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Proc. Natl. Acad. Sci. USA Vol. 83, pp. 6925-6929, September 1986 Efficient introduction of DNA into human hemopoietic cells by encapsidation in simian 40 pseudovirions (bone marrow / transfer//simian virus 40 vector/) 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high levels of virus-encoded large tumor (T) achieved in COS (monkey kidney) cells, which express SV40 antigen (A.O., unpublished data).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 . It carried the pseudovirions to transmit DNA. SV40 (on), to facilitate replication of the plasmid in the COS cells. The SV40 were METHODS supplied in trans by a helper SV40 virus. The bacterial chloramphenicol acetyltransferase gene cat was used as a model 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 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. 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 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 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 . 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 (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, (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 -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 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 ( T4) that was mixed with the cell cells). Therefore, 8 of24 cells (or 33%) incorporated pSO3cat lysates. The titer of the pSV2cat/SV40 virion mixture was DNA. The number ofradioactive signals was not reduced by computed to be 2 x 108 "full" particles per ml. treating the cells with trypsin or by repeated washings with Encapsidation of pSV2cat was inferred from transmission EDTA before they were spotted onto nitrocellulose, indicat- of the cat gene into fresh cultures of COS cells (Fig. 2A). ing that the plasmid DNA was contained within the cells and Duplicate cultures were each infected by incubation with 0.5 not in pseudovirions adsorbed to the cell surface. ml ofthe cell lysate. One set was incubated for 5 days to allow new virion particles to form, and the other was harvested after 48 hr and assayed for CAT activity (Fig. 2A, infection RESULTS cycle 1). The virus and pseudovirus were thus repassaged in Encapsidation of Plasmid DNA as SV40 Pseudovirions. The COS cells several times. The results, summarized in Fig. 2A, experimental design included encapsidation ofthe plasmid in show that the cat gene was not transmitted in the absence of SV40 coat proteins and infection of the hemopoietic cells the helper SV40. Therefore, we concluded that encapsidation In the was taking place and was obligatory for the process. It can be with the plasmid-containing pseudovirion preparation. observed that CAT activity was rapidly declining with every initial experiments, we used as a model for encapsidation and infection. Analysis of Hirt supernatants (20) gene transmission the plasmid pSV2cat (ref. 14 and Fig. 1), subsequent in obtained from the infected cultures revealed that SV40 was constructed for expression of the bacterial cat gene the infection. Hirt supernatants of the cells eukaryotic cells by fusion to SV40 expression signals (18). overtaking were as follows. COS cells were The experiments performed A B 100 _ 100 _ EcoRl (Cla I/Hpa II) Hilndlll U>~

