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Efficient gene transfer by fiber-mutant adenoviral vectors containing RGD peptide
Biochimica et Biophysica Acta 1568 (2001) 13^20
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E¤cient gene transfer by ¢ber-mutant adenoviral vectors containing RGD peptide Naoya Koizumi a;b , Hiroyuki Mizuguchi a; *, Tetsuji Hosono a , Akiko Ishii-Watabe a , Eriko Uchida a , Naoki Utoguchi b , Yoshiteru Watanabe b , Takao Hayakawa a a
Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan b Department of Pharmaceutics and Biopharmaceutics, Showa Pharmaceutical University, Tokyo 194-8543, Japan Received 19 April 2001 ; received in revised form 31 July 2001; accepted 1 August 2001
Abstract One of the hurdles to adenovirus (Ad)-mediated gene transfer is that Ad vectors mediate inefficient gene transfer into cells lacking in the primary receptors, Coxsackievirus and adenovirus receptor (CAR). We previously developed a fiber-mutant Ad vector containing the ArgGly-Asp (RGD)-containing peptide motif on the HI loop of the fiber knob, and showed that the mutant vector had enhanced gene transfer activity to human glioma cells, which showed little CAR expression, compared to the vector containing wild type fiber. In this study, the feasibility of the Ad vector containing RGD peptide on the fiber knob was examined in a wide variety of cell types: CAR-positive or -negative human tumor cells, mouse cells, and leukemia cells. The mutant vector infected the cells, which lacked CAR expression but showed Kv integrin expression, about 10^1000 times more efficiently than the vector containing wild type fiber via an RGD-integrin (KvL3 and KvL5)-dependent, CAR-independent cell entry pathway. The results of this study indicate that Ad vector containing RGD peptide on the fiber knob could be of great utility for gene therapy and gene transfer experiments. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : Gene therapy; Adenovirus vector; Fiber ; Retargeting
1. Introduction Recombinant adenovirus (Ad) vectors are potentially useful for gene transfer to a wide variety of cells and tissues both in vitro and in vivo. They can be grown easily to high titer, and can transfer the genes to both diving and nondividing cells [1,2]. There are, however, some limitations associated with the use of Ad vectors. One such disadvantage is that Ad vectors result in ine¤cient gene transfer to cells lacking in the primary Ad receptors, Coxsackievirus and adenovirus receptor (CAR) [3^6]. The initial step of Ad infection involves at least two sequential steps. The ¢rst step is the attachment of the virus to the cell surface through binding of the knob domain of the ¢ber to CAR [3,7]. Following attachment, the viral internalization into the cells occurs by the interaction of RGD (Arg-Gly-Asp) motifs of penton base with integrin receptors, KvL3 and KvL5, expressed on most cell types
* Corresponding author. Fax: +81-3-3700-9084. E-mail address : [email protected] (H. Mizuguchi).
[8,9]. Therefore, the interaction of the ¢ber knob with CAR on the cell is the key mediator by which Ad vectors enter the cells. Modi¢cation of ¢ber protein is an attractive strategy for overcoming the limitations imposed by the CAR dependence of Ad infection [10]. We and other groups reported that an Ad vector containing RGD peptide, which binds with high a¤nity to integrins (KvL3 and KvL5) on the cell surface, on the HI loop or the C-terminal portion of the ¢ber protein mediated not only CAR-dependent gene delivery but also CAR-independent, RGD-integrin (KvL3 and KvL5)-dependent gene delivery [11^13]. However, enhanced gene transfers by Ad vector containing RGD peptide have been examined in only limited types of cells. In this study, we compared the usefulness of an Ad vector containing RGD peptide on the ¢ber knob with that of an Ad vector containing wild type ¢ber with respect to their potential e¤cacy for gene transfer using several types of human cells (CAR-positive or -negative), mouse cells, and human leukemia cells (14 types in total). Data suggest that an Ad vector containing RGD peptide on the ¢ber knob could be of great utility for e¤cient gene transfer to a wide variety of cell types.
0304-4165 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 1 ) 0 0 1 9 4 - 5
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2. Materials and methods
2.3. Adenovirus-mediated gene transduction
2.1. Cells
The cells (1U104 cells) were seeded into a 96-well dish. On the following day, they were transduced with Ad-L2 or AdRGD-L2 (300 or 3000 vector particles (VP)/cell) for 1.5 h. After 48 h in culture, luciferase productions in the cells were measured using a luciferase assay system (PicaGene LT2.0 ; Toyo Inki, Tokyo, Japan). We repeated all transfection experiments and obtained similar results.
SK HEP-1 cells (an endothelial cell line derived from human liver) [14] were cultured in Dulbecco's modi¢ed Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). Human glioma cell lines, LN319 (anaplastic astrocytoma), LN444 (glioblastoma multiforme), LNZ308 (glioblastoma multiforme), and SF295 cells (glioblastoma multiforme) [15] (kindly provided by Dr. M. Tada, Hokkaido University, Hokkaido, Japan) were cultured in DMEM supplemented with 10% FCS. MS1 cells (mouse pancreatic islet endothelial cell line, ATCC, CRL2279) were cultured in Dulbecco's modi¢ed Eagle's medium supplemented with 5% FCS. L (Lcl1D, mouse skin ¢broblast; Human Science Research Resources Bank (HSRRB), JCRB0722) and NIH3T3 cells (mouse embryo ¢broblast; HSRRB, JCRB0615) were cultured in minimum essential medium (MEM) supplemented with 10% FCS. B16BL6 cells and colon26 cells (kindly provided by Dr. T. Mayumi, Osaka University, Osaka, Japan) were cultured in MEM supplemented with 10% FCS or in RPMI 1640 medium supplemented with 10% FCS, respectively. KG-1a cells (human bone marrow acute myelogenous leukemia cells, obtained from Dainippon, Osaka, Japan) were cultured in Iscove's modi¢ed Dulbecco's medium supplemented with 20% FCS. Y-79 cells (human retinoblastoma cells, obtained from Dainippon) were cultured in RPMI 1640 medium supplemented with 15% FCS. Ramos cells (Burkitt lymphoma cells, HSRRB, JCRB9119) and K-562 cells (chronic myelogenous leukemia cells from blast crisis, HSRRB, JCRB0019) were cultured in RPMI 1640 medium supplemented with 10% FCS.
