pa317 Retrovirus Packaging Cells
Authors: A MILLER
Journal: Molecular Therapy (2002)

Designed in 1985 to produce retroviral vectors for use in humans, PA317 cells [1Miller A.D. Buttimore C. Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production.Mol. Cell. Biol. 1986; 6: 2895-2902Crossref PubMed Scopus (1145) Google Scholar] were used in the first approved gene transfer and gene therapy trials starting in 1989 [2Rosenberg S.A. Gene transfer into humans—immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction.N. Engl. J. Med. 1990; 323: 570-578Crossref PubMed Scopus (1082) Google Scholar, 3Blaese R.M. T lymphocyte-directed gene therapy for ADA-SCID: initial trial results after 4 years.Science. 1995; 270: 475-480Crossref PubMed Scopus (1202) Google Scholar] and are still being used in clinical trials today. Although no longer considered state-of-the-art, PA317 cells and vectors produced from these cells have been the most thoroughly tested of any retroviral vector system in human clinical trials, and have exhibited an outstanding safety record. Development of retroviral vectors for laboratory gene transfer and for human gene therapy applications was stimulated by an appreciation of several key features of retrovirus biology. First, retroviruses integrate into the host cell genome in a precise manner with respect to the viral genome such that the viral genetic information is preserved. This contrasts with DNA transfection, for example, during which DNA is integrated but in a haphazard manner and with unpredictable copy number. Second, the acutely transforming, replication-incompetent retroviruses provided naturally occurring examples of viral gene transfer vectors. These viruses acquire one or more oncogenic genes from the host organism and replicate in the presence of “helper” virus, a replication-competent virus that provides all the viral functions necessary for transmission of the replication-defective oncogenic virus. Indeed, some oncogenic viruses of this type encode only the oncogenic protein in the absence of any viral proteins, showing that all required viral proteins can be supplied in trans by another retrovirus. It was a simple step to propose replacement of the oncogenes in such viruses with other genes of interest. Together these observations led to the development of retrovirus vectors as a high-fidelity system for transfer of any gene of interest. The last step in the process was the development of retrovirus packaging cell lines that supplied all of the virus proteins in trans, obviating the need for helper virus and resulting in vectors that could transfer a gene into a target cell without further replication or production of viral proteins. This last step was critical for the development of vectors for use in humans, and lack of helper virus production is a primary criterion by which packaging cell lines are judged. Early retrovirus packaging cells were engineered by using helper viruses (Fig. 1A) that were rendered replication-incompetent by removal of sequences between the 5′-long terminal repeat (LTR) and the gag gene–called ψ sequences–that are required for packaging of the viral RNA into virions (Fig. 1B) [4Mann R. Mulligan R.C. Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus.Cell. 1983; 33: 153-159Abstract Full Text PDF PubMed Scopus (1117) Google Scholar, 5Cone R.D. Mulligan R.C. High-efficiency gene transfer into mammalian cells: generation of helper-free recombinant retrovirus with broad mammalian host range.Proc. Natl. Acad. Sci. USA. 1984; 81: 6349-6353Crossref PubMed Scopus (252) Google Scholar, 6Miller A.D. Law M.F. Verma I.M. Generation of helper-free amphotropic retroviruses that transduce a dominant-acting, methotrexate-resistant dihydrofolate reductase gene.Mol. Cell. Biol. 1985; 5: 431-437Crossref PubMed Scopus (217) Google Scholar]. Although the removal of the ψ sequences was designed to prevent packaging and transmission of the helper virus, this strategy was not completely successful, resulting in the generation of new “packaging cells” at low frequency in the cell population that was the target of viral gene transfer [1Miller A.D. Buttimore C. Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production.Mol. Cell. Biol. 1986; 6: 2895-2902Crossref PubMed Scopus (1145) Google Scholar]. More troubling was the problem of recombination between the deleted helper viral genome and the retroviral vectors that were introduced into the packaging cells (Fig. 1B). Recombination resulted in the production of replication-competent helper virus, an unacceptable passenger in vector preparations for human use. This problem became particularly bothersome when it was discovered that the packaging signal in the Moloney murine leukemia virus (MoMLV) actually extended into the gag region of the virus [7Armentano D. Effect of internal viral sequences on the utility of retroviral vectors.J. Virol. 1987; 61: 1647-1650PubMed Google Scholar, 8Bender M.A. Palmer T.D. Gelinas R.E. Miller A.D. Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region.J. Virol. 