H>50 - 50 - E pSO3cat tz z 4072 bp (a O L -0a o

(Hae II) BarnHI 2 T 1 2 Hfae 11 Hiindlll T 1 Infection cycle FIG. 1. Structure of carrying the cat gene. pSV2cat (14) was kindly provided by C. Gorman. pSO3cat was constructed from FIG. 2. Encapsidation of the plasmids with SV40 as a helper. The pBRM (ref. 17; obtained from J. Hartman), a derivative of pBR322 experiments are described in detail in the text. Logarithmically (BamHI-EcoRI); pBR322 (EcoRI-Cla I); SV40 (Hpa II-HindIII); growing COS cells (25-cm2 cultures) were transfected as follows. (A) and pSV2cat (HindIII-BamHI). pBR322 sequence is represented by *, pSV2cat and SV40 DNA (0.5 ,g each); o, pSV2cat (1 ug); A, SV4O and SV40 sequence, by - *. Tn9 is represented by a thick DNA (1 ,ug). (B) *, pSO3cat and SV40 DNA (0.5 jig each); o, line. Direction of of cat and the ampicillin-resistance pS03cat (1 ,g); A, SV40 DNA (1 ,ug). CAT activity is expressed as gene amp are shown. Parentheses indicate restriction sites that were the percentage of chloramphenicol acetylated during 1 hr by an destroyed in constructing the plasmid. bp, Base pairs. extract of 106 cells. T, transfection. Downloaded by guest on September 28, 2021 Genetics: Oppenheirn et al. Proc. Natl. Acad. Sci. USA 83 (1986) 6927 infected in the second cycle contained more than 80% SV40 Infection of Human Bone Marrow Cells. Several human DNA and little pSV2cat DNA (results not shown). bone marrow samples were infected with the same virion Infection of hemopoietic cells-MEL (murine erythroleu- mixture and analyzed for CAT activity (Fig. 4) and the kemia), K562, and human bone marrow cells-showed that number of cells that incorporated plasmid DNA (Fig. 5). The the cat gene was introduced into those cells by the pSV2cat results are summarized in Table 1. Although differences pseudovirus. However, transmission was not very efficient, between different bone marrow samples were observed, it as deduced from CAT activity in transient-expression exper- can be seen that about 30% ofthe cells incorporated pSO3cat iments. Only 2-5% of the chloramphenicol substrate became DNA. In addition, CAT activity in these transient-expression acetylated during a 1-hr incubation at 370C with an extract of assays was reasonably high: 6-40% of the chloramphenicol 106 cells. substrate was acetylated during 1 hr at 37°C. The results To improve the efficiency of gene transmission, a new suggest that this vector can be used for transient-expression plasmid carrying the cat gene was constructed, pSO3cat (Fig. studies in human bone marrow cells. 1). pBR322 sequences that may interfere with replication initiated at the SV40 origin of replication (ori) (16) were DISCUSSION removed by transferring the cat gene and SV40 regulatory signals into pBRM (17). In addition, some SV40 late se- The studies reported here describe encapsidation of plasmid quences were removed and the orientation ofcat with respect DNA as SV40 pseudovirions and the use of this approach to to the vector sequences was reversed. As will be described introduce DNA into human hemopoietic cells. Our results below, pSO3cat was significantly more efficient than demonstrate that the plasmid pSO3cat is significantly more pSV2cat in transmission of cat. This could be due to any one efficient than pSV2cat in supporting the expression of cat and of the differences between the two plasmids. To find out in gene transmission. Additional experiments are required to whether replication of pSO3cat was more efficient, we establish which DNA signals are responsible for this differ- transfected COS cells with each of the two plasmids, at two ence. The pSO construct reported here may be superior to different DNA levels, and assayed for CAT after 48 hr. The pSV2 (18) in the expression of other genes as well. rationale was that a higher starting level of pSV2cat DNA In pSO3cat, the cat gene is transcribed from the SV40 early might compensate for its poor replication. However, the promoter, which has been found to be several times less results (not shown) indicated that this was not the case. This efficient than the Rous sarcoma virus (RSV) long terminal question was not further investigated. repeat (LTR) promoter in a variety of cells (14). It will be Encapsidation of pSO3cat was studied by gene transmis- interesting to find whether combining the RSV promoter with sion in COS cells as described for pSV2cat (Fig. 2B). The new the pSO construct will increase gene transcription even plasmid appeared satisfactory and was used in subsequent further. experiments. Since CAT activity was declining after the first The results demonstrate that pSO is an efficient vector for infection cycle, the first viral mixture obtained from the introducing genetic material into human hemopoietic cells. In transfected COS cells was used in infection of hemopoietic the experiments described here, 30-40% of the hemopoietic cells. cells became infected. The results in Fig. 3 and Table 1 Infection of K562 Cells. Logarithmic cultures of K562 cells suggest that the efficiency of the vector can be improved by were infected with viral stocks as described in Methods. The increasing the multiplicity of infection and may approach the number of cells that incorporated plasmid DNA was assayed theoretical 100%. With this method, one could study the by in situ hybridization of individual cells immobilized on nitrocellulose membrane filter. The number of cells that incorporated plasmid DNA, as well as total CAT activity in _ CM 1,3-diacetate the transient expression assay, increased with the multiplic- ity of infection (Fig. 3). At the highest multiplicity used in these experiments, more than 40% of the cells that were infected incorporated the plasmid DNA. The efficiency ofthe infection probably could be. improved by increasing the multiplicity of infection and by optimizing the conditions for adsorption. Similar results (not shown) were obtained with MEL cells. -CM 3-acetate 60 60~ -CM 1-acetate

>0'=40 40~ *** _-CM..

