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Efficient Introduction of Plasmid DNA Into Human Hemopoietic Cells by Encapsidation in Simian Virus 40 Pseudovirions

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Efficient Introduction of Plasmid DNA Into Human Hemopoietic Cells by Encapsidation in Simian Virus 40 Pseudovirions Proc. Natl. Acad. Sci. USA Vol. 83, pp. 6925-6929, September 1986 Genetics Efficient introduction of plasmid DNA into human hemopoietic cells by encapsidation in simian virus 40 pseudovirions (bone marrow cell/gene transfer/transient expression/simian virus 40 vector/gene therapy) ARIELLA OPPENHEIM, AVIVA PELEG, EITAN FIBACH, AND ELIEZER A. RACHMILEWITZ Department of Hematology, Hadassah University Hospital, Jerusalem, Israel 91120 Communicated by Helen M. Ranney, May 15, 1986 ABSTRACT Introduction ofDNA into human hemopoietic promoter, into the hemopoietic cells was observed, in com- cells is required for the study of regulatory mechanisms parison with the calcium phosphate precipitation technique. operating in these cells, as well as for possible procedures of However, that SV40 vector system was found to generate gene therapy. However, with hemopoietic cells the convention- wild-type SV40 virions with high frequency and was not al technique of calcium phosphate precipitation is inefficient. further investigated. The pathway of encapsidation of plasmid DNA as simian virus SV40 can efficiently infect human lymphoid and erythroid 40 (SV40) pseudovirions for the introduction of new genetic cells; 10-25% of the cells, infected at a multiplicity of 40, material was therefore investigated. Encapsidation was expressed high levels of virus-encoded large tumor (T) achieved in COS (monkey kidney) cells, which express SV40 antigen (A.O., unpublished data). In the present communi- large tumor (T) antigen constitutively. The vector, pSO, was cation, we describe an efficient method that utilizes SV40 introduced to the COS cells by DNA transfection. It carried the pseudovirions to transmit DNA. SV40 origin of replication (on), to facilitate replication of the plasmid in the COS cells. The SV40 capsid proteins were METHODS supplied in trans by a helper SV40 virus. The bacterial chloramphenicol acetyltransferase gene cat was used as a model Cell Culture and Viral Stocks. COS (monkey kidney) cells, for gene transmission. After encapsidation, the pseudovirions which are constitutive for SV40 T antigen (12), were cultured were used in infection of the human erythroleukemic cell line in Dulbecco's modified Eagle's medium with 10% fetal K562 and of normal human bone marrow cells. The results bovine serum (FBS). Logarithmically growing cultures were demonstrate that the cat gene can be transmitted with high used in transfection and infection experiments. Transfections efficiency. Over 40% of the infected K562 cells and 30% of the were performed by the DEAE-dextran method (13). During infected bone marrow cells were observed to contain plasmid transfection, one culture was treated with DEAE-dextran in DNA 48 hr after infection. Moreover, the results suggest that parallel to the others, but without DNA (mock transfection). the efficiency of gene transmission by this vector can be Infections were performed by incubating the cells with viral improved and so may approach the theoretical 100%. stocks for 90 min at 37°C, followed by the addition of fresh medium. For CAT assays the cells were harvested by A plethora of studies on the regulation of inserted genes in a trypsinization after 48 hr. variety of cultured cells has contributed enormously to the The inducible human erythroleukemic cell line K562 was understanding of gene regulation in eukaryotic cells in recent cultured in alpha medium with 10% FBS. For infection years. However, the lack of an adequate method for intro- experiments, logarithmically growing cells (5 x 105 per ml) duction ofcloned genes into hemopoietic cells in general, and were collected by centrifugation. Virus stock (or a lysate human bone marrow cells in particular, has hindered the prepared from mock-transfected cells, for mock infection) investigation of regulatory mechanisms in these cells. was added and allowed to adsorb for 2 hr at 37°C. The cells With hemopoietic cells, the conventional technique of were then centrifuged and fresh medium (with 10% FBS) was calcium phosphate precipitation (1) is inefficient for intro- added. Cells were counted and harvested after 48 hr. duction of DNA, as only 1 in 105 cells becomes transfected Human bone marrow cells were obtained from four healthy (2). Therefore, methods that are more efficient with donors for marrow transplantation, designated A-D. The hemopoietic cells have been developed. Protoplast fusion has cells were separated on a Ficoll-Hypaque gradient and increased the frequency of transfection in some cell lines to monocytes were removed by adherence to plastic. The cells 10-4 (3). With electroporation (electric field-mediated trans- were infected with viral stock as described for the K562 cells and harvested 48 hr later. fer), DNA is introduced at an efficiency of 10-3 to 10-4 (4). Viral stocks