2.4. Flow cytometry The cells (5U105 cells) from human origin were labeled with mouse monoclonal antibody RmcB [3] (kindly provided by Dr. J.M. Bergelson, Children's Hospital of Philadelphia, PA, USA) for human CAR detection, mouse anti-human integrin KvL3 (LM609; Chemicon International, Temecula, CA, USA), or mouse anti-human integrin KvL5 (P1F6; Gibco BRL, Gaithersburg, MD, USA). Then, the cells were incubated with FITC-conjugated goat anti-mouse IgG second antibody (Pharmingen, San Diego,
2.2. Plasmid and virus Luciferase-expressing Ad vectors, Ad-L2 and AdRGDL2, have been constructed previously by an improved in vitro ligation method [13,16,17]. The cytomegalovirus (CMV) promoter-driven luciferase gene derived from the pGL3-Control (Promega, Madison, WI, USA) was inserted into the E1 deletion region of the Ad genome. Ad-L2 contains wild type ¢ber, while AdRGD-L2 contains mutant ¢ber containing the RGD peptide, CDCRGDCFC, in the HI loop of the ¢ber knob. Viruses (Ad-L2 and AdRGD-L2) were prepared as described previously [16] and puri¢ed by CsCl2 step gradient ultracentrifugation followed by CsCl2 linear gradient ultracentrifugation. Determination of virus particle titer and infectious (plaque forming unit : PFU) titer was accomplished spectrophotometrically by the method of Maizel et al. [18] and by the method of Kanegae et al. [19], respectively. The PFU to particle ratio was 1:8 for Ad-L2 and 1:23 for AdRGD-L2.
Fig. 1. Flow cytometric analysis of CAR and integrin (KvL3 and KvL5) expression in several human adherent cells. Cells were incubated with anti-CAR (RmcB), anti-KvL3 (LM609) and anti-KvL5 (P1F6) integrin monoclonal antibodies. Then, the cells were incubated with FITC-conjugated goat anti-mouse IgG second antibody and analyzed by £ow cytometry. (A) SK HEP-1; (B) LN319; (C) LN444 ; (D) LNZ308 ; (E) SF295. Flow cytometric analysis of CAR and integrin expression in SK HEP-1 and LN444 cells has been described previously [13].
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Fig. 2. Comparison of luciferase production in several human adherent cells transduced by Ad-L2 and AdRGD-L2. SK HEP-1 (A), LN319 (B), LN444 (C), LNZ308 (D), and SF295 (E) cells were transduced with 300 or 3000 VP/cell of Ad-L2 and AdRGD-L2 for 1.5 h. Forty-eight hours later luciferase production was measured by luminescent assay. All data represent the mean þ S.D. of four experiments. n.d., not detectable. Luciferase production in SK HEP-1 and LN444 cells has described previously [13].
CA, USA) and analyzed by £ow cytometry with a Cyto ACE-150 Auto Cell Screener (Jasco, Tokyo, Japan). 2.5. Reverse transcription (RT)-polymerase chain reaction (PCR) analysis Total RNA of the cells from mouse origin was isolated using an RNeasy total RNA kit (Qiagen, Valencia, CA, USA). RT was carried out using a SuperScript Preampli¢cation System for First Strand cDNA Synthesis (Life Technologies, Rockville, MD, USA) according to the instructions of the manufacturer. In brief, 1 Wg total RNA was treated with DNase I. DNase I-treated RNA was annealed to 0.5 Wg oligo(dT)12ÿ18 at 70³C for 10 min, and then chilled on ice. RT was performed for 50 min at 42³C in a reaction mixture containing 2 Wl 10UPCR bu¡er, 2 Wl 25 mM MgCl2 , 1 Wl 10 mM dNTP mix, 2 Wl 0.1 M DTT, and 200 U superscript II reverse transcriptase. PCR ampli¢cation of the mouse CAR, mouse integrin receptor (Kv, L3, L5) transcripts and GAPDH was performed in 100 Wl of the reaction mixture containing 1 Wl of RT material, 2.5 U Ampli Taq DNA polymerase, 1.5 mM MgCl2 and 0.2 mM dNTP using GeneAmp PCR Core Reagents (Perkin Elmer, Norwalk, CT, USA). The sequences of the primers for ampli¢cation of the di¡erent receptors are as follows [20]: CAR: forward, 5P-tga tca ttt tgt att ctg ga-3P; reverse, 5P-tta aca aga acg gtc agc ag-3P; integrin Kv: forward, 5P-cca gcc tgg gat tgt aga ag-3P; reverse, 5P-act cca gtg ggt cat ctt tg-3P; integrin L3: forward, 5P-tct ggc tgt gag tcc tgt gt-3P; reverse, 5P-gcc tca ctg
act ggg aac tc-3P; integrin L5: forward, 5P-tcg tgt gaa gaa tgc ctg tt-3P; reverse, 5P-gct gga ctc tca atc tca cc-3P; GAPDH : forward, 5P-acc aca gtc cat gcc atc ac-3P; reverse, 5P-tcc acc acc ctg ttg ctg ta-3P. The following parameters were used: CAR: 45 s at 94³C, 60 s at 50³C, and 90 s at 72³C for 40 cycles ; integrin Kv, L3, L5: 45 s at 94³C, 60 s at 53³C, and 90 s at 72³C for 40 cycles; GAPDH : 45 s at 94³C, 60 s at 50³C, and 90 s at 72³C for 30 cycles. The expected PCR product sizes were : CAR, 211 bp; integrin Kv, 105 bp; integrin L3, 115 bp; integrin L5, 126 bp; GAPDH, 452 bp. The PCR product was electrophoresed in 2.0% agarose gel. To ensure the quality of the procedure, RT-PCR was also performed on the sample using speci¢c primers for GAPDH. 2.6. Recombinant ¢ber knob Recombinant Ad type 5 ¢ber knob protein was expressed in baculovirus with an N-terminal hexahistidine tag. The ¢ber knob-coding sequence was synthesized by PCR on pAdHM4 [16] that contained the E1/E3-deleted Ad genome, with the following primers: forward, 5P-agt cga att cac ttt gtg gac cac acc agc tcc-3P (EcoRI site is underlined); reverse, 5P-agt cgc ggc cgc tta ttc ttg ggc aat gta tga aa-3P (NotI site is underlined). PCR products were digested with EcoRI and NotI, and then cloned into the EcoRI-NotI site of pAcHLT-A, which is a baculovirus transfer vector (PhaMingen, San Diego, CA, USA). Recombinant Ad type 5 ¢ber knob protein was expressed in
