Gene transfer and expression of a non-viral polycation-based ... - Nature

Gene Therapy (1999) 6, 1774–1778  1999 Stockton Press All rights reserved 0969-7128/99 $15.00 http://www.stockton-press.co.uk/gt

BRIEF COMMUNICATION

Gene transfer and expression of a non-viral polycation-based vector in CD4+ cells RL Puls1 and RF Minchin1,2 1

Department of Pharmacology, University of Western Australia; and 2Laboratory for Cancer Medicine, Royal Perth Hospital, Perth, Western Australia

CD4-selective targeting of an antibody-polycation-DNA complex was investigated. The complex was synthesized with the anti-CD4 monoclonal antibody B-F5, polylysine268 (pLL) and either the pGL3 control vector containing the luciferase reporter gene or the pGeneGrip vector containing the green fluorescent protein (GFP) gene. B-F5-pLLDNA complexes inhibited the binding of 125I-B-F5 to CD4+ Jurkat cells, while complexes synthesised either without BF5 or using a non-specific mouse IgG1 antibody had little or no effect. Expression of the luciferase reporter gene was achieved in Jurkat cells using the B-F5-pLL-pGL3 complex and was enhanced in the presence of PMA. Negligible luciferase activity was detected with the non-specific antibody complex in Jurkat cells or with the B-F5-pLL-pGL3

complex in the CD4− K-562 cells. Using complexes synthesised with the pGeneGrip vector, the transfection efficiency in Jurkat and K-562 cells was examined using confocal microscopy. More than 95% of Jurkat cells expressed GFP and the level of this expression was markedly enhanced by PMA. Negligible GFP expression was seen in K-562 cells or when B-F5 was replaced by a nonspecific antibody. Using flow cytometry, fluorescein-labelled complex showed specific targeting to CD4+ cells in a mixed cell population from human peripheral blood. These studies demonstrate the selective transfection of CD4+ T-lymphoid cells using a polycation-based gene delivery system. The complex may provide a means of delivering anti-HIV gene therapies to CD4+ cells in vivo.

Keywords: CD4; HIV; gene transfer

CD4, an integral membrane glycoprotein found on the surface of cells of lymphoid origin acts as a co-receptor in the T cell receptor complex, recognising foreign antigen in the context of MHC class II structures.1 CD4 also has been identified as the primary cellular receptor for HIV2,3 and interaction of the HIV coat protein gp120 with this receptor is the first step in cell infection. The coreceptor CCR-5 or CXCR-4 then acts to trigger fusion of the virus with the cell membrane.4–6 A number of strategies have been proposed to render CD4+ cells resistant to HIV infection. These include the expression of HIV specific antisense and ribozyme constructs,7,8 expression of transdominant or dominant negative mutant forms of Tat, Rev or Gag9–11 and recently, expression of intrakines targeting the co-receptors in CD4 cells.12 While each of these approaches holds considerable therapeutic potential, the delivery of genetic material to CD4+ cells in vivo remains a major limitation. CD4+ cells have been targeted using Moloney murine leukemia virus and vesicular stomatitis virus with their respective Env elements replaced with recombinant gp160.13,14 CD4+ cells also have been targeted with liposomes incorporating gp12015 and specific anti-CD4 antibodies on their surface.16–20 These liposomes have

Correspondence: RF Minchin, Department of Pharmacology, University of Western Australia, Nedlands 6907, Western Australia Received 15 January 1999; accepted 16 June 1999

