Enhancement of adenoviral transduction with polycationic ... - Nature

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Enhancement of adenoviral transduction with polycationic liposomes in vivo Sang Goo Lee,1,2 Seong Jun Yoon,1 Chun Dong Kim,3 Kyungjin Kim,2 Dong Soo Lim,4 Young Il Yeom,4 Myung-Whun Sung,3 Dae Seog Heo,1,5,6 and Noe Kyeong Kim6 1

Cancer Research Center, Departments of 6Internal Medicine and 3Otolaryngology, College of Medicine, School of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul, Korea; 5 Clinical Research Institute, Seoul National University Hospital, Seoul, Korea; and 4Korea Research Institute of Bioscience and Biotechnology, Tae-jeon, Korea. 2

Although the high transfection efficiency with adenovirus in vitro is well documented, it is still not clear whether adenoviral vectors are effective in vivo in solid tumor models. In our preliminary experiment, transduction of tumor tissue was limited to just around the injection site after intratumoral injection of the adenoviral vector. To improve the transduction efficiency in vivo, we tried a combination of adenoviral vector and liposome in our animal model. Adenovirus carrying human placental alkaline phosphatase (AdALP) and Lipofectamine or 1,3-di-oleoyloxy-2-(6-carboxyspermyl)-propylamide were used as a marker gene and the cationic liposome, respectively. A ⬎15-fold increase in the transfection efficiency was observed in CT26 tumor cell lines with the combination of AdALP adenovirus carrying murine granulocyte-macrophage colony-stimulating factor (AdmGM-CSF), and liposome compared with adenovirus alone, showing the feasibility of the combination treatment. In the animal model, with the combination of liposome and AdALP, deeper and wider distribution of the marker gene in the tumor mass was shown. We conclude that the limitations of direct application of adenoviral vectors in a solid tumor model could be overcome by the use of cationic liposomes. This approach will facilitate the more effective delivery of adenoviral vectors in a clinical trial setting. Cancer Gene Therapy (2000) 7, 1329 –1335

Key words: Gene therapy; adenovirus; liposome; in vivo.

T

hrough an understanding of the basic biology of the cellular events that lead to tumorigenesis, many advances have been developed in cancer therapy. Gene therapy is a technique by which different genes are transferred into cells to replace or augment the function of an absent or faulty wild-type gene.1,2 To date, many clinical protocols have been approved, and their results have been encouraging. The key step in the gene therapy is the introduction of foreign genetic materials into the target cells. Transduction of adenovirus results in the transient expression of a foreign gene, and the expression level is very high even in resting target cells. There have been many studies designed to improve virusmediated gene therapy, for example by modifying the vector through the attachment of ligand for cellular receptors, incorporation of chimeric envelope glycoprotein, or chemically coupled ligands.3 Moreover, biochemical reagents such as polybrene and diethylaminoethyl-dextran have been used to facilitate the entry of virus particles into the target cells.4 –9

Because of its simplicity, the use of liposomes to introduce foreign genes into cells has become increasingly popular.10 Moreover, many kinds of liposomal preparations are available.4,10,11 Positive charges on the cationic liposomes facilitate the association with negatively charged nucleic acids as well as with membrane phospholipids.4 Cationic lipid receptors have also been reported to facilitate endosomal uptake of lamella liposome particles,4,12 which themselves have been used to infect resistant cells with DNA viruses and retroviruses13,14 and with adenoviruses.15–20 In our preliminary studies on the direct introduction of an adenoviral vector into a solid tumor, we showed that transduction was limited around the needle tract in vivo. In the present study, we aimed to enhance the transduction efficiency of the adenoviral vector in vivo into the solid tumor by introducing it in combination with polycationic liposomes. We report here the enhancement of transduction efficiency and dispersion rate in vitro and in vivo by the application of this combination technique.

Received October 15, 1999; accepted June 25, 2000. Address correspondence and reprint requests to Dr. Dae Seog Heo, Department of Internal Medicine, Seoul National University Hospital, 28 Yungon-dong, Chongno-gu, Seoul, 110-744, Korea. E-mail address: [email protected]

MATERIALS AND METHODS

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Cell lines All of the cell lines used in this study were generously provided by G. J. Nabel (University of Michigan, Ann Arbor, Mich),

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LEE, YOON, KIM, ET AL: ENHANCEMENT OF ADENOVIRAL TRANSDUCTION IN VIVO

T. L. Whiteside (University of Pittsburgh, Pittsburgh, Penn), and J. G. Park (Korean Cell Line Bank, Seoul National University, Seoul, Korea). CT26, HEK293, and SCCVII/SF cell lines were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10 mg/mL gentamicin, 10% fetal bovine sera, 2 mM L-glutamine, and 3.024 g/L sodium bicarbonate. HeLa and PCI-13 cell lines were maintained in RPMI 1640 supplemented as described above. All cell lines were maintained at 37°C in a humidified 5% CO2, 95% air atmosphere.