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baculovirus-infected Sf9 cells according to the manufacturer's instructions, and puri¢ed by chromatography on NiNTA-Sepharose. Recombinant Ad type 5 ¢ber knob protein containing RGD peptide was prepared similarly using pAdHM15-RGD [13] as a template of PCR. 3. Results 3.1. Human adherent cells We evaluated Ad vector containing RGD peptide on the ¢ber knob as a gene delivery vehicle for several types of human glioma cells (LN319, LN444, LNZ308, and SF295) and SK HEP-1 cells, in comparison with Ad vector containing wild type ¢ber. First, the expression levels of CAR, KvL3 and KvL5 integrins on each cell surface were examined by £ow cytometry analysis (Fig. 1). Signi¢cant amounts of CAR were detected on SK HEP-1 and
LN319 cells (Fig. 1A,B). In contrast, no or very low levels of CAR were detected on LN444, LNZ308, and SF295 cells (Fig. 1C^E). LN319 and LN444 cells expressed KvL5 integrin and no or very low levels of KvL3 integrin (Fig. 1B,C). SK HEP-1, LNZ308, and SF295 cells expressed both KvL3 and KvL5 integrins (Fig. 1A,D,E). Ad-L2, the Ad vector containing wild type ¢ber, mediated e¤cient gene transfer to SK HEP-1 and LN319 cells (Fig. 2A,B). In contrast, luciferase production in other cell lines (LN444, LNZ308, and SF295) transduced by Ad-L2 was over 2^3 log orders lower than that in SK HEP-1 and LN319 cells transduced by Ad-L2 (Fig. 2C^E). These results suggest that Ad vector containing wild type ¢ber mediated less e¤cient gene transfer to CAR-negative cells. On the other hand, AdRGD-L2, the Ad vector containing RGD peptide on the ¢ber knob, mediated e¤cient luciferase expression even in the CAR-negative cell lines (LN444, LNZ308, and SF295). LN444, LNZ308, and SF295 cells transduced with Ad-L2 and AdRGD-L2 re-
Fig. 3. Inhibition of transduction of Ad-L2 and AdRGD-L2 to SK HEP-1 and LN444 cells by recombinant ¢ber knob. SK HEP-1 (A) and LN444 (B) cells were preincubated with recombinant wild type ¢ber knob (A-1, A-2, B-1) or recombinant ¢ber knob containing RGD peptide (B-2) (0, 0.4, 2, 10 Wg/ml) for 10 min. Then, the cells were transduced with 300 VP/cell of Ad-L2 (A-1) or AdRGD-L2 (A-2, B-1, B-2) for 0.5 h in the presence of recombinant ¢ber knob. Forty-eight hours later luciferase production was measured by luminescent assay. All data represent the mean þ S.D. of four experiments.
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Fig. 4. RT-PCR analysis of levels of CAR and integrin (Kv, L3, and L5) expression in mouse cells. Total RNA was isolated from MS1, NIH3T3, L, B16BL6, and colon26 cells, and then RT-PCR analysis was performed as described in Section 2. Lanes: 1, MS1; 2, NIH3T3; 3, L; 4, B16BL6; 5, colon26; 6, water; 7, DNA marker.
sulted in 2^4 log orders di¡erence in luciferase production (Fig. 2). In contrast, SK HEP-1 and LN319 cells transduced with Ad-L2 and AdRGD-L2 showed similar levels of luciferase production (less than 1 log order di¡erence). RGD peptide sequence inserted on the ¢ber knob binds with high a¤nity to both KvL3 and KvL5 integrins [21,22]. LN444, LNZ308, and SF295 cells expressed KvL5 (and KvL3) integrin. Therefore, the presence of RGD peptide on the ¢ber knob was required for e¤cient transduction of these CAR-de¢cient cells. To demonstrate the e¡ect of RGD peptide on the ¢ber knob on e¤cient transduction to CAR-de¢cient cells more
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clearly, a blocking experiment of Ad-mediated gene transfer with recombinant wild type ¢ber knob or recombinant ¢ber knob containing RGD peptide was performed (Fig. 3). Recombinant wild type ¢ber knob inhibited Ad-L2mediated luciferase production in SK HEP-1 cells, which express CAR, in a dose-dependent manner, but not AdRGD-L2-mediated luciferase production, suggesting that the AdRGD-L2 infects SK HEP-1 cells via a CARindependent pathway as well (Fig. 3A). Furthermore, recombinant wild type ¢ber knob did not inhibit AdRGDL2-mediated gene transfer in LN444 cells, which do not express CAR, while recombinant ¢ber knob containing RGD peptide inhibited AdRGD-L2-mediated gene transfer in LN444 cells (Fig. 3B). Taken together, these results suggest that AdRGD-L2 infects the cells via not only a CAR-dependent pathway, but also an RGD-integrin-dependent pathway. 3.2. Mouse cells Next, we examined the utility of an Ad vector containing RGD peptide on the ¢ber knob for gene transfer to mouse cells. Most mouse cells are known to be transduced by Ad vectors less e¤ciently than human cells. Five cell lines, MS1, NIH3T3, L, B16BL6, and colon26, were used. First, we examined the mRNA levels of CAR, Kv-integrin, L3-integrin, and L5-integrin on each cell by RT-PCR analysis (Fig. 4). We used RT-PCR analysis for mouse cells due to the lack of appropriate antibodies to detect the mouse CAR and integrin. CAR mRNA was detectable in MS1 cells. In contrast, no CAR mRNA was detected
Fig. 5. Comparison of luciferase production in mouse cells transduced by Ad-L2 and AdRGD-L2. MS1 (A), NIH3T3 (B), L (C), B16BL6 (D), and colon26 (E) cells were transduced with 300 or 3000 VP/cell of Ad-L2 and AdRGD-L2 for 1.5 h. Forty-eight hours later luciferase production was measured by luminescent assay. All data represent the mean þ S.D. of four experiments. n.d., not detectable.
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Fig. 6. Flow cytometric analysis of CAR and integrin expression in human leukemia cells. Cells were incubated with anti-CAR (RmcB), antiKvL3 (LM609) and anti-KvL5 (P1F6) integrin monoclonal antibodies. Then, the cells were incubated with FITC-conjugated goat anti-mouse IgG second antibody and analyzed by £ow cytometry. (A) K-562; (B) Y79; (C) Ramos; (D) KG-1a.