been used to deliver anti-cancer agents,16–18 as well as oligonucleotides19,20 to CD4+ cells. The present study characterises an anti-CD4 antibodypolycation-DNA complex that selectively targets CD4+ cells. Polycation-DNA complexes are relatively simple to produce and have been shown to deliver exogenous genes to a number of target cells.21–23 Using luciferase assays in cell homogenates and green fluorescent protein (GFP) localisation by confocal microscopy, we found that the antibody–DNA complex was selectively taken up and expressed in the CD4+ cells. These findings may provide a means of delivery in vivo for anti-HIV gene therapies. The anti-CD4 antibody used in this study was B-F5, a mouse anti-human mAb of the IgG1 isotype that recognises an epitope between amino acids 37 and 105 of the extracellular domain of the CD4 molecule.24 B-F5 inhibits gp120 binding and rosette formation between CD4+ COS cells and Raji B cells.25 The antibody has been used in clinical trials for the treatment of autoimmune disorders such as rheumatoid arthritis,26,27 severe active multiple sclerosis,24 Crohn’s disease28 and as prophylactic therapy to prevent rejection following transplantation.29 Preliminary studies in this laboratory with B-F5 showed that it specifically bound to CD4 receptors on human Jurkat cells, CCRF-CEM cells and peripheral blood lymphocytes. Following binding, it was slowly internalised over 12–24 h (data not shown). The rate of binding and internalisation was similar to that described for a number of other anti-CD4 antibodies.30,31 Moreover, the rate of internalisation was significantly enhanced following

CD4-directed gene transfer RL Puls and RF Minchin

phorbol ester treatment. It has been previously reported that the cytoplasmic domain of CD4 interacts with p56lck of lymphoid cells, inhibiting constitutive internalisation of CD4 by inhibiting its aggregation in coated pits.30,32,33 Phorbol esters disrupt CD4-p56lck complexes increasing the rate of CD4 endocytosis.34,35 B-F5 was conjugated to poly(l)lysine268 (pLL) using the heterobifunctional linker, N-succinimidyl 3-(2pyridyldithio) propionate (SPDP) as previously described.22,23 The resulting B-F5-pLL conjugate was combined with plasmid reporter DNA to form a B-F5pLL-DNA complex. The plasmid used was either the pGL3 control vector (Promega, Sydney, Australia) which contains the firefly luciferase gene, or the pGeneGrip vector (Gene Therapy Systems, San Diego, CA, USA) which contains the GFP gene. Ferkol and Perales36,37 devised a multistep process for the formation of polycation-DNA complexes that involved increasing NaCl concentration, leading to the gradual condensation of DNA into compact unimolecular DNA-pLL complexes of uniform size and structure. Condensation of DNA has been reported to afford greater transfection efficiency38,39 and can protect the DNA from nuclease degradation. This technique was used to make complexes in the present study. pLL-DNA complexes were formed at a 1:10 ratio of DNA to pLL and the concentration of NaCl required to achieve dissipation of unimolecular complexes (0.8–1.1 m, as determined by UV spectroscopy) was similar to that observed by Perales and colleagues.40 The final complex consisted of 40 molecules of B-F5 and 10 molecules of pLL for each molecule of DNA. To determine whether B-F5 in the complex retained CD4 receptor recognition, competition binding studies were performed using Jurkat cells. The B-F5 conjugate and complex significantly inhibited 125I-B-F5 binding to a similar extent as that seen with unlabelled antibody (Figure 1). By contrast, a conjugate or complex synthesised with a non-specific murine antibody of isotype IgG1 did not affect 125I-B-F5 binding. These results show that modification of B-F5 to form complexes did not markedly affect CD4 binding. The B-F5-DNA complex containing the luciferase reporter gene (pGL3 control vector) was transiently transfected into the CD4− K-562 cell line and the CD4+ Jurkat cell-line using DEAE-dextran41 to demonstrate expression of the complex. Under these conditions, expression of the reporter gene was slightly greater in K-562 cells (460 000 ± 70 000 luciferase activity units/106 cells) than in Jurkat cells (320 000 ± 10 000 luciferase activity units/106 cells). This experiment showed that the complex was able to be expressed in each cell line. The complex was then added to the cells in the absence of DEAE-dextran and the level of luciferase gene expression was determined 48 h later (Figure 2). Little or no expression of the B-F5 complex was seen in the K-562 cells whereas significant expression was measured in the Jurkat cells. In addition, complexes constructed either without antibody or with a non-specific antibody showed only background levels of expression in the Jurkat cells. When cells were treated with phorbol myristate acetate (PMA) and the B-F5 complex, expression was significantly elevated in the Jurkat cells but not in the K-562 cells. Finally, PMA had no effect on the level of luciferase expression from constructs containing no antibody or the non-specific antibody. These experiments show that the B-F5-pLL-DNA complex was