Adenoviral vectors and preparation of adenovirusliposome complexes The adenoviral vectors used in this study, AdALP and AdmGM-CSF, were obtained from Dr. Nabel (University of Michigan) and D. S. Lim (Korea Research Institute of Bioscience and Biotechnology, Korea Institute of Science and Technology, Tae-jeon, Korea), respectively. Adenovirus was purified and titrated as described previously.21 Lipofectamine (Life Technologies, Gaithersburg, Md) and 1,3-di-oleoyloxy-2(6-carboxyspermyl)-propylamide (DOSPER) (Boehringer Mannheim, Indianapolis, Ind) were used as cationic liposomes. Lipofectamine reagent is a 3:1(wt/wt) liposome formulation of the polycationic lipid 2,3-dioleyloxy-N-(2[sperminecarboxamido]ethyl)-N,N-dimethyl-1-propanaminium trifluoroacetate and the neutral lipid dioleoyl phosphatidylethanolamine in membrane-filtered water. DOSPER is a modified liposome carrying four cations. Adenoviruses delivering human placental alkaline phosphatase or murine granulocyte-macrophage colony-stimulating factor (GM-CSF) were titrated to 1–20 multiplicities of infection (MOIs)/200 ␮L Opti-modified Eagle’s medium (Life Technologies). Next, 10 ␮L of liposomes (10 ␮g) were added to the solution. The solution was left at 4°C for 5–10 minutes for complex formation to occur. After aspiration of the culture media, adenovirus-liposome complexes were applied to the target cells (1 ⫻ 106 cells/well) and incubated at 37°C for 2 hours. The medium was then removed and the cells were washed once with culture medium. The cells were then cultured for 24 hours before being examined for the expression of the gene delivered by the adenoviral vectors.

Animals and in vivo studies Female BALB/c mice were obtained from Japan SLC (Haruna Breeding Branch, Tokyo, Japan). All mice were housed in laminar flow animal isolation hoods under specific pathogenfree conditions and used at 6 –7 weeks of age. CT26 tumors were established in vivo by injecting 1 ⫻ 106 cells into the right flanks of BALB/c mice in 50 ␮L of phosphate-buffered saline (PBS) via a 26-gauge needle. When the tumor reached ⬃1 cm in diameter (2 weeks after CT26 cell inoculation), we injected the adenovirus containing human placental alkaline phosphatase with or without polycationic liposomes. Adenovirus delivering human placental alkaline phosphatase was titrated to 1 ⫻ 109 plaque-forming units (PFU) per 50 ␮L of PBS. Next, 10 ␮L of liposomes (10 ␮g) was mixed with 50 ␮L of adenovirus solution. The solution was left at 4°C for 5–10 minutes for complex formation to occur. For adenovirus/liposome complex transfer, a 26-gauge needle was used to deliver the complex to the center of the tumor nodule. Animals were sacrificed 48 hours later, and the tissues were prepared for alkaline phosphatase staining.

Reverse transcriptase polymerase chain reaction (RT-PCR) RNA was prepared from CT26, HEK293, and HeLa cell lines using Trizol RNA extraction reagent (Life Technologies). Total RNA (1 ␮g) was used to synthesize cDNA with 250 U of Moloney murine leukemia virus (Life Technologies) in a 20-␮L reaction volume. A total of 1 ␮L of the cDNA reaction mixture was used in the PCR amplification, which contained the following in 1 ␮L of 10⫻ PCR buffer (Boehringer Mannheim): 10 pmol of each primer, 10 mM of deoxynucleotide triphosphate, and 2.5 U of Taq polymerase. The amplification conditions used were: 35 cycles at 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute. Amplification was performed using a Perkin-Elmer GeneAmp PCR System 9600 (Perkin-Elmer, Norwalk, Conn). The sequences of the oligonucleotide primers used were as follows: 5⬘-GGCATTGTGGTCTACAGCCT-3⬘ (sense) and 5⬘-CCGTAGACCCTGCTCGAATA-3⬘ (antisense) for murine GM-CSF (mGM-CSF).

Enzyme-linked immunosorbent assay (ELISA) for GM-CSF Levels of cytokine production from the cloned cells were measured in triplicate by using standard ELISA kits 24 hours after infection according to the manufacturer’s instructions (Endogen, Woburn, Mass).