1987; 61: 1639-1646PubMed Google Scholar]. Vectors containing the 5’ end of MoMLV up to and including a portion of the gag gene were good substrates for recombination with the ψ-deleted retrovirus present in the packaging cells, requiring only a single homologous recombination between the genomes to generate fully replication-competent virus. The approach that we took to solve this problem was to remove additional elements essential for retrovirus replication from the genome of the helper virus in the packaging cells (Fig. 1C), and to design complementary vectors that would minimize the chances of recombination with the integrated genome in the packaging lines. The retrovirus genome used to make PA317 cells was modified by removal of the packaging signal and a portion of the 5′-LTR. We also replaced the 3′-LTR and the origin of second-strand synthesis with an SV40 polyadenylation signal [1Miller A.D. Buttimore C. Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production.Mol. Cell. Biol. 1986; 6: 2895-2902Crossref PubMed Scopus (1145) Google Scholar]. Even though RNA transcribed from this construct might be packaged into virions, many signals required for reverse transcription and integration were missing, thus preventing transmission. Complementary vectors were made to avoid overlap with the multiply deleted viral genome in the PA317 cells, and included the LNL6 vector [8Bender M.A. Palmer T.D. Gelinas R.E. Miller A.D. Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region.J. Virol. 1987; 61: 1639-1646PubMed Google Scholar] used in the first human gene transfer trial [2Rosenberg S.A. Gene transfer into humans—immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction.N. Engl. J. Med. 1990; 323: 570-578Crossref PubMed Scopus (1082) Google Scholar] and the LASN vector [9Hock R.A. Miller A.D. Osborne W.R.A. Expression of human adenosine deaminase from various strong promoters after gene transfer into human hematopoietic cell lines.Blood. 1989; 74: 876-881PubMed Google Scholar] used in the first approved gene therapy trial [3Blaese R.M. T lymphocyte-directed gene therapy for ADA-SCID: initial trial results after 4 years.Science. 1995; 270: 475-480Crossref PubMed Scopus (1202) Google Scholar]. Ultimately, the widely used “LN” series of retroviral vectors [10Miller A.D. Rosman G.J. Improved retroviral vectors for gene transfer and expression.Biotechniques. 1989; 7: 980-990PubMed Google Scholar] were developed, which required at least one recombination in a short region of homology and one nonhomologous recombination event between these vectors and the retroviral genome in the PA317 cells to generate replication-competent virus (Fig. 1E). PA317 cells were available widely by request, and were the first packaging line available through the American Type Culture Collection. Although other lines had been developed, easy access likely led the Rosenberg, Anderson, and Blaese groups to propose using PA317 cells to produce a vector designed to mark and track T cells after their introduction into patients. But there was a problem. They were using the N2 vector [7Armentano D. Effect of internal viral sequences on the utility of retroviral vectors.J. Virol. 1987; 61: 1647-1650PubMed Google Scholar], which had significant overlap with the retroviral genome in the PA317 cells. The result was the contamination of the vector preparations with helper virus at a low but detectable frequency. Although Anderson proposed using the vector stocks anyway–citing the lack of disease in monkeys receiving large quantities of replication-competent amphotropic retrovirus [11Cornetta K. Amphotropic murine leukemia retrovirus is not an acute pathogen for primates.Hum. Gene Ther. 1990; 1: 15-30Crossref PubMed Scopus (155) Google Scholar]–the Recombinant DNA Advisory Committee (RAC) that reviewed human gene therapy trials did not approve the proposal. However, my group had designed a vector called LNL6 [8Bender M.A. Palmer T.D. Gelinas R.E. Miller A.D. Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region.J. Virol. 1987; 61: 1639-1646PubMed Google Scholar] that, like N2, carried the neo gene but, unlike N2, never led to the production of helper virus in PA317 cells. Anderson and I agreed in 1988 to collaborate and use PA317 cells producing the LNL6 vector for the marking trial, which was completed without any evidence of replication-competent virus in the vector stocks or in the patients. A similar story unfolded in the trial designed to transfer adenosine deaminase (ADA) into T cells of patients with severe combined immunodeficiency caused by ADA deficiency. The ADA vector the NIH group was working with, called SAX, was a derivative of the N2 vector, and preclinical tests of the approach were plagued by contamination with replication-competent virus [12Kantoff P.W. Expression of human adenosine deaminase in nonhuman primates after retrovirus-mediated gene transfer.J. Exp. Med. 1987; 166: 219-234Crossref PubMed Scopus (133) Google Scholar]. As it turns out, my group and Bill Osborne's group at the University of Washington had developed PA317 cells that produced a vector encoding ADA, called LASN, that had never produced helper virus and synthesized large amounts of ADA in hematopoietic cell lines and in skin fibroblasts transduced with the vector [9Hock R.A. Miller A.D. Osborne W.R.A. Expression of human adenosine deaminase from various strong promoters after gene transfer into human hematopoietic cell lines.Blood. 