1775

Figure 1 Binding of 125I-B-F5 in the presence of various antibody conjugates and DNA complexes. The conjugates and complexes were synthesized as follows: a 10-fold molar excess of SPDP in dimethylformamide (DMF) was added to 3.23 nmol B-F5 or non-specific antibody (1 mg/ml in PBS, pH 7.4) and incubated at 37°C for 30 min. This solution was dialysed overnight against 5 l PBS at 4°C. pLL (average MW approximately 57 000) in HEPES-buffered saline (150 mm NaCl, 10 mm HEPES, pH 8.0) was incubated with a 10-fold excess of SPDP in DMF at 37°C for 45 min. SPDP-modified pLL was desalted and reduced using a ReduceImm Reducing Column (Pierce Chemical, Rockford, IL, USA) to give pLLSH. SPDP-modified B-F5 (or non-specific antibody) and pLL-SH (molar ratio of 4:1) were incubated at room temperature overnight on a rotating wheel. Aliquots of resulting solutions were analysed by SDS-PAGE. The DNA complex was made by a NaCl gradient method36,37,40 and the ratio of pGL3 control vector: pLL was 1:10 for all complexes. Briefly, aliquots of pGL3 control vector were slowly added to the conjugate solution with mixing and allowed to stand at room temperature for several minutes. Microlitre aliquots of 4 m NaCl were added to the turbid mixture in the same manner, until the solution achieved clarity. Binding of 125I-B-F5 (0.5 nm) at 37°C was investigated in the presence of various conjugate and complex solutions. After 4 h, aliquots of cells were centrifuged, washed twice with PBS at 37°C and 125I activity of the pellets determined by ␥ counting. Data are presented as mean ± s.e.m., n = 3. NSIgG, nonspecific antibody.

selectively expressed in the CD4+ cells and that this specificity was determined by the anti-CD4 antibody. To determine the extent of cell transfection by the antibody–DNA complex, a construct containing the GFP gene was used. Cells were treated as above and examined by confocal microscopy. Figure 3A shows the level of expression in Jurkat cells of a complex consisting of the reporter vector and pLL. Little or no expression was observed compared with the complex containing B-F5 (Figure 3B). Similarly, when B-F5 was replaced with a non-specific antibody, the level of GFP expression was markedly decreased (Figure 3D). The percentage of cells expressing GFP following treatment with the B-F5 complex was greater than 95%. When cells were treated with the B-F5 complex and PMA, GFP expression was not only enhanced, but it was also observed much earlier (Figure 3C). Finally, the B-F5 complex did not express in K-562 cells even in the presence of PMA (Figure 3E). Dual-color flow cytometric analysis was used to determine the binding specificity of the complex to primary lymphocytes in peripheral blood. For these experiments, the B-F5-pLL conjugate was labelled with fluorescein iso-


1776

Figure 2 Transfection of Jurkat and K-562 cells with various DNA complexes. Cells (1.0 × 107) were transfected in RPMI 1640 containing 20 mm HEPES (RPMI/HEPES) at 37°C with 5% CO2 in the presence and absence of 100 ng/ml PMA. At 1 and 4 h, additional RPMI/HEPES was added to the transfecting incubations. At 12 h, pelleted cells were washed and resuspended in RPMI 1640 supplemented with 10% FCS. At 48 h after transfection, luciferase activity of cell lysates was determined by single photon counting on a liquid scintillation counter. Cell viability was greater than 90% under all conditions. Data are presented as mean ± s.e.m. of three independent experiments. NSIgG, non-specific antibody.