Alkaline phosphatase assay After adenoviral transduction, cells were fixed with 4% paraformaldehyde fixatives for 10 minutes at 4°C. To eliminate the activity of endogenous alkaline phosphatase, cells were incubated in a 65°C water bath for 1 hour. The exogenous virally transmitted, heat-stable human placental alkaline phosphatase was then developed by incubation with substrate nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) solution (Bio-Rad, Hercules, Calif). Solid tumor tissue sections were treated as described above.

RESULTS

Direct introduction of an adenoviral vector into an established solid tumor In our preliminary study of adenoviral transduction into an established syngeneic tumor model (CT26 colon adenocarcinoma in BALB/c mice), we found that only a minor region around the injection site was infected. Several layers (⬍10 layers) of tumor cells around the needle tract were stained with NBT/BCIP alkaline phosphatase substrate solution (Fig 1B). Uninjected perpendicular tissue section showed no staining (Fig 1A).

Differences of adenoviral susceptibility between cell lines We screened for the transduction efficiency of various cell lines by using adenovirus carrying human placental alkaline phosphatase as a marker gene. When adenovirus was introduced into each plate containing four different cell lines (HEK293, PCI-13, SCCVII/SF, and CT26), considerable differences in the efficiency of gene transduction were observed (Fig 2). Each cell line (PCI13, SCCVII/SF, and CT26) was exposed to AdALP for 1

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Figure 2. Differential expression of alkaline phosphatase activity in various cell lines. The cells (A, CT26; B, SCCVII/SF; C, PCI-13; D, HEK293) were exposed to AdALP for 1 hour at various titers (mock (0) and the following MOIs: 10, 20, and 50); ALP activity was examined 24 hours later. HEK293 cells were infected at the following MOIs: mock (0), 5, 10, and 20. Figure 1. Direct adenoviral vector infection shows limited transfection around the needle tract. A total of 1 ⫻ 109 PFU per 50 ␮L of AdALP was injected intratumorally. A, control; B, AdALP injected into CT26 tumors in BALB/c mice. At 2 days postinjection, tumor masses were sectioned perpendicular to the injection axis and screened for alkaline phosphatase activity as described in Materials and Methods. The dark blue stain shows the transduced tumor cells.

hour at various titers (MOIs of 0,10, 20, and 50), and alkaline phosphatase activity was examined 24 hours later. HEK293 was infected at MOIs of 0, 5, 10, and 20. At these MOIs, however, HEK293 (an adenoviruspackaging cell line) showed the strongest expression of human placental alkaline phosphatase. Even at an MOI of 5, 100% of the cells expressed the marker gene and the level was very high. With a very small amount of adenovirus, the expression of the marker gene reached its plateau (data not shown). PCI-13, a cell line derived from human squamous cell carcinoma of the head and neck also stained strongly. However, a murine squamous cell carcinoma of the head and neck cell line, SCCVII/SF showed only moderate staining, and a murine colon adenocarcinoma cell line, CT26, showed minimal staining by infection with AdALP. To correlate the differences between cell lines above, we screened the expression level of Coxsackievirus adenovirus receptors (CAR) by RT-PCR. CT26 and SCCVII/SF cells showed an extremely low level of murine CAR; however, the expression level of human CAR was very high in HEK293 and PCI-13 cells. CAR mRNA levels paralleled the viral sensitivity in the cell lines above (data not shown). To establish an efficient protocol for the solid tumor model, we needed to enhance the efficiency of transduction even in the transduction-resistant cell lines, such as CT26. Therefore, we attempted to improve the transduction rate in vitro and in vivo.

Enhancement of adenoviral transduction efficiency in vitro To enhance the transduction and expression of a foreign gene delivered by an adenovirus, we combined the adenovirus with a polycationic liposome and tested this