1989; 74: 876-881PubMed Google Scholar, 13Palmer T.D. Hock R.A. Osborne W.R.A. Miller A.D. Efficient retrovirus-mediated transfer and expression of a human adenosine deaminase gene in diploid skin fibroblasts from an adenosine deaminase-deficient human.Proc. Natl. Acad. Sci. USA. 1987; 84: 1055-1059Crossref PubMed Scopus (107) Google Scholar, 14Palmer T.D. Rosman G.J. Osborne W.R.A. Miller A.D. Genetically modified skin fibroblasts persist long after transplantation but gradually inactivate introduced genes.Proc. Natl. Acad. Sci. USA. 1991; 88: 1330-1334Crossref PubMed Scopus (408) Google Scholar]. In 1989, Mike Blaese and I agreed to collaborate on the human trial. Ken Culver, working in Blaese's lab, Bill Osborne, and I performed tests that showed LASN actually made more ADA in T cells than did the original SAX vector, and following additional safety studies, the trial went ahead. Again, no helper virus was detected in the vector stocks made for the clinical trial or in the two patients receiving the vector. After these initial trials, PA317 cells became a standard line in which to produce retroviral vectors for clinical application. The Fred Hutchinson Cancer Research Center had patented the cell line, but made it available to all academic researchers and to companies under nonexclusive licenses for a modest fee. One of these initial licenses was to Genetic Therapy Inc. (GTI; now a part of Novartis), the first gene therapy company, which produced the vectors used in the first gene marking trial and in the first gene therapy trial–in both cases from vector-producing cell lines that I supplied. In one of the most remarkable tests of the safety of packaging cells, work begun at the NIH and at GTI using the PA317 cells and a vector designed to kill brain tumor cells ultimately led to phase I, phase II, and the first phase III gene therapy trials, sponsored by Novartis, which involved direct injection of live vector-producing PA317 cells into the brains of patients! Until this time, vector stocks for use in humans and for transduction of cells modified ex vivo for return to patients could be screened for the presence of helper virus before delivery to human subjects. Direct injection of the PA317 vector-producing cells meant that the subject could not be protected from chance helper virus generation after delivery of the cells, and that lack of helper virus exposure depended on the vector system not producing helper virus. While this method for treating brain cancer was ultimately not successful, none of over 170 patients with glioblastoma in these clinical trials showed any evidence for the production of replication-competent virus [15Shand N. A phase 1-2 trial of gene therapy for recurrent glioblastoma multiforme by tumor transduction with the herpes simplex thymidine kinase gene followed by ganciclovir.Hum. Gene Ther. 1999; 10: 2325-2335Crossref PubMed Scopus (241) Google Scholar, 16Rainov N.G. A phase III clinical evaluation of herpes simplex virus type 1 thymidine kinase and ganciclovir gene therapy as an adjuvant to surgical resection and radiation in adults with previously untreated glioblastoma multiforme.Hum. Gene Ther. 2000; 11: 2389-2401Crossref PubMed Scopus (571) Google Scholar]. In the most extensive characterization reported so far [17Otto E. Characterization of a replication-competent retrovirus resulting from recombination of packaging and vector sequences.Hum. Gene Ther. 1994; 5: 567-575Crossref PubMed Scopus (105) Google Scholar], the GTI group found three helper-virus-positive lots of G1Na vector that had been harvested on consecutive days from a single culture of PA317/G1Na producer cells. In other production runs, 171 lots of vector (855 liters) from PA317 cells containing various vectors, including 42 lots containing the G1Na vector, had tested negative for helper virus, so the rate of helper production is very low. Analysis of the recombinant helper virus was consistent with a mechanism for recombination involving two homologous recombinations between the G1Na vector and the packaging genome to restore the 5′- and 3′-LTRs and adjacent sequences to the two ends of the contiguous gag, pol, and env sequences present in the PA317 cells. Even these results may overestimate the rate of helper virus production by PA317 cells containing appropriate vectors. For example, the 3’ end of the LN series of vectors was designed such that there is no homology with the 3’ end of the packaging genome in PA317 cells. G1Na is a copy of the LN vector made for proprietary reasons, and is identical to LN with the exception of different linker sequences flanking the neo gene in the vector. Unfortunately, the linkers used at the 3’ end generated an 8 of 10 base pair region of homology with the 3’ end of the packaging genome in PA317 cells, and the 3’ recombination described above occurred in this region. In one study [12Kantoff P.W. Expression of human adenosine deaminase in nonhuman primates after retrovirus-mediated gene transfer.J. Exp. Med. 1987; 166: 219-234Crossref PubMed Scopus (133) Google Scholar, 18Cornetta K. No retroviremia or pathology in long-term follow-up of monkeys exposed to a murine amphotropic retrovirus.Hum. Gene Ther. 1991; 2: 215-219Crossref PubMed Scopus (70) Google Scholar], bone marrow cells from four cynomolgus monkeys were exposed for 2 hours to a retroviral vector preparation that contained over 106 colony-forming units of a vector encoding ADA along with an approximately equal amount of amphotropic replication-competent retrovirus that arose through vector/packaging cell recombination. The cells were re-infused after 1000-rad lethal irradiation of the recipients. These animals showed no evidence of pathology or retroviremia during a 2- to 3-year follow-up. In another study [19Donahue R.E. Helper virus induced T cell lymphoma in nonhuman primates after retroviral mediated gene transfer.J. Exp. Med. 1992; 176: 1125-1135Crossref PubMed Scopus (481) Google Scholar], bone