in NIH3T3, L, B16BL6, or colon26 cells under the conditions used here. All cell lines expressed Kv integrin, L3 integrin, and L5 integrin, suggesting that the cells should possess KvL3 and KvL5 integrins as heterodimers on their cellular surface. Luciferase production in MS1 cells following transduction with Ad-L2 was moderate, but the luciferase production in NIH3T3, L, B16BL6, and colon26 cells was slight due to the lack of CAR expression in these cells (Fig. 5). On the other hand, AdRGD-L2-mediated luciferase expression was 2^3 log orders more e¤cient than Ad-L2mediated luciferase expression in NIH3T3, L, B16BL6, and colon26 cells. AdRGD-L2 also mediated more e¤cient luciferase expression in MS1 cells. These results suggested that RGD peptide on the ¢ber knob also binds with high a¤nity to KvL3 or KvL5 integrin on mouse cells, and that Ad vector containing RGD peptide on the ¢ber knob would be of great utility for e¤cient gene transfer to mouse-derived CAR-de¢cient cells. 3.3. Human leukemia cells Most leukemia (suspension) cells are not e¤ciently transduced by conventional Ad vectors containing wild type ¢ber. We compared the gene transfer activity of Ad-L2 and AdRGD-L2 in four kinds of human leukemia
cells, K-562, Y79, Ramos, and KG-1a cells (Fig. 7). K-562, Y79, and Ramos cells expressed CAR (Fig. 6). As a result, Ad-L2 mediated moderate levels of luciferase expression in these types of cells. Their activities, however, were 2^3 log orders less than those in the human adherent cells (Figs. 2 and 7). K-562 cells expressed a moderate amount of KvL3 or KvL5 integrins, while Y79 cells expressed a moderate amount of KvL5 integrins. As a result, K-562 and Y79 cells transduced with AdRGD-L2 expressed an approx. 10-fold greater amount of luciferase than those transduced with Ad-L2, but this amount was still less than that in human adherent cells transduced with AdRGD-L2. Ramos cells expressed only slight amounts of KvL3 or KvL5 integrins. There was only a 3-fold di¡erence in luciferase production between Ramos cells transduced with Ad-L2 and those transduced with AdRGD-L2. KG1a cells did not express CAR, but expressed small amounts of KvL3 integrin. AdRGD-L2 mediated small levels of luciferase expression in KG-1a cells, while Ad-L2 did not. These results suggest that AdRGD-L2 improved the e¤ciency of Ad-mediated gene transfer to human leukemia cells expressing KvL3 or KvL5 integrin, but this e¡ect was not su¤ciently high compared with the transduction e¤ciency to adherent cells. 4. Discussion Expression of cellular surface Ad receptor, CAR, is an important determinant for e¤cient Ad-mediated gene de-
Fig. 7. Comparison of luciferase production in human leukemia cells transduced by Ad-L2 and AdRGD-L2. K-562 (A), Y79 (B), Ramos (C), and KG-1a (D) cells were transduced with 300 or 3000 VP/cell of AdL2 and AdRGD-L2 for 1.5 h. Forty-eight hours later luciferase production was measured by luminescent assay. All data represent the mean þ S.D. of four experiments. n.d., not detectable.
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livery [3^6]. Ad vector mediated ine¤cient gene transfer to cells lacking in CAR expression. This barrier can be overcome by the alteration of Ad ¢ber protein [10]. We and others developed an Ad vector containing RGD peptide on the ¢ber knob, which targets Kv (KvL3 and KvL5) integrin on the cellular surface, and showed that the mutant vector had expanded tropism [11^13]. In this study, we examined the utility of Ad vector containing RGD peptide on the ¢ber knob for gene therapy and gene transfer experiments in various types of cells (a total of 14 cell lines). The results clearly showed that Ad vector containing wild type ¢ber mediated less e¤cient gene transfer to the CAR-negative cells, while Ad vector containing RGD peptide on the ¢ber knob mediated about 100^1000 times more e¤cient gene transfer to the CAR-negative but Kv integrin-positive human adherent cells (Fig. 2). This enhanced e¡ect was also observed in mouse cells (Fig. 5). It has been reported that several types of cells and tissues, e.g. smooth muscle cells, bronchial epithelium and skeletal muscle cells, do not readily allow e¤cient introduction of the transgene by Ad vector due to their ine¤cient expression of CAR [10]. If these types of cells express KvL3 or KvL5 integrins, they would be expected to allow e¤cient transduction by Ad vector containing RGD peptide on the ¢ber knob. However, in leukemia cells, which are known to resist e¤cient transduction by Ad vector, the enhancement of transduction by use of a ¢ber-modi¢ed vector was smaller than that in adherent cells (Fig. 7). The luciferase production in leukemia cells transduced with ¢ber-modi¢ed vector containing RGD peptide was also lower than that in adherent cells. Moreover, no luciferase production occurred in HL-60 cells (acute promyelotic leukemia cells) transduced with Ad-L2 or AdRGD-L2 (data not shown). These results suggest that an Ad vector containing RGD peptide on the ¢ber knob has great potential for e¤cient transduction to most CAR-negative cells, but does not adequately enhance transduction to some leukemia cells. The RGD peptide motif is thus considered inadequate for ¢ber modi¢cation for transduction to these types of leukemia cells. We previously reported a simple method for construction of a ¢ber-mutant Ad vector [13]. In our system, foreign DNAs corresponding to the peptide of interest can be introduced into the HI loop coding region of the ¢ber knob of the Ad genome by one step in vitro ligation. Modi¢cation of the ¢ber knob by a peptide having high a¤nities to the molecules on the leukemia cell might lead to more e¤cient transduction. These studies are underway. The ¢ber-mutant Ad vector has several advantages compared to the conventional Ad vectors for use in transduction. When the vector is injected into CAR-negative cells or animal tissues in vivo, such as CAR-negative tumor cells, smooth muscle cells, bronchial epithelium, skeletal muscle cells, etc., the transduction can be enhanced and the side e¡ects lessened by reducing the injected dose of
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the vector. Furthermore, the use of ¢ber-mutant Ad vectors broadens the spectrum of possible Ad vector applications. Gene transfer experiments could be performed even in cells which do not readily lend themselves to transduction by the conventional Ad vectors. In summary, this study suggests that an Ad vector containing RGD peptide on the ¢ber knob mediated e¤cient transgene expression even in CAR-negative (but Kv integrin-positive) cells. Our study indicates the potential e¤cacy of the Ad vector containing RGD peptide on the ¢ber knob in gene therapy and gene transfer experiments. Acknowledgements We would like to thank Mr. Jun Murai and Ms. Nobuko Heishi for their technical assistance. This work was supported by grants from the Ministry of Health, Labour and Welfare of Japan and by a Grant-in-Aid for Scienti¢c Research on Priority Areas (C).
