Efficient Infection of a Human T-Cell Line and of Human Primary
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Proc. Natl. Acad. Sci. USA Vol. 93, pp. 11842-11847, October 1996 Medical Sciences Efficient infection of a human T-cell line and of human primary peripheral blood leukocytes with a pseudotyped retrovirus vector SANJAI SHARMA*, MARK CANTWELLt, THOMAS J. Kippst, AND THEODORE FRIEDMANN*t Departments of *Pediatrics and tMedicine, University of California School of Medicine at San Diego, La Jolla, CA 92093-0634 Communicated by Helen M. Ranney, Alliance Pharmaceutical Corp., San Diego, CA, July 29, 1996 (received for review April 26, 1996) ABSTRACT Peripheral blood lymphocytes (PBLs) are an permit highly efficient infection of CD4+ T cells and other important target for gene transfer studies aimed at human classes of lymphocytes in vitro, and possibly in vivo. Studies gene therapy. However, no reproducibly efficient methods are toward this goal have included a number of cell culturing currently available to transfer foreign, potentially therapeutic manipulations to improve transduction efficiency, but such genes into these cells. While vectors derived from murine approaches have limited utility for many potential applica- retroviruses have been the most widely used system, their low tions, including in vivo gene transfer. More efficient applica- infection efficiency in lymphocytes has required prolonged in tion of retroviruses to gene transfer into lymphocytes would vitro culturing and selection after infection to obtain useful also benefit from improved retrovirus vector technology, numbers of genetically modified cells. We previously reported including the preparation of vectors to much higher titers than that retroviral vectors pseudotyped with vesicular stomatitis currently possible and the design of vectors able to infect G glycoprotein (VSV-G) envelope can infect a wide variety of quiescent cells. cell types and can be concentrated to titers of greater than 109 Our laboratory has recently taken a step toward overcoming infectious units/ml. In this present study, we examined the one of these technical barriers-i.e., that of low retrovirus vector ability of amphotropic and pseudotyped vectors expressing a titers. We have shown that the env gene product of Moloney murine cell surface protein, B7-1, to infect the human T-cell murine leukemia virus (MoMLV) can be replaced by the G line Jurkat or human blood lymphocytes. Limiting dilution glycoprotein of vesicular stomatitis virus (VSV-G) (12, 13) to analysis of transduced Jurkat cells demonstrated that the produce vectors with a markedly enhanced host range and pseudotyped vector is significantly more efficient in infecting expanded tissue tropism. Because the cellular receptors for T cells than an amphotropic vector used at the same multiplicity VSV-G, possibly including phosphatidylserine, phosphatidylino- of infection (moi). To identify the transduction efficiency on sitol, and GM3 ganglioside (14, 15) appear to be very abundant PBLs, we examined the levels of cell surface expression of the and ubiquitous membrane components of most mammalian cells, B7-1 surface marker 48 to 72 hr after infection. The transduction the mechanisms of cell entry and infection with the pseudotyped efficiency ofPBLs with the pseudotyped vector increased linearly vectors are entirely different from those associated with tradi- with increasing moi to a maximum of approximately 16-32% at tional amphotropic retrovirus vectors. Most important, however, an moi of40. This relatively high efficiency ofinfection ofa T-cell is the fact that the VSV-G pseudotyped virus can be concentrated line and of blood lymphocytes with VSV-G pseudotyped virus to high titers, thereby permitting the use of much higher values of demonstrates that such modified pseudotyped retrovirus vectors multiplicity of infection (moi) than have been previously possible. may be useful reagents for studies of gene therapy for a variety In the present study, we present evidence that a T-cell line and of genetic or neoplastic disorders. primary PBLs are readily susceptible to infection with pseudotyped virus and a high percentage of cells express the T lymphocytes are relatively long-lived (1) cells of the hema- transgene. topoietic system that regulate immune responses to infectious or neoplastic disease. T-cell function is impaired in a variety of MATERIALS AND METHODS genetic or acquired diseases that affect lymphocytes, resulting in immunodeficiency, severe chronic infections, or neoplasia. Plasmids. The cDNA encoding the murine immune acces- Since most circulating lymphocytes are T cells, the peripheral sory molecule B7-1 (muB7) cDNA, a gift from James Allison blood lymphocyte compartment is an important target for (University of