combination in the CT26 cell line. When confluent CT26 cells were infected with AdALP at different MOIs from 0 to 20, the expression of alkaline phosphatase increased as the dose of virus increased (Fig 3A). However, the rate of transduction is not satisfactory enough to use adenoviral vectors as therapeutic material for such a resistant cell line. With DOSPER and an MOI of 1, staining could be observed, and a much higher expression level of alkaline phosphatase was seen at the higher MOIs than was observed without DOSPER (Fig 3, A and C). When the virus was combined with Lipofectamine (Fig 3B), the synergistic effect was slightly reduced relative to that of DOSPER (Fig 3C). The transduction enhancement was most dramatic in the transduction-resistant cell lines, such as the CT26 cell line (see Fig 5B). There was, however, no enhancement of the transduction of HEK293 cells by the liposome combination. To measure the enhancing effect of DOSPER on the expression of a transferred mGM-CSF gene quantitatively, we checked cytokine production with an ELISA method. CT26, HeLa, and HEK293 cells were infected with the fixed titer of 10 MOIs of AdmGM-CSF, and the dose of DOSPER was varied (0 –20 ␮g/␮l). With each dose increase of DOSPER, the concentration of mGM-CSF (measured 24 hours after the infection) increased proportionally to the level of DOSPER, especially in CT26 cells. A slight decrease was observed at 20 ␮L of DOSPER (Fig 4A). The expression level of mGM-CSF mRNA was determined by RT-PCR, and replicated the result of mGM-CSF expression status (Fig 4B). Expression of mGM-CSF mRNA was a result of enhanced adenoviral transduction of AdmGM-CSF. The same phenomenon was seen in the transductioncompetent cell line HeLa (data not shown). At a 10-␮L fixed concentration of DOSPER (10 ␮g) per 200 ␮L of infection medium, the production of mGM-CSF increased with higher MOIs of adenovirus in the presence of liposomes, compared with gene transfer without DOSPER in CT26 cells (Fig 5B).22

Polycationic liposomes enhance gene transfer in vivo When histologic sections were made perpendicular to the needle direction, transgene expression was found to

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Figure 3. In vitro enhancement of adenoviral vector infection. CT26 colon adenocarcinoma cells were infected with AdALP alone (A), with AdALP in combination with Lipofectamine (B), or with AdALP with DOSPER (C). A total of 1 ⫻ 106 CT26 cells were seeded at various viral titers (0, 1, 2, 5, 10, and 20 MOIs) of AdALP and were exposed for 1 hour; ALP activity was examined 24 hours later.

be confined to a small area around the needle tract in the CT26 solid tumors established in BALB/c mice (Figs 1B and 6D). Under the microscope, the transduced and stained region comprised only a few layers of cells in vivo (Fig 1B). As seen in in vitro experiments, the addition of DOSPER resulted in a remarkable enhancing effect on gene transfer in vivo (Fig 6F) compared with transfection with the adenovirus alone or with the control groups (Fig 6, A–D). None of the control groups (saline alone, Ad⌬E1 alone, Lipofectamine alone, DOSPER alone, Ad⌬E1 ⫹ Lipofectamine, and Ad⌬E1 ⫹ DOSPER) showed any positive signals for alkaline phosphatase activity. We assume that the positive signals shown in

Figure 6, E and F, are derived from the transduction of AdALP. Although strong staining of alkaline phosphatase and extensive necrosis of the tumor were seen in the animals treated with AdALP/DOSPER, animals treated with AdALP/Lipofectamine did not show such a marked enhancing effect (Fig 6E). We could observe the same enhancing activity of liposomes on the SCCVII/SF tumor in C3H mice (data not shown). By combining adenoviruses with polycationic liposomes, especially with DOSPER, we enhanced not only the transduction and expression but also the penetration and diffusion of the virus-liposome complexes.

Figure 4. Dose effect of polycationic liposomes in vitro. The production of mGM-CSF protein (A) and the expression level of mGM-CSF mRNA (B) were determined by ELISA and RT-PCR, respectively. CT26 cells were infected with AdmGM-CSF as described above. The viral titer was kept constant at an MOI of 10; various doses (2, 5, 10, and 20 ␮L) of DOSPER were used. mGM-CSF production was determined by ELISA (Endogen), and the mRNA level was determined by RT-PCR.



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Figure 5. Enhancement of mGM-CSF production after AdmGM-CSF infection in combination with polycationic liposomes (DOSPER) in vitro. HEK293 (A) and CT26 (B) cells were infected with or without DOSPER (10 ␮L) at various viral titers (0 –20 MOIs). The level of mGM-CSF production was determined by ELISA as described above.

DISCUSSION The most important factor in the application of gene therapy is the efficiency of gene transfer in vivo. Several studies have also shown that the choice of vector is important. Even though adenovirus vector has relatively good transduction efficiency, we observed that some cell lines were slightly resistant to adenoviral transduction in vitro, and that the expression of the foreign gene was limited to the vicinity of the needle tracts of the solid tumor in an animal model, such as the CT26/BALB/c model. Although multiple injections into the target organ or tissue increased the delivery of the therapeutic gene a little, the result was far from satisfactory (data not shown). As we determined the expression level of CAR in the cell lines, the CAR mRNA expression in the CT26 cell lines was hardly detectable. We observed significant differences between cell lines in the intensity of CAR PCR products. The CAR mRNA level paralleled the adenoviral sensitivity. So we can suggest that the low infectivity and expression of foreign gene transferred by adenoviral vector is primarily dependent upon the expression level of CAR in the target cells.23,24 Previous reports have described gene transfer systems that combine viral and nonviral approaches.5–9 Because it is known that liposome can increase the titer of ecotropic and amphotropic retroviral systems,11 we combined adenovirus with a cationic polymer to increase the efficiency of gene transfer to CT26 tumor cells, which are very resistant to adenoviral transduction. We observed a significant enhancing effect of DOSPER on the expression of transgenes in vitro and in vivo. In our experiments, Lipofectamine showed a lower enhancing effect, which suggests that there are differences in the ability of various cationic molecules to enhance gene transfer. Although our goal was not to perform a detailed analysis of the relationship between cationic lipid structure and expression, it appears that, in general, polyvalent lipids are more effective than monovalent lipids. It has been