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E1/E4 deleted recombinant adenovirus vector, Hum. Gene Ther. 10 (1999) 2013^2017. J.V.J. Maizel, D.O. White, M.D. Schar¡, The polypeptides of adenovirus. I. Evidence for multiple protein components in the virion and a comparison of types 2, 7A, and 12, Virology 36 (1968) 115^125. Y. Kanegae, M. Makimura, I. Saito, A simple and e¤cient method for puri¢cation of infectious recombinant adenovirus, Jpn. J. Med. Sci. Biol. 47 (1994) 157^166. J.C. van Deutekom, B. Cao, R. Pruchnic, T.J. Wickham, I. Kovesdi, J. Huard, Extended tropism of an adenoviral vector does not circumvent the maturation-dependent transducibility of mouse skeletal muscle, J. Gene Med. 1 (1999) 393^399. E. Koivunen, B. Wang, E. Ruoslahti, Phage libraries displaying cyclic peptides with di¡erent ring sizes: ligand speci¢cities of the RGDdirected integrins, Biotechnology 13 (1995) 265^270. R. Pasqualini, E. Koivunen, E. Ruoslahti, Alpha v integrins as receptors for tumor targeting by circulating ligands, Nat. Biotechnol. 15 (1997) 542^546.
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E¤cient gene transfer by ¢ber-mutant adenoviral vectors containing RGD peptide Naoya Koizumi a;b , Hiroyuki Mizuguchi a; *, Tetsuji Hosono a , Akiko Ishii-Watabe a , Eriko Uchida a , Naoki Utoguchi b , Yoshiteru Watanabe b , Takao Hayakawa a a
Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan b Department of Pharmaceutics and Biopharmaceutics, Showa Pharmaceutical University, Tokyo 194-8543, Japan Received 19 April 2001 ; received in revised form 31 July 2001; accepted 1 August 2001
Abstract One of the hurdles to adenovirus (Ad)-mediated gene transfer is that Ad vectors mediate inefficient gene transfer into cells lacking in the primary receptors, Coxsackievirus and adenovirus receptor (CAR). We previously developed a fiber-mutant Ad vector containing the ArgGly-Asp (RGD)-containing peptide motif on the HI loop of the fiber knob, and showed that the mutant vector had enhanced gene transfer activity to human glioma cells, which showed little CAR expression, compared to the vector containing wild type fiber. In this study, the feasibility of the Ad vector containing RGD peptide on the fiber knob was examined in a wide variety of cell types: CAR-positive or -negative human tumor cells, mouse cells, and leukemia cells. The mutant vector infected the cells, which lacked CAR expression but showed Kv integrin expression, about 10^1000 times more efficiently than the vector containing wild type fiber via an RGD-integrin (KvL3 and KvL5)-dependent, CAR-independent cell entry pathway. The results of this study indicate that Ad vector containing RGD peptide on the fiber knob could be of great utility for gene therapy and gene transfer experiments. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : Gene therapy; Adenovirus vector; Fiber ; Retargeting
1. Introduction Recombinant adenovirus (Ad) vectors are potentially useful for gene transfer to a wide variety of cells and tissues both in vitro and in vivo. They can be grown easily to high titer, and can transfer the genes to both diving and nondividing cells [1,2]. There are, however, some limitations associated with the use of Ad vectors. One such disadvantage is that Ad vectors result in ine¤cient gene transfer to cells lacking in the primary Ad receptors, Coxsackievirus and adenovirus receptor (CAR) [3^6]. The initial step of Ad infection involves at least two sequential steps. The ¢rst step is the attachment of the virus to the cell surface through binding of the knob domain of the ¢ber to CAR [3,7]. Following attachment, the viral internalization into the cells occurs by the interaction of RGD (Arg-Gly-Asp) motifs of penton base with integrin receptors, KvL3 and KvL5, expressed on most cell types
* Corresponding author. Fax: +81-3-3700-9084. E-mail address : [email protected] (H. Mizuguchi).
[8,9]. Therefore, the interaction of the ¢ber knob with CAR on the cell is the key mediator by which Ad vectors enter the cells. Modi¢cation of ¢ber protein is an attractive strategy for overcoming the limitations imposed by the CAR dependence of Ad infection [10]. We and other groups reported that an Ad vector containing RGD peptide, which binds with high a¤nity to integrins (KvL3 and KvL5) on the cell surface, on the HI loop or the C-terminal portion of the ¢ber protein mediated not only CAR-dependent gene delivery but also CAR-independent, RGD-integrin (KvL3 and KvL5)-dependent gene delivery [11^13]. However, enhanced gene transfers by Ad vector containing RGD peptide have been examined in only limited types of cells. In this study, we compared the usefulness of an Ad vector containing RGD peptide on the ¢ber knob with that of an Ad vector containing wild type ¢ber with respect to their potential e¤cacy for gene transfer using several types of human cells (CAR-positive or -negative), mouse cells, and human leukemia cells (14 types in total). Data suggest that an Ad vector containing RGD peptide on the ¢ber knob could be of great utility for e¤cient gene transfer to a wide variety of cell types.
0304-4165 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 1 ) 0 0 1 9 4 - 5
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2. Materials and methods
2.3. Adenovirus-mediated gene transduction
2.1. Cells
The cells (1U104 cells) were seeded into a 96-well dish. On the following day, they were transduced with Ad-L2 or AdRGD-L2 (300 or 3000 vector particles (VP)/cell) for 1.5 h. After 48 h in culture, luciferase productions in the cells were measured using a luciferase assay system (PicaGene LT2.0 ; Toyo Inki, Tokyo, Japan). We repeated all transfection experiments and obtained similar results.
SK HEP-1 cells (an endothelial cell line derived from human liver) [14] were cultured in Dulbecco's modi¢ed Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). Human glioma cell lines, LN319 (anaplastic astrocytoma), LN444 (glioblastoma multiforme), LNZ308 (glioblastoma multiforme), and SF295 cells (glioblastoma multiforme) [15] (kindly provided by Dr. M. Tada, Hokkaido University, Hokkaido, Japan) were cultured in DMEM supplemented with 10% FCS. MS1 cells (mouse pancreatic islet endothelial cell line, ATCC, CRL2279) were cultured in Dulbecco's modi¢ed Eagle's medium supplemented with 5% FCS. L (Lcl1D, mouse skin ¢broblast; Human Science Research Resources Bank (HSRRB), JCRB0722) and NIH3T3 cells (mouse embryo ¢broblast; HSRRB, JCRB0615) were cultured in minimum essential medium (MEM) supplemented with 10% FCS. B16BL6 cells and colon26 cells (kindly provided by Dr. T. Mayumi, Osaka University, Osaka, Japan) were cultured in MEM supplemented with 10% FCS or in RPMI 1640 medium supplemented with 10% FCS, respectively. KG-1a cells (human bone marrow acute myelogenous leukemia cells, obtained from Dainippon, Osaka, Japan) were cultured in Iscove's modi¢ed Dulbecco's medium supplemented with 20% FCS. Y-79 cells (human retinoblastoma cells, obtained from Dainippon) were cultured in RPMI 1640 medium supplemented with 15% FCS. Ramos cells (Burkitt lymphoma cells, HSRRB, JCRB9119) and K-562 cells (chronic myelogenous leukemia cells from blast crisis, HSRRB, JCRB0019) were cultured in RPMI 1640 medium supplemented with 10% FCS.