U 20 20

0 A~~~~~~ 0 1 2 3 4 0.2 0.4 1.0 FIG. 4. CAT activity in human bone marrow cells. Bone marrow Multiplicity of infection samples from donors C and D (Table 1) were harvested 48 hr after infection. Extracts of 106 cells were incubated with [14C]chlor- FIG. 3. Effect ofmultiplicity ofinfection (ml ofvirus stock per 106 amphenicol (CM) and acetyl-CoA for 1 hr at 37°C. The reaction cells) on infection of K562 cells. CAT activity (e) is expressed as % mixture was analyzed by TLC followed by autoradiography. Lanes chloramphenicol acetylated during 1 hr by an extract of 106 cells. The 1 and 2: mock-infected and infected bone marrow cells, respectively, percentage of cells with pSO3cat DNA of the total cells that were from donor D. Lane 3: infected bone marrow cells from donor C. infected (A, % infected cells) was assayed by in situ hybridization as Lane 4: control CAT extract from , prepared by in Fig. 6. sonicating E. coli cells harboring cat in Tn9. Downloaded by guest on September 28, 2021 6928 Genetics: Oppenheirn et al. Proc. Natl. Acad. Sci. USA 83 (1986)

Cells per SV40 sequences have beep used in many different vectors. spot Extrapolation of previous results suggests that genes that will AWOL- .dailk, be carried by pSO will not undergo excessive rearrangements W:Wv''"",6, .i -1000 after introduction into eukaryotic cells. In particular, intron - 500 and flanking sequences will not be destabilized. This is most O 00* important in view of the findings that regulatory signals 0 0 w.a. -:90LAk. - 250 sometimes reside within introns (21-24). In addition, avail- able information suggests that SV40 pseudovirions will have M. 40 * .014,.. - 125 a wide, perhaps unlimited, host range. i -62 The prokaryotic pBRM DNA can be removed from the plasmid before introduction into the COS cells for encapsida- ;4.9 - 31 tion. In several cases, prokaryotic vector sequences have been found to interfere with regulatory processes in eukary- - 16 otic cells (16, 26). This procedure will increase the -8 capacity of the vector, which is limited by size requirement for encapsidation in SV40 to about 5.5 kbp. The only part of C D c D the vector essential for the pseudoviral preparation is the mock- mock- + pSO3cat + pSO3cat SV40 ori region, which is contained in a fragment of '200 bp, infected infected (0.5 ml) (2.0 ml) allowing additional cloning space of =5 kbp. In the present construct, pSO3cat, the SV40 enhancer and expression signals are included to facilitate expression of the bacterial cat gene in eukaryotic cells. However, in constructs that carry eukaryotic genes, these DNA sequences could be *^s *411-31 removed. Under these conditions, when the vector part will include only the SV40 ori, we anticipate minimal interference of vector signals with the regulation of the cloned gene. Thus this vector will be most suitable for studies on the regulation of normal genes in hemopoietic cells. Efficient introduction of DNA into human bone marrow cells is also a prerequisite for gene therapy of thalassemia, FIG. 5. Analysis of the number of infected cells by in situ enzyme deficiencies. Our hybridization. Human bone marrow cells (donors C and D) were sickle cell anemia, and some harvested 48 hr after infection, counted, diluted in triplicate, and method may be suitable for this purpose when a helper-free spotted on nitrocellulose. (Upper) The filter was hybridized to pseudoviral preparation