectors produced in murine and canine producer cells are sensitive to inactivation by human complement. [9][10][11] The primary trigger of complement attack in these instances is human antibody recognition of the a-1,3-galactosyl (a-Gal) epitope, which is presented on cells from lower mammals but not humans or Old World primates. 7,8,12 An example of an envelope protein that induces complement attack is VSV-G. VSV-G-pseudotyped viral vectors, regardless of producer cell type or vector type, will be inactivated by human serum complement, while vectors pseudotyped with other envelope glycoproteins such as the MLV amphotropic envelope (Ampho) or RD114 are resistant to inactivation.…”

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

“…Furthermore, murine retroviral sequences that are present in murine packaging cells can be selectively packaged into retroviral particles (Torrent et al 1994) increasing the possibility of generating RCRs. 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).…”

“…This gives them a significant advantage over transient transfection or infection systems, such as baculovirus or adenovirus systems, where generation of large volumes of characterized supernatants is difficult (Rigg et al 1996).…”

Transgene amplification and persistence after delivery of retroviral vector and packaging functions with E1/E4-deleted adenoviruses

“…Such features are commonly used to construct safe and efficient retroviral packaging cell lines. 19,20 It is generally assumed that recombinant adenoviruses resulting in viral DNA less than a net genome size of 105% of that of the wild-type Ad5 genome length are genetically stable and grow to normal titers. 21 Transfection of the pAdGAG/POL and pAdTK/ENV backbones in IGRP2 generated the corresponding AdGAG/POL and AdTK/ENV adenoviruses.…”