were prepared by transfection of COS cells as Retroviral vectors (5, 6) were reported to be very efficient in described in Results. For high-titer stocks, part of the introducing genetic material into murine (7, 8) and human medium was removed after 3 days and the cultures were hemopoietic stem cells (9, 10). incubated in 2.0 ml of medium per 25-cm2 culture flask for 2 The use of simian virus 40 (SV40) virions carrying the more days. Virus stocks were harvested by freeze-thaw (25). bacterial chloramphenicol acetyltransferase (CAT; acetyl- Mock-transfected cultures were processed in parallel. CoA:chloramphenicol 03-acetyltransferase, EC 2.3.1.28) CAT Assays. CAT assays were performed essentially as gene cat to transmit genetic material into hemopoietic cells described by Gorman et al. (14). The cells were sonicated in was explored by Karlsson et al. (11), who also investigated 0.25 M Tris/HCl buffer at pH 7.8. The extract (of 0.5 x 106 the use of adenovirus vectors. A great increase in the cells) was incubated in the presence of [14C]chloramphenicol efficiency of introduction of the bacterial cat gene, fused to (0.25 ,uCi; 1 Ci = 37 GBq) and 0.5 mM acetyl-CoA at 37°C for the Rous sarcoma virus (RSV) long terminal repeat (LTR) 30 or 60 min, in 0.25 M Tris/HCl buffer (pH 7.5) in a total volume of 180 ,ul. To separate the products from the sub- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: CAT, chloramphenicol acetyltransferase; cat, gene in accordance with 18 U.S.C. §1734 solely to indicate this fact. encoding CAT; SV40, simian virus 40; bp, base pair(s). 6925 Downloaded by guest on September 28, 2021 6926 Genetics: Oppenheirn et al. Proc. Natl. Acad. Sci. USA 83 (1986) strate, the reaction mixture was fractionated by TLC. Ra- cotransfected with pSV2cat and SV40 DNA by the DEAE- dioactivity in the spots was quantitated in a liquid scintillation dextran procedure (13). It was expected that pSV2cat DNA counter. The assays were performed at the linear range for would replicate in these cells and would become encapsid- enzyme activity and were later normalized to percent acetyla- ated in the SV40 capsid proteins supplied by the helper viral tion catalyzed by an extract of 106 cells during a 60-min DNA. Virion mixture, presumably containing pSV2cat incubation. pseudovirions and wild-type SV40, was harvested 5 days In Situ Hybridization. To assay for the number of cells that later. The virion mixture was analyzed as detailed below and incorporated plasmid DNA, individual cells were immobi- used to transmit the cat gene into the hemopoietic cells by lized on nitrocellulose membrane filter for in situ hybridiza- infection. tion as follows. The infected cells were harvested 48 hr after To determine whether pSV2cat becomes encapsidated, infection, washed in Dulbecco's phosphate-buffered saline logarithmically growing COS cells (25-cm2 cultures) were without Ca2l and Mg2+ (PBS), and counted. The cells were transfected in duplicate as follows: (a) pSV2cat plus SV40 serially diluted in serological plates, and known aliquots were DNA (0.5 ,ug each), (b) pSV2cat (1 ,ug), (c) SV40 DNA (1 ,g), spotted in triplicate onto nitrocellulose membrane filter. The and (d) mock transfection (i.e., no DNA). After 48 hr, one of nitrocellulose filter was washed in PBS and processed for in each pair ofduplicate cultures was harvested and assayed for situ hybridization of DNA. This was accomplished by gentle CAT enzymatic activity. As expected, CAT activity was cell lysis and DNA denaturation in alkali followed by neu- observed only in cultures a and b. The other duplicate culture tralization and baking at 80'C under reduced pressure (15). was incubated for 5 days to allow for DNA replication and The filters were hybridized to nick-translated pML2 (16) and encapsidation. These cultures were harvested by repeated not to pSO3cat, since the latter plasmid contains SV40 freeze-thaw to break up the cells and liberate viral particles. sequences (see Fig. 1) and would have cross-hybridized to When the cell lysates were viewed under the electron the SV40 helper. pML2 did not cross-hybridize to the SV40 microscope, SV40-like particles were observed only in the helper (data not shown). cultures transfected with SV40 and with pSV2cat plus SV40. Autoradiography was performed by exposing the filter to Curiously, more empty capsids were observed in the SV40 x-ray film. Radioactive signals visualized on the film repre- transfection as compared with the cotransfection by pSV2cat sent single cells (or clumps of cells) that contain pSO3cat and SV40. Empty SV40 capsids were reported to constitute DNA. The percentage of cells that incorporated plasmid a large part of the SV40 viral stock prepared in monkey DNA was computed by counting the signals. For example, in kidney cells (19). For estimation of the titer, virion particles Fig. 5 Lower there are a total of 8 signals (2 + 3 + 3) in the were counted at several dilutions together with a standard of 3 replicate spots that contain 8 cells each (altogether, 24 known titer (bacteriophage T4) that was mixed with the cell cells).
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