2.4. Flow cytometry The cells (5U105 cells) from human origin were labeled with mouse monoclonal antibody RmcB [3] (kindly provided by Dr. J.M. Bergelson, Children's Hospital of Philadelphia, PA, USA) for human CAR detection, mouse anti-human integrin KvL3 (LM609; Chemicon International, Temecula, CA, USA), or mouse anti-human integrin KvL5 (P1F6; Gibco BRL, Gaithersburg, MD, USA). Then, the cells were incubated with FITC-conjugated goat anti-mouse IgG second antibody (Pharmingen, San Diego,
2.2. Plasmid and virus Luciferase-expressing Ad vectors, Ad-L2 and AdRGDL2, have been constructed previously by an improved in vitro ligation method [13,16,17]. The cytomegalovirus (CMV) promoter-driven luciferase gene derived from the pGL3-Control (Promega, Madison, WI, USA) was inserted into the E1 deletion region of the Ad genome. Ad-L2 contains wild type ¢ber, while AdRGD-L2 contains mutant ¢ber containing the RGD peptide, CDCRGDCFC, in the HI loop of the ¢ber knob. Viruses (Ad-L2 and AdRGD-L2) were prepared as described previously [16] and puri¢ed by CsCl2 step gradient ultracentrifugation followed by CsCl2 linear gradient ultracentrifugation. Determination of virus particle titer and infectious (plaque forming unit : PFU) titer was accomplished spectrophotometrically by the method of Maizel et al. [18] and by the method of Kanegae et al. [19], respectively. The PFU to particle ratio was 1:8 for Ad-L2 and 1:23 for AdRGD-L2.
Fig. 1. Flow cytometric analysis of CAR and integrin (KvL3 and KvL5) expression in several human adherent cells. Cells were incubated with anti-CAR (RmcB), anti-KvL3 (LM609) and anti-KvL5 (P1F6) integrin monoclonal antibodies. Then, the cells were incubated with FITC-conjugated goat anti-mouse IgG second antibody and analyzed by £ow cytometry. (A) SK HEP-1; (B) LN319; (C) LN444 ; (D) LNZ308 ; (E) SF295. Flow cytometric analysis of CAR and integrin expression in SK HEP-1 and LN444 cells has been described previously [13].
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Fig. 2. Comparison of luciferase production in several human adherent cells transduced by Ad-L2 and AdRGD-L2. SK HEP-1 (A), LN319 (B), LN444 (C), LNZ308 (D), and SF295 (E) cells were transduced with 300 or 3000 VP/cell of Ad-L2 and AdRGD-L2 for 1.5 h. Forty-eight hours later luciferase production was measured by luminescent assay. All data represent the mean þ S.D. of four experiments. n.d., not detectable. Luciferase production in SK HEP-1 and LN444 cells has described previously [13].
CA, USA) and analyzed by £ow cytometry with a Cyto ACE-150 Auto Cell Screener (Jasco, Tokyo, Japan). 2.5. Reverse transcription (RT)-polymerase chain reaction (PCR) analysis Total RNA of the cells from mouse origin was isolated using an RNeasy total RNA kit (Qiagen, Valencia, CA, USA). RT was carried out using a SuperScript Preampli¢cation System for First Strand cDNA Synthesis (Life Technologies, Rockville, MD, USA) according to the instructions of the manufacturer. In brief, 1 Wg total RNA was treated with DNase I. DNase I-treated RNA was annealed to 0.5 Wg oligo(dT)12ÿ18 at 70³C for 10 min, and then chilled on ice. RT was performed for 50 min at 42³C in a reaction mixture containing 2 Wl 10UPCR bu¡er, 2 Wl 25 mM MgCl2 , 1 Wl 10 mM dNTP mix, 2 Wl 0.1 M DTT, and 200 U superscript II reverse transcriptase. PCR ampli¢cation of the mouse CAR, mouse integrin receptor (Kv, L3, L5) transcripts and GAPDH was performed in 100 Wl of the reaction mixture containing 1 Wl of RT material, 2.5 U Ampli Taq DNA polymerase, 1.5 mM MgCl2 and 0.2 mM dNTP using GeneAmp PCR Core Reagents (Perkin Elmer, Norwalk, CT, USA). The sequences of the primers for ampli¢cation of the di¡erent receptors are as follows [20]: CAR: forward, 5P-tga tca ttt tgt att ctg ga-3P; reverse, 5P-tta aca aga acg gtc agc ag-3P; integrin Kv: forward, 5P-cca gcc tgg gat tgt aga ag-3P; reverse, 5P-act cca gtg ggt cat ctt tg-3P; integrin L3: forward, 5P-tct ggc tgt gag tcc tgt gt-3P; reverse, 5P-gcc tca ctg
act ggg aac tc-3P; integrin L5: forward, 5P-tcg tgt gaa gaa tgc ctg tt-3P; reverse, 5P-gct gga ctc tca atc tca cc-3P; GAPDH : forward, 5P-acc aca gtc cat gcc atc ac-3P; reverse, 5P-tcc acc acc ctg ttg ctg ta-3P. The following parameters were used: CAR: 45 s at 94³C, 60 s at 50³C, and 90 s at 72³C for 40 cycles ; integrin Kv, L3, L5: 45 s at 94³C, 60 s at 53³C, and 90 s at 72³C for 40 cycles; GAPDH : 45 s at 94³C, 60 s at 50³C, and 90 s at 72³C for 30 cycles. The expected PCR product sizes were : CAR, 211 bp; integrin Kv, 105 bp; integrin L3, 115 bp; integrin L5, 126 bp; GAPDH, 452 bp. The PCR product was electrophoresed in 2.0% agarose gel. To ensure the quality of the procedure, RT-PCR was also performed on the sample using speci¢c primers for GAPDH. 2.6. Recombinant ¢ber knob Recombinant Ad type 5 ¢ber knob protein was expressed in baculovirus with an N-terminal hexahistidine tag. The ¢ber knob-coding sequence was synthesized by PCR on pAdHM4 [16] that contained the E1/E3-deleted Ad genome, with the following primers: forward, 5P-agt cga att cac ttt gtg gac cac acc agc tcc-3P (EcoRI site is underlined); reverse, 5P-agt cgc ggc cgc tta ttc ttg ggc aat gta tga aa-3P (NotI site is underlined). PCR products were digested with EcoRI and NotI, and then cloned into the EcoRI-NotI site of pAcHLT-A, which is a baculovirus transfer vector (PhaMingen, San Diego, CA, USA). Recombinant Ad type 5 ¢ber knob protein was expressed in
