Following in Vivo Electroporation'

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[CANCERRESEARCH56, 1050-1055, March 1, 19961 High-Efficiency in Vivo Gene Transfer Using Intraarterial Plasmid DNA Injection following in Vivo Electroporation'

Toni NiShi,2 Kimio Yoshizato, Shigeo Yamashiro, Hideo Takeshima, Kyoichi Sato, Kazuya Hamada, Isao Kitamura, Teizo Yoshimura, Hideyuki Saya, Jun-ichi Kuratsu, and Yukitaka Ushio Departments of Neurosurgery IT. N., K. Y., S. Y., K. S., K. H., 1. K., J. K., Y. U.] and Tumor Genetics and Biology [H. S.], Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto 860, Japan, and Immunopathology Section, Laboratory of Immunobiology, National Cancer institute, Frederick, Maryland 21702 (T. Y.j

ABSTRACT Electroporation or electrical permeabilization of cell membranes has been developed for the purpose of achieving highly efficient in A novel method for high-efficiency and region-controlled in vivo gene vitro gene and/or drug transfer (6â€”9).This system provides markedly transfer was developed by combining in vivo electroporation and intraar higher efficiency transfer compared with other nonviral transfer sys terinl plasmid DNA injection. A mammalian expression plasmid for the tems, including cationic liposomes. Application of the electroporation Escherichia coli lacZ gene (driven with a SV4O early promoter) was injected into the internal carotid artery of rats whose brain tumors (from to cultured cells is well established, but the use of in vivo electropo prior inoculation) had been electroporated between two electrodes. The ration has received little attention (10, 11). Recently, the electrical lacZ gene was efficiently transferred and expressed in the tumor cells 3 permeabilization method has been applied to in vivo drug transfers for daysafterplasmidInjection.However,neitheranygenetransfersnorany cancer treatment, and a clinical trial has been started (12, 13). This elevatedlacZactivitywas detectedin tissuesoutsidethe electrodes.The method is called electrochemotherapy or electric impulse chemotherapy. plasmid was not transferred without electroporation. Human monocyte In our study, a mammalian expression plasmid carrying the bacte chemoattractant protein-i eDNA was also transferred by this method, and rial lacZ gene was intraarterially injected into rats immediately after in its long-lasting (3 weeks) expression was confirmed by using the Epstein vivo electroporation (between a pair of electrodes) of rat brain tumor Barr virus episomal replicon system. The expressed monocyte chemoat cells that had been inoculated into their brains 10 days earlier. The tractant protein-i protein was functional, as evident by the presence of a injected plasmid was efficiently incorporated into tumor tissues and large number of monocytes in the tumor tissue. This method, â€œelectro. gene therapy,â€•whichdoes not requireviral genes or particles,allows found to provide the expression of the lacZ protein in the cells. genesto be transferredandexpressedindesiredorgansor tissues,andit Sections from a wide variety of tissues from treated animals were may lead to the development of a new type of highly effective gene analyzed for evidence of toxic effects. We found no treatment-related therapy. toxic reactions nor increased lacZ activity in any organs other than those between the electrodes in treated animals. We have also tested INTRODUCTION for long-lasting expression of the human cDNA encoding the MCP-l,3 a cytokine that gathers macrophages (14â€”16),in the EBV episomal Many types of methods and techniques for in vivo gene transfer replicon vector. Immunohistochemical analysis revealed that human have been developed, and some of them have already been applied in MCP-l was expressed in inoculated tumor cells for at least 3 weeks. clinical trials. In most cases, genes are transferred into tissues by the This method will allow us to easily deliver the gene of interest to infectivity of viral particles. The retroviral system, which is the most desired tissues or organs. Many applications, including gene therapy widely accepted gene transfer method to date, can achieve highly of malignant solid tumors and of metabolic disorders, will become efficient integration, providing the potential for permanent gene cx possible. We feel this method could be named â€œelectro-genetherapy.â€• pression. However, the system has some major disadvantages, such as the typically low titer, instability of the viral vector obtained, and the requirement for target cell division for integration and expression (1). MATERIALS AND METHODS Although the adenovirus system can provide more efficient gene Rat Brain Tumor Model. C6 rat glioma cells (1 X i0@;American Type transfer and stability of virus, the difficulties in control of target cells Culture Collection, Rockville, MD) were stereotactically inoculated into the and of re-administrations necessitated by the strong antigenicity of the right striatum of Wistar rats weighing 150â€”200g.In all procedures, animal virus are serious problems (2). Furthermore, there are some common care was in accordance with institutional guidelines. drawbacks in viral gene transfer systems. Particularly, the quality Carotid Artery Cannulation, Electroporation, and Plasmid Injection. control of viral particles for in vivo administration is laborious and On the tenth day after inoculation, the tumor-beating rats were randomized into expensive work (3). four experimental groups designated: E+P+(ic), n = 8; E+P+(iv), n = 3; If an efficient and specific transfer method could be developed, E+Pâ€”,n = 8; and Eâ€”P+,n = 7; where E = electrical pulse, P = plasmid, naked plasmid DNA is an ideal system for gene transfer. A plasmid and Â±indicates presence or absence, respectively. The animals of the E+P+(ic) group received a run of electrical pulses, followed by intracarotid mediated method would be economical and easy because use of this plasmid DNA injection. The E+P+(iv) group received iv. injection of the system obviates the necessity to construct viral vectors, establish same amount ofplasmid DNA following electroporation instead of intraarterial clones of producer cells, and assess viral titers and the presence of injection. The E+Pâ€” group received electrical pulses without plasmid injec replication-competent helper virus, which has been known to activate tion, and the Eâ€”P+group received intraarterial plasmid injection only. passive oncogenes. Also, the transfer procedure could be easily re Rats were anesthetized by inhalation of halothane. For intraarterial injec peated because naked plasmid DNA has little antigenicity to the host tion, a microcatheter connected to a 2.5-ml syringe was cannulated into the body (4, 5). right internal carotid artery through the external carotid artery. The pterygo palatine artery, a major branch of the internal carotid artery, was coagulated to Received 9/25/95; accepted 1/3/96. concentrate injected plasmid into the brain circulation. The animals in the The costs of publication of this article were defrayed in part by the payment of page E+P+(iv) and E+Pâ€”groups received the same operation without plasmid charges. This article must therefore be hereby marked advertisement in accordance with injection. 18 U.S.C. Section 1734 solely to indicate this fact. I This work was supported in part by the prize money of the first Hoshino Award and Grants-in-Aid from the Ministry of Education, Science and Culture ofJapan (07557095). 3 The abbreviations used are: MCP-l, monocyte chemoattractant protein-l; H & E, 2 To whom requests for reprints should be addressed. Phone: 96-373-5219; Fax: hematoxylin and rosin; X-Gal, 5-bromo-4-chloro-3-indolyl-@-o-galactoside; EBV, 96-371-8064; E-mail: tnishiÂ®gpo.kumamoto-u.ac.jp. Epstein-Barr virus. 1050