becomes available. For this, a helper nick-translated pML2 DNA and autoradiographed on x-ray film for plasmid that will supply the necessary SV40 functions in 2 days. (Lower) The bottom-right portion of a 7-day exposure of the trans but will not be encapsidated, and therefore will not be same filter, magnified. The numerical results appear in Table 1. contained in the pseudoviral preparation, is needed. It is anticipated that the use of SV40 will facilitate rigorous regulation of foreign genes introduced into hemopoietic cells control and routine screening for absence of SV40 virions in by relying on transient-expression studies. Tissue-specific the helper-free preparations, to ensure the level of safety expression of genes in different hemopoietic lineages could required for gene therapy. be explored in conjunction with appropriate the need for selection in tissue We wish to thank Ms. A. Treves and M. Yanuv for assistance in techniques. By avoiding some of the experiments. This research was supported by the Harry culture, the complications that usually arise in interpretation and Jenny Klein Fellowship in Medical Research and by the Joel of the results due to random integration ofthe incoming DNA Ostrowicz Foundation. into the host would be avoided. In long-term experiments, we anticipate that the plasmid will integrate at 1. Graham, F. L. & van der Eb, A. J. (1973) 52, random chromosomal locations, as has been observed before 456-467. for other SV40 vectors. 2. Miller, G., Wertheim, P., Wilson, G., Robinson, J., Geelen, J. L. M. C., van der Noordaa, J. & van der Eb, A. J. (1979) Proc. Natl. Acad. Sci. USA 76, 949-953. Table 1. Infection of human bone marrow cells with 3. Oi, V. T., Morrison, S. L., Herzenberg, L. A. & Berg, P. pSO3cat pseudovirus (1983) Proc. Natl. Acad. Sci. USA 80, 825-829. Pseudoviral % 4. Potter, H., Weir, L. & Leder, P. (1984) Proc. Natl. Acad. Sci. % with USA 81, 7161-7165. preparation, ml cells chloramphenicol 5.' Joyner, A., Keller, G., Phillips, R. A. & Bernstein, A. (1983) Donor per 106 cells pSO3cat DNA acetylated Nature (London) 305, 556-558. A 0 0 0 6. Cepko, C. L., Roberts, B. E. & Mulligan, R. C. (1984) Cell 37, 0.4 (mock) 0 0 1053-1062. 0.4 (pSO3cat) 3 6 7. Williams, D. A., Lemischka, I. R., Nathan, D. G. & Mulligan, R. C. (1984) Nature (London) 310, 476-480. B 2.0 (mock) 0 0 8. Dick, J. E., Magli, M. C., Huszar, D., Phillips, R. A. & 2.0 (pSO3cat) 23 8 Bernstein, A. (1985) Cell 42, 71-79. 9. Gruber, H. E., Finley, K. D., Hershberg, R. M., Katzman, C 0.5 (mock) 0 0 S. S., Laikind, P. K., Seegmiller, J. E., Friedmann, T., Yee, 0.5 (pSO3cat) 4 9 J. K. & Jolly, D. J. (1985) Science 230, 1057-1061. 10. Hock, R. A. & Miller, A. D. (1986) Nature (London) 230, D 2.0 (mock) 0 0 275-277. 2.0 (pSO3cat) 33 41 11. Karlsson S., Humphries, R. K., Gluzman, Y. & Nienhuis, Experiments are described in the text. Chloramphenicol acetyla- A. W. (1985) Proc. Natl. Acad. Sci. USA 82, 158-162. tion was assayed for 1 hr at 370C with an extract of 106 cells. The 12. Gluzman, Y. (1981) Cell 23, 175-182. percentage of cells that incorporated pSO3cat DNA was calculated 13. McCutchan, J. H. & Pagano, J. S. (1968) J. Natl. Cancer Inst. from in situ hybridization experiments, as shown in Fig. 5. 41, 351-357. Downloaded by guest on September 28, 2021 Genetics: Oppenheim et al. Proc. Nati. Acad. Sci. USA 83 (1986) 6929

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