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baculovirus-infected Sf9 cells according to the manufacturer's instructions, and puri¢ed by chromatography on NiNTA-Sepharose. Recombinant Ad type 5 ¢ber knob protein containing RGD peptide was prepared similarly using pAdHM15-RGD [13] as a template of PCR. 3. Results 3.1. Human adherent cells We evaluated Ad vector containing RGD peptide on the ¢ber knob as a gene delivery vehicle for several types of human glioma cells (LN319, LN444, LNZ308, and SF295) and SK HEP-1 cells, in comparison with Ad vector containing wild type ¢ber. First, the expression levels of CAR, KvL3 and KvL5 integrins on each cell surface were examined by £ow cytometry analysis (Fig. 1). Signi¢cant amounts of CAR were detected on SK HEP-1 and
LN319 cells (Fig. 1A,B). In contrast, no or very low levels of CAR were detected on LN444, LNZ308, and SF295 cells (Fig. 1C^E). LN319 and LN444 cells expressed KvL5 integrin and no or very low levels of KvL3 integrin (Fig. 1B,C). SK HEP-1, LNZ308, and SF295 cells expressed both KvL3 and KvL5 integrins (Fig. 1A,D,E). Ad-L2, the Ad vector containing wild type ¢ber, mediated e¤cient gene transfer to SK HEP-1 and LN319 cells (Fig. 2A,B). In contrast, luciferase production in other cell lines (LN444, LNZ308, and SF295) transduced by Ad-L2 was over 2^3 log orders lower than that in SK HEP-1 and LN319 cells transduced by Ad-L2 (Fig. 2C^E). These results suggest that Ad vector containing wild type ¢ber mediated less e¤cient gene transfer to CAR-negative cells. On the other hand, AdRGD-L2, the Ad vector containing RGD peptide on the ¢ber knob, mediated e¤cient luciferase expression even in the CAR-negative cell lines (LN444, LNZ308, and SF295). LN444, LNZ308, and SF295 cells transduced with Ad-L2 and AdRGD-L2 re-
Fig. 3. Inhibition of transduction of Ad-L2 and AdRGD-L2 to SK HEP-1 and LN444 cells by recombinant ¢ber knob. SK HEP-1 (A) and LN444 (B) cells were preincubated with recombinant wild type ¢ber knob (A-1, A-2, B-1) or recombinant ¢ber knob containing RGD peptide (B-2) (0, 0.4, 2, 10 Wg/ml) for 10 min. Then, the cells were transduced with 300 VP/cell of Ad-L2 (A-1) or AdRGD-L2 (A-2, B-1, B-2) for 0.5 h in the presence of recombinant ¢ber knob. Forty-eight hours later luciferase production was measured by luminescent assay. All data represent the mean þ S.D. of four experiments.
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Fig. 4. RT-PCR analysis of levels of CAR and integrin (Kv, L3, and L5) expression in mouse cells. Total RNA was isolated from MS1, NIH3T3, L, B16BL6, and colon26 cells, and then RT-PCR analysis was performed as described in Section 2. Lanes: 1, MS1; 2, NIH3T3; 3, L; 4, B16BL6; 5, colon26; 6, water; 7, DNA marker.
sulted in 2^4 log orders di¡erence in luciferase production (Fig. 2). In contrast, SK HEP-1 and LN319 cells transduced with Ad-L2 and AdRGD-L2 showed similar levels of luciferase production (less than 1 log order di¡erence). RGD peptide sequence inserted on the ¢ber knob binds with high a¤nity to both KvL3 and KvL5 integrins [21,22]. LN444, LNZ308, and SF295 cells expressed KvL5 (and KvL3) integrin. Therefore, the presence of RGD peptide on the ¢ber knob was required for e¤cient transduction of these CAR-de¢cient cells. To demonstrate the e¡ect of RGD peptide on the ¢ber knob on e¤cient transduction to CAR-de¢cient cells more
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clearly, a blocking experiment of Ad-mediated gene transfer with recombinant wild type ¢ber knob or recombinant ¢ber knob containing RGD peptide was performed (Fig. 3). Recombinant wild type ¢ber knob inhibited Ad-L2mediated luciferase production in SK HEP-1 cells, which express CAR, in a dose-dependent manner, but not AdRGD-L2-mediated luciferase production, suggesting that the AdRGD-L2 infects SK HEP-1 cells via a CARindependent pathway as well (Fig. 3A). Furthermore, recombinant wild type ¢ber knob did not inhibit AdRGDL2-mediated gene transfer in LN444 cells, which do not express CAR, while recombinant ¢ber knob containing RGD peptide inhibited AdRGD-L2-mediated gene transfer in LN444 cells (Fig. 3B). Taken together, these results suggest that AdRGD-L2 infects the cells via not only a CAR-dependent pathway, but also an RGD-integrin-dependent pathway. 3.2. Mouse cells Next, we examined the utility of an Ad vector containing RGD peptide on the ¢ber knob for gene transfer to mouse cells. Most mouse cells are known to be transduced by Ad vectors less e¤ciently than human cells. Five cell lines, MS1, NIH3T3, L, B16BL6, and colon26, were used. First, we examined the mRNA levels of CAR, Kv-integrin, L3-integrin, and L5-integrin on each cell by RT-PCR analysis (Fig. 4). We used RT-PCR analysis for mouse cells due to the lack of appropriate antibodies to detect the mouse CAR and integrin. CAR mRNA was detectable in MS1 cells. In contrast, no CAR mRNA was detected
Fig. 5. Comparison of luciferase production in mouse cells transduced by Ad-L2 and AdRGD-L2. MS1 (A), NIH3T3 (B), L (C), B16BL6 (D), and colon26 (E) cells were transduced with 300 or 3000 VP/cell of Ad-L2 and AdRGD-L2 for 1.5 h. Forty-eight hours later luciferase production was measured by luminescent assay. All data represent the mean þ S.D. of four experiments. n.d., not detectable.