thiocyanate (FITC) and used to produce a complex (FITC complex). Human peripheral blood mononuclear cells (PBMC) were incubated with the complex and with phycoerythrin-labelled anti-human CD4 antibody (PE-CD4) which specifically labels CD4+ primary lymphocytes. The PE-CD4 used was clone Q4120 (Sigma Chemical, St Louis, MO, USA) which bound to a different epitope to B-F5, as determined by competition assays (data not shown). Figure 4a shows the flow cytometry of PMBC in the absence of either complex or PE-CD4. In the presence of PE-CD4 (Figure 4b), approximately 11% of the cells showed specific binding of the antibody. A similar proportion of cells specifically bound FITC-complex (Figure 4c). When PBMC were incubated with both FITC-complex and PE-CD4 (Figure 4d), nearly all of the CD4+ cells bound the complex, as shown by the shift in the cell population from the upper left quadrant to the upper right quadrant. In the present study, a CD4 receptor targeted polycationic gene complex was constructed using the monoclonal antibody B-F5. The complex was shown to target CD4+ cells selectively with a transfection efficiency of greater than 95%. Moreover, the complex showed specific binding to CD4+ primary lymphocytes in PBMC. These investigations are the first demonstrating the selective transfection of CD4+ T lymphoid cells using a polycationmediated gene delivery system. The data also suggest

Figure 3 Confocal laser scanning microscopy of Jurkat and K-562 cells transfected with various DNA complexes. Transfections were conducted as described in Figure 2 except the pGL3 control vector was replaced with the pGeneGrip vector. Aliquots of cells were removed at 12 and 48 h and cytospun on to glass slides. The slides were air dried for approximately 60 min at room temperature in the dark. The cells were mounted in PVA biopsy mounting medium (40 mm Tris phosphate buffer, pH 9, 25% glycerol w/v, 0.1% chlorbutanol w/v in polyvinyl alcohol). Confocal laser scanning microscopy (MRC-1024; BioRad, Hercules, CA, USA) using a Nikon Plan Apo 60/1.4 oil immersion lens was employed to detect fluorescence. The 488 nm line of a 250 mW Argon Ion laser was used to excite the cells, and emission was detected following passage through a 522/35 nm band pass filter. (A) Jurkat cells treated with pLL-DNA complex; (B) Jurkat cells treated with B-F5 complex; (C) Jurkat cells treated with B-F5 complex and PMA; (D) Jurkat cells treated with non-specific antibody complex; (E) K-562 cells treated with B-F5 complex and PMA.


5

6 7

8

9

10 11

12 Figure 4 Targeting of primary CD4+ lymphocytes in peripheral blood mononuclear cells (PBMC). Dual color flow cytometry was used to determine the degree of targeting of complex to CD4+ cells in PBMC. B-F5pLL conjugate was labeled with fluorescein by incubating the conjugate with a 10-fold molar excess (with respect to pLL) of fluorescein isothiocyanate in 0.1 m Na2CO3 for 30 min at room temperature. After desalting on G25 resin, the conjugate was used to produce a complex (FITCcomplex) as described in Figure 1. To detect CD4+ lymphocytes, the cells were incubated with phycoerythrin-antihuman-anti-CD4 antibody (PECD4). This antibody (clone Q4120) did not affect the binding of B-F5 to CD4. PBMC were prepared by centrifugation of whole blood on Ficoll– Plaque. The cells were washed and resuspended in PBS containing 1% FCS. PE-CD4 and FITC-complex were added to the cells at 4°C for 30 min. The cells were washed three times with PBS containing 1% FCS and resuspended in PBS. Flow cytometry was performed using a Coulter Epics cell sorter (Beckman Coulter, Fullerton, CA, USA). A total of 10 000 events was collected and the data were gated to exclude contaminating red blood cells if they were present. The results are representative of two independent experiments. Fluoresence intensity is displayed on a logarithmic scale.

13

14

15

16

17

18

19

that pharmacological enhancement of CD4 internalisation may facilitate expression of transgenes delivered to CD4+ cells in this manner.