reported that the neutral colipid, dioleoyl phosphatidylethanolamine, is also important.25 Cationic molecules used for the transformation of target cells, such as polybrene, Ca2⫹, and diethylaminoethyl-dextran have been shown to increase the efficiency of adenoviral transduction.11 Each molecule is thought to work by the same mechanism as a cation donor between the cell membrane and the adenoviral envelope.26 A combination of cationic polymers with adenovirus has been tried to transfer transgenes to differentiated airway epithelium by others.4 They showed an increase in adenovirus uptake and transgene expression in cells that were inefficiently infected by adenovirus alone. Poor binding and cellular uptake might be critical barriers to adenovirus-mediated gene transfer in cells that do not express CAR, which binds adenovirus fiber.4,27,28 Complexes of adenovirus with cationic molecules were thought to interact with the cell surface through charge association.4 So the complexes are thought to bypass the normal adenoviral infection pathway through CAR binding of adenovirus fiber. Cationic molecules may act by neutralizing the negative charge found on both the cell membrane and the viral envelope, thus facilitating envelope glycoprotein-receptor interactions in the absence of charge repulsion.4,29 The combination of cationic molecules and adenovirus in which the transgene is encoded in adenovirus DNA seems to use the cationic molecule to enhance adenovirus uptake into poorly infected cells and then uses adenovirus proteins to facilitate the steps that ultimately lead to gene transfer. If this were the case, cationic liposome would be expected to substitute for the envelope glycoprotein that normally mediates infection of specific cells. Adenovirus in complex with liposome can facilitate the adenovirus-dependent processes such as escape from the endosome and entry into the nucleus.30 The system we describe here may be of value in gene transfer for cells in which infection is limited because the cells lack CARs. It may also be possible to use a lower titer of adenovirus with this system by enhancing gene

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Figure 6. Enhancement of adenoviral vector transduction in vivo. For controls, untreated (A), Ad⌬E1 ⫹ Lipofectamine (B), and Ad⌬E1 ⫹ DOSPER (C) were injected. AdALP only (D), AdALP ⫹ Lipofectamine (E), and AdALP ⫹ DOSPER (F) were employed to determine the effect. At 2 days after inoculation of AdALP (1 ⫻ 109 PFU per 50 ␮L of PBS) intratumorally, the masses were assessed for alkaline phosphatase activity after cryosection followed by hematoxylin and eosin staining. Diffuse and dark-blue staining with NBT/BCIP can be seen in the combination treatment groups (E,F). The filled arrow indicates the site of needle injection center. The open arrow indicates the extent of adenoviral transduction and diffusion in vivo. The sections were photographed with a surgical microscope at ⫻40 magnification. Scale bar indicates 1 mm.

transfer efficiency.27,28 Because adenovirus could be coated with cationic molecules that replace the cell binding and internalization function of the virus, the application of this technique to the cancers of the upper aerodigestive tract could be therapeutic.4 In addition, this coating might shield the virus from neutralizing antibodies that can prevent effective gene transfer upon repeated administration.4 However, it is likely that the system we describe here offers no advantage for cells that are already easily infected by adenovirus alone, as was the case of HEK293 cells. In summary, this work demonstrates that a complex between an adenovirus containing a transgene and a cationic liposome, such as DOSPER, can enhance gene transfer to an adenovirus-resistant cancer cell line in vitro and in vivo. With the improvement in the efficiency of gene transfer, it may be possible not only to facilitate the expression of therapeutic genes but also to deliver a lower titer of vector and, as a result, to reduce toxicity and immune responses. The consequent increase in the therapeutic index could enhance the development of successful gene therapy against cancers. ACKNOWLEDGMENTS This work was supported by a grant from the Korean Ministry of Science and Technology (G7 Project (8-1-55)) and in part by BK21 Human Life Sciences.

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