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Fig. 6. Flow cytometric analysis of CAR and integrin expression in human leukemia cells. Cells were incubated with anti-CAR (RmcB), antiKvL3 (LM609) and anti-KvL5 (P1F6) integrin monoclonal antibodies. Then, the cells were incubated with FITC-conjugated goat anti-mouse IgG second antibody and analyzed by £ow cytometry. (A) K-562; (B) Y79; (C) Ramos; (D) KG-1a.
in NIH3T3, L, B16BL6, or colon26 cells under the conditions used here. All cell lines expressed Kv integrin, L3 integrin, and L5 integrin, suggesting that the cells should possess KvL3 and KvL5 integrins as heterodimers on their cellular surface. Luciferase production in MS1 cells following transduction with Ad-L2 was moderate, but the luciferase production in NIH3T3, L, B16BL6, and colon26 cells was slight due to the lack of CAR expression in these cells (Fig. 5). On the other hand, AdRGD-L2-mediated luciferase expression was 2^3 log orders more e¤cient than Ad-L2mediated luciferase expression in NIH3T3, L, B16BL6, and colon26 cells. AdRGD-L2 also mediated more e¤cient luciferase expression in MS1 cells. These results suggested that RGD peptide on the ¢ber knob also binds with high a¤nity to KvL3 or KvL5 integrin on mouse cells, and that Ad vector containing RGD peptide on the ¢ber knob would be of great utility for e¤cient gene transfer to mouse-derived CAR-de¢cient cells. 3.3. Human leukemia cells Most leukemia (suspension) cells are not e¤ciently transduced by conventional Ad vectors containing wild type ¢ber. We compared the gene transfer activity of Ad-L2 and AdRGD-L2 in four kinds of human leukemia
cells, K-562, Y79, Ramos, and KG-1a cells (Fig. 7). K-562, Y79, and Ramos cells expressed CAR (Fig. 6). As a result, Ad-L2 mediated moderate levels of luciferase expression in these types of cells. Their activities, however, were 2^3 log orders less than those in the human adherent cells (Figs. 2 and 7). K-562 cells expressed a moderate amount of KvL3 or KvL5 integrins, while Y79 cells expressed a moderate amount of KvL5 integrins. As a result, K-562 and Y79 cells transduced with AdRGD-L2 expressed an approx. 10-fold greater amount of luciferase than those transduced with Ad-L2, but this amount was still less than that in human adherent cells transduced with AdRGD-L2. Ramos cells expressed only slight amounts of KvL3 or KvL5 integrins. There was only a 3-fold di¡erence in luciferase production between Ramos cells transduced with Ad-L2 and those transduced with AdRGD-L2. KG1a cells did not express CAR, but expressed small amounts of KvL3 integrin. AdRGD-L2 mediated small levels of luciferase expression in KG-1a cells, while Ad-L2 did not. These results suggest that AdRGD-L2 improved the e¤ciency of Ad-mediated gene transfer to human leukemia cells expressing KvL3 or KvL5 integrin, but this e¡ect was not su¤ciently high compared with the transduction e¤ciency to adherent cells. 4. Discussion Expression of cellular surface Ad receptor, CAR, is an important determinant for e¤cient Ad-mediated gene de-
Fig. 7. Comparison of luciferase production in human leukemia cells transduced by Ad-L2 and AdRGD-L2. K-562 (A), Y79 (B), Ramos (C), and KG-1a (D) cells were transduced with 300 or 3000 VP/cell of AdL2 and AdRGD-L2 for 1.5 h. Forty-eight hours later luciferase production was measured by luminescent assay. All data represent the mean þ S.D. of four experiments. n.d., not detectable.
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livery [3^6]. Ad vector mediated ine¤cient gene transfer to cells lacking in CAR expression. This barrier can be overcome by the alteration of Ad ¢ber protein [10]. We and others developed an Ad vector containing RGD peptide on the ¢ber knob, which targets Kv (KvL3 and KvL5) integrin on the cellular surface, and showed that the mutant vector had expanded tropism [11^13]. In this study, we examined the utility of Ad vector containing RGD peptide on the ¢ber knob for gene therapy and gene transfer experiments in various types of cells (a total of 14 cell lines). The results clearly showed that Ad vector containing wild type ¢ber mediated less e¤cient gene transfer to the CAR-negative cells, while Ad vector containing RGD peptide on the ¢ber knob mediated about 100^1000 times more e¤cient gene transfer to the CAR-negative but Kv integrin-positive human adherent cells (Fig. 2). This enhanced e¡ect was also observed in mouse cells (Fig. 5). It has been reported that several types of cells and tissues, e.g. smooth muscle cells, bronchial epithelium and skeletal muscle cells, do not readily allow e¤cient introduction of the transgene by Ad vector due to their ine¤cient expression of CAR [10]. If these types of cells express KvL3 or KvL5 integrins, they would be expected to allow e¤cient transduction by Ad vector containing RGD peptide on the ¢ber knob. However, in leukemia cells, which are known to resist e¤cient transduction by Ad vector, the enhancement of transduction by use of a ¢ber-modi¢ed vector was smaller than that in adherent cells (Fig. 7). The luciferase production in leukemia cells transduced with ¢ber-modi¢ed vector containing RGD peptide was also lower than that in adherent cells. Moreover, no luciferase production occurred in HL-60 cells (acute promyelotic leukemia cells) transduced with Ad-L2 or AdRGD-L2 (data not shown). These results suggest that an Ad vector containing RGD peptide on the ¢ber knob has great potential for e¤cient transduction to most CAR-negative cells, but does not adequately enhance transduction to some leukemia cells. The RGD peptide motif is thus considered inadequate for ¢ber modi¢cation for transduction to these types of leukemia cells. We previously reported a simple method for construction of a ¢ber-mutant Ad vector [13]. In our system, foreign DNAs corresponding to the peptide of interest can be introduced into the HI loop coding region of the ¢ber knob of the Ad genome by one step in vitro ligation. Modi¢cation of the ¢ber knob by a peptide having high a¤nities to the molecules on the leukemia cell might lead to more e¤cient transduction. These studies are underway. The ¢ber-mutant Ad vector has several advantages compared to the conventional Ad vectors for use in transduction. When the vector is injected into CAR-negative cells or animal tissues in vivo, such as CAR-negative tumor cells, smooth muscle cells, bronchial epithelium, skeletal muscle cells, etc., the transduction can be enhanced and the side e¡ects lessened by reducing the injected dose of
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the vector. Furthermore, the use of ¢ber-mutant Ad vectors broadens the spectrum of possible Ad vector applications. Gene transfer experiments could be performed even in cells which do not readily lend themselves to transduction by the conventional Ad vectors. In summary, this study suggests that an Ad vector containing RGD peptide on the ¢ber knob mediated e¤cient transgene expression even in CAR-negative (but Kv integrin-positive) cells. Our study indicates the potential e¤cacy of the Ad vector containing RGD peptide on the ¢ber knob in gene therapy and gene transfer experiments. Acknowledgements We would like to thank Mr. Jun Murai and Ms. Nobuko Heishi for their technical assistance. This work was supported by grants from the Ministry of Health, Labour and Welfare of Japan and by a Grant-in-Aid for Scienti¢c Research on Priority Areas (C).
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