20 21

Acknowledgements We wish to thank John Wijdenes of Diaclone, Besanc¸on, France for his generous gift of the antibody, B-F5. This work was supported in part by the National Health and Medical Research Council of Australia, the Raine Medical Research Foundation and the AIDS Trust of Australia.

22

23

24

References 1 Janeway CA Jr. The T cell receptor as a multicomponent signalling machine: CD4/CD8 co-receptors and CD45 in T cell activation. Annu Rev Immunol 1992; 10: 645–674. 2 Dalgleish AG et al. The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature 1984; 312: 763–767. 3 Klatzmann D et al. T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV. Nature 1984; 312: 767–768. 4 Feng Y, Broder CC, Kennedy PE, Berger EA. HIV-1 entry cofac-

25

26

tor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science 1996; 272: 872–877. Alkhatib G et al. CC CKR5: a RANTES, MIP-1␣, MIP-1␤ receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 1996; 272: 1955–1958. Deng H et al. Identification of a major co-receptor for primary isolates of HIV-1. Nature 1996; 381: 661–666. Chang HK et al. Block of HIV-1 infection by a combination of antisense tat RNA and TAR decoys: a strategy for control of HIV-1. Gene Therapy 1994; 1: 208–216. Sun L-Q et al. Ribozyme-mediated suppression of Moloney murine leukemia virus and human immunodeficiency virus type 1 replication in permissive cell lines. Proc Natl Acad Sci USA 1994; 91: 9715–9719. Trono D, Feinberg MB, Baltimore D. HIV-1 Gag mutants can dominantly interfere with the replication of the wild-type virus. Cell 1989; 59: 113–120. Escaich S et al. RevM10-mediated inhibition of HIV-1 replication in chronically infected T cells. Hum Gene Ther 1995; 6: 625–634. Bonyhadi ML et al. RevM10-expressing T cells derived in vivo from transduced human hematopoietic stem-progenitor cells inhibit human immunodeficiency virus replication. J Virol 1997; 71: 4707–4716. Bai X et al. Genetic co-inactivation of macrophage- and T-tropic HIV-1 chemokine coreceptors CCR-5 and CXCR-4 by intrakines. Gene Therapy 1998; 5: 984–994. Schnierle BS et al. Pseudotyping of murine leukemia virus with the envelope glycoproteins of HIV generates a retroviral vector with specificity of infection for CD4-expressing cells. Proc Natl Acad Sci USA 1997; 94: 8640–8645. Johnson JE, Schnell MJ, Buonocore L, Rose JK. Specific targeting to CD4+ cells of recombinant vesicular stomatitis viruses encoding human immunodeficiency virus envelope proteins. J Virol 1997; 71: 5060–5068. Schreier H, Moran P, Caras IW. Targeting of liposomes to cells expressing CD4 using glycophosphatidylinositol-anchored gp120. J Biol Chem 1994; 269: 9090–9098. Zarling JM et al. Inhibition of HIV replication by pokeweed antiviral protein targeted to CD4+ cells by monoclonal antibodies. Nature 1990; 347: 92–95. Suzuki H, Zelphati O, Hildebrand G, Leserman L. CD4 and CD7 molecules as targets for drug delivery from antibody-bearing liposomes. Exp Cell Res 1991; 193: 112–119. Yemul S et al. Phototoxic liposomes coupled to an antibody that alone cannot modulate its cell-surface antigen kill selected target cells. Cancer Immunol Immunother 1990; 30: 317–322. Morelli D et al. A monoclonal antibody extends the half-life of an anti-HIV oligodeoxynucleotide and targets it to CD4+ cells. Nucleic Acids Res 1995; 23: 4603–4607. Selvam MP et al. Inhibition of HIV replication by immunoliposomal antisense oligonucleotide. Antiviral Res 1996; 33: 11–20. Wu GY, Wu CH. Receptor-mediated in vitro gene transformation by a soluble DNA carrier system. J Biol Chem 1987; 262: 4429–4432. Wagner E et al. Transferrin-polycation conjugates as carriers for DNA uptake into cells. Proc Natl Acad Sci USA 1990; 87: 3410– 3414. Edwards RJ, Carpenter DS, Minchin RF. Uptake and intracellular trafficking of asialoglycoprotein-polylysine-DNA complexes in isolated rat hepatocytes. Gene Therapy 1996; 3: 937–940. Racadot E et al. Treatment of multiple sclerosis with anti-CD4 monoclonal antibody. A preliminary report on B-F5 in 21 patients. J Autoimmunity 1993; 6: 771–786. Piatier-Tonneau D et al. Characterisation of 18 workshop antiCD4 mAb: epitope mapping to CD4 mutants and effects on CD4-HLA class II interaction. In: Schlossman SF et al (eds). Leukocyte Typing: V, vol 1, Oxford University: Oxford, 1995, pp 476–478. Racadot E, Wijdenes J, Wendling D. Immunological follow-up of 17 patients with rheumatoid arthritis treated in vivo with an anti-T CD4+ monoclonal antibody (B-F5). Clin Exp Rheumatol 1992; 10: 365–374.