marrow cells from 10 rhesus monkeys were transduced using various protocols with a retroviral vector contaminated with replication-competent virus, and 3 died of lymphoma at ≈ 6 months after transplantation due to virus exposure. The three animals that developed lymphoma were pretreated with either 5-fluorouracil or recombinant human stem cell factor to stimulate hematopoietic cell replication, marrow was harvested and CD34+ hematopoietic cells were purified, the cells were exposed to vector for > 3 days with daily additions of fresh vector-containing medium, and the cells were re-infused after 1000-rad lethal irradiation of the animals. The vector preparation was estimated to contain ≈ 104 infectious helper viruses per ml, and over the course of exposure, the cells were exposed to over 106 infectious helper viruses. The more aggressive transduction protocol and the severe immunosuppression in these animals due to the use of lethal irradiation and a T-cell depleted marrow graft likely explains the difference in results between the two studies. This study shows that recombinant amphotropic retroviruses that arise in packaging cells can cause disease in primates, but under conditions of gross helper virus contamination that would not go undetected in vector preparations destined for human use. Of interest is the fact that replication-competent retrovirus has not been detected in any human gene transfer subject so far (personal communication, Office of Biotechnology Activities, NIH). Even before the PA317 cells were used in clinical trials, “split” packaging cell lines were available in which the gag-pol and env genes were separated such that three recombination events between these expression constructs and a retroviral vector were required to generate replication-competent virus (Fig. 1D). Theoretically this should reduce the rate of helper virus generation compared with that of PA317 cells, in which only two recombination events are required. However, production of replication-competent retrovirus has been documented in split packaging cells [20Chong H. Vile R.G. Replication-competent retrovirus produced by a ‘splitfunction’ third generation amphotropic packaging cell line.Gene Ther. 1996; 3: 624-629PubMed Google Scholar, 21Chong H. Starkey W. Vile R.G. A replication-competent retrovirus arising from a split-function packaging cell line was generated by recombination events between the vector, one of the packaging constructs, and endogenous retroviral sequences.J. Virol. 1998; 72: 2663-2670Crossref PubMed Google Scholar], so even this system does not guarantee safety from helper virus contamination. Compounding this situation is the existence of many endogenous retroviral elements in cells that can provide substrates for recombination to generate replication-competent viruses. Indeed, the helper virus that arose from the split packaging cells described above involved recombination between endogenous retroviral elements, the 3’ end of the retroviral vector, and only one of the packaging DNAs, that encoding the Env protein [21Chong H. Starkey W. Vile R.G. A replication-competent retrovirus arising from a split-function packaging cell line was generated by recombination events between the vector, one of the packaging constructs, and endogenous retroviral sequences.J. Virol. 1998; 72: 2663-2670Crossref PubMed Google Scholar]. Thus while the split packaging design and other theoretical improvements in packaging cells have occurred since the development of PA317 cells, many factors contribute to the frequency of helper virus generation in vector-producing packaging cells, and all cell lines must be continuously evaluated for potential helper virus production. It cannot be overemphasized that the vector used with a particular packaging cell line is a key factor determining the propensity for helper virus production, and many reports of helper virus production from PA317 and other packaging lines are simply due to the use of inappropriate vectors that have large regions of homology with viral DNA in the packaging cells. Other improvements in packaging cell design include the use of envelope proteins from viruses other than the amphotropic virus used in PA317 cells to promote improved vector entry into specific cell types. In addition, while many packaging cell lines have been constructed by using mouse cells, these cells contain many endogenous elements related to and capable of recombining with the murine retroviruses used to make packaging lines. Endogenous retrovirus elements in cells from species other than rodents are less closely related, thus the potential for recombination leading to helper virus generation should be reduced in packaging lines derived using such cells. It is interesting to consider the parentage and construction of the PA317 cells given their widespread use in human gene therapy trials. They were made from an NIH 3T3 thymidine kinase negative cell line of uncertain parentage obtained from cell stocks at the Molecular Biology and Virology Lab at the Salk Institute. The original cells were cultured in Plexiglas “hoods” designed for bacterial use and which had no HEPA-filtered airflow to control contamination. Even so, bacterial or fungal contamination of the cultures was rare. Conditions were nowhere near current Good Manufacturing Practices (cGMP) standards. In spite of this, the PA317 cells have passed all of the myriad tests for adventitious agents, etc., required for use of the cells for human gene therapy.