California, Berkeley), was subcloned into genetic modification. Unfortunately, T cells are refractory to pBluescript polylinker. A HindIII-XbaI fragment with the most current viral and non-viral gene transfer techniques. Only muB7 cDNA was then blunt- ended and inserted into the retrovirus vectors have been found useful in transferring blunt-ended BamHI site of vector plasmid pLPONL (16), foreign, potentially therapeutic genes into a variety of lym- which expresses the neomycin phosphotransferase gene from phocytes in vitro, including peripheral blood lymphocytes the Moloney retroviral 5' long terminal repeat by virtue of the (PBLs) and tumor-infiltrating lymphocytes (2-8). This has led presence of a poliovirus ribosome reentry sequence (PO). The to several clinical gene therapy studies involving gene transfer resulting plasmid was designated pLPONL-muB7. into such cells (6-11). Retrovirus Vector Preparation. Vector pLPONL-muB7 The recent results with adenosine deaminase deficiency plasmid DNA was transfected into the packaging cell line (ADA) reconstitution studies demonstrated that a limited PA317 (17) by -using the established calcium phosphate trans- degree of gene transfer and prolonged gene expression in fection method (18). Forty-eight hours after transfection, the lymphocytes are possible with current retroviral vectors. How- culture medium containing amphotropic LPONL-muB7 virus ever, prolonged periods ofin vitro culture of infected cells were was used to infect 293GP cells, which express high levels of required to obtain useful numbers of genetically modified cells Moloney murine leukemia virus gag and pol (12). After because of the poor transduction efficiencies of retroviruses. This mandates improvements in gene transfer technology to Abbreviations: PBL, peripheral blood lymphocyte; VSV-G, G glyco- protein of vesicular stomatitis virus; moi, multiplicity of infection; muB7, murine immune accessory molecule B7-1; IL-2, interleukin 2; The publication costs of this article were defrayed in part by page charge FACS, fluorescence-activated cell sorter; PE, phycoerythrin; FITC, payment. This article must therefore be hereby marked "advertisement" in fluorescein isothiocyanate; TE, transduction efficiency. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 11842 Downloaded by guest on October 2, 2021 Medical Sciences: Sharma et al. Proc. Natl. Acad. Sci. USA 93 (1996) 11843 selection in G418-containing medium (400 ,tg/ml) for 2 weeks, FACScan (Becton Dickinson). Other antibodies used to char- colonies were picked and the clones (muB7/293GP cells) acterize the phenotype of infected cells included an anti-CD3 expressing high levels of the muB7 transgene, as assessed by antibody [clone UCHT-1, fluorescein isothiocyanate (FITC) flow cytometric analysis (see below), were pooled. conjugate, Sigma], an anti-CD4 antibody (clone Q4120, FITC The preparation of high-titer VSV-G-pseudotyped LPONL- conjugate, Sigma), and an anti-CD45RO antibody (clone muB7 vector was carried out by methods described previously UCHL-1 FITC conjugate, Sigma). Cells were stained with (13). The muB7/293GP cells were transfected with plasmid propidium iodide (1 ,ug/ml) to label dead cells, allowing for pHCMV-G expressing VSV-G from the human cytomegalo- their exclusion from FACS analysis. virus (HCMV) promoter (13) by using the calcium phosphate Limiting Dilution Analysis of Infected Cells. For limiting method. Culture media were collected at 48 and 72 hr after dilution analysis (19), cells were infected with the amphotropic transfection, filtered through a 0.45-,um pore diameter filter, and pseudotyped vectors at an moi of 1 as described above, and and concentrated by ultracentrifugation as described (13). after 3 days the cells were replated in complete medium at a Virus was titered on 208F cells by infection with virus dilutions density of 100, 25, 10, or 1 cell per well in 96-well plates. in the presence of Polybrene (8 ,ug/ml) followed by G418 Duplicate cultures either were maintained in complete growth selection (400 ,g/ml) starting 24 hr after infection. G418- medium or were grown under selective conditions consisting of resistant colonies were counted 10-12 days after infection. complete medium containing G418 at 800 ,ug/ml. Wells with Aliquots of virus stocks were frozen at -70°C. To obtain viable cells were counted 2 weeks after plating. retrovirus enveloped with an amphotropic envelope, plasmid Efficiency of Transduction. The transduction efficiency was pHCMV-ampho-env was constructed by excising the BamHI calculated by dividing the negative natural log of the G418- fragment VSV-G cDNA from pHCMV-G and replacing it with selected fraction of wells that did not contain cells by the a 2.8-kb EagI fragment from plasmid PA14 (from A. Rein, number of cells plated in that