1777


1778

27 Wendling D, Racadot E, Morel-Fourrier B, Wijdenes J. Treatment of rheumatoid arthritis with anti-CD4 monoclonal antibody. Open study of 25 patients with the B-F5 clone. Clin Rheumatol 1992; 11: 542–547. 28 Canva-Delcambre V et al. Treatment of severe Crohn’s disease with anti-CD4 monoclonal antibody. Aliment Pharmacol Ther 1996; 10: 721–727. 29 Dantal J et al. Anti-CD4 MoAb therapy in kidney transplantation – a pilot study in early prophylaxis of rejection. Transplant. 1996; 62: 1502–1506. 30 Pelchen-Matthews A et al. The protein tyrosine kinase p56lck inhibits CD4 endocytosis by preventing entry of CD4 into coated pits. J Cell Biol 1992; 117: 279–290. 31 Morel P, Vincent C, Wijdenes J, Revillard J-P. Down-regulation of cell surface CD4 molecule expression induced by anti-CD4 antibodies in human T lymphocytes. Cell Immunol 1992; 145: 287–298. 32 Rudd CE et al. The CD4 antigen is complexed in detergent lysates to a protein tyrosine kinase (pp58) from human T lymphocytes. Proc Natl Acad Sci USA 1988; 85: 5190–5194. 33 Veillette A, Bookman MA, Horak EM, Bolen JB. The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56lck. Cell 1988; 55: 301–308. 34 Acres BR, Conlon PJ, Mochizuki DY, Gallis B. Rapid phos-

35

36

37

38

39

40

41

phorylation and modulation of the T4 antigen on cloned helper T cells induced by phorbol myristate acetate or antigen. J Biol Chem 1986; 261: 16210–16214. Hoxie JA et al. Transient modulation and internalisation of the T4 antigen induced by phorbol esters. J Immunol 1986; 137: 1194–1201. Perales JC et al. Gene transfer in vivo: sustained expression and regulation of genes introduced into the liver by receptor-targeted uptake. Proc Natl Acad Sci USA 1994; 91: 4086–4090. Ferkol T et al. Gene transfer into the airway epithelium of animals by targeting the polymeric immunoglobulin receptor. J Clin Invest 1995; 95: 493–502. Lee RJ, Huang L. Folate-targeted, anionic liposome-entrapped polylysine-condensed DNA for tumour cell-specific gene transfer. J Biol Chem 1996; 271: 8481–8487. Vitiello L et al. Condensation of plasmid DNA with polylysine improves liposome-mediated gene transfer into established and primary muscle cells. Gene Therapy 1996; 3: 396–404. Perales JC et al. Biochemical and functional characterisation of DNA complexes capable of targeting genes to hepatocytes via the asialoglycoprotein receptor. J Biol Chem 1997; 272: 7398–7407. McCutchan JH, Pagano JS. Enhancement of the infectivity of Simian virus 40 deoxyribonucleic acid with diethyl-aminoethyldextran. J Natl Cancer Inst 1968; 41: 351–356.