After cannulation, the heads of the rats were fixed onto a stereotaxic as a candidate transgene for the gene therapy of brain tumors. The mammalian operating table. The head skin was cut and retracted. One each burr hole was expression vector pCEP4 (Invitrogen, San Diego, CA) was used in this study. opened in front and behind of the burr hole used for cell inoculation. A pair of This vector system includes discrete DNA elements, the EBV origin of stainless steel electrodes, 0.5 cm long and 0.5 mm in diameter, coated with replication, and EBV nuclear antigen-l . Because these elements together gold (Unique Medical Imada Co., Tokyo, Japan), was inserted into the brain confer an episomal replicon capacity to DNA circles in an array of human cell through the burr holes, and the resistance between the electrodes was measured types, this vector not only offers an expeditious means for achieving amplifi with a BTX 500 optimizer (BTX, Inc., San Diego, CA). The average distance cation of transfected genes but also bypasses cis effects upon promoter func of the electrodes was 0.5 cm. A series of eight electrical pulses with pulse tion at chromosomal integration sites. The 700-bp fragment of human MCP-l length ranging from 95â€”99p.s(17â€”19)wasdelivered with a standard square cDNA was ligated into the unique BamHI/HindIII sites of the pCEP4 vector wave electroporator BTX T820 (BTX, Inc., San Diego, CA) in groups (pCEP4/hMCP-l) with T4 DNA ligase (New England Biolabs, Beverly, MA). E+P+(ic), E+P+(iv), and E+Pâ€” as shown in Fig. I. The voltage was Thirty ,@gof the resulting plasmid pCEPIhMCP-l were transferred into five adjusted to deliver 0.5 mA of current between the electrodes based on the tumor-bearing rats in the same manner with pCHl 10. results of the premeasured resistivity. The average voltage was 300 V/0.5 cm. Immunohistochemistry of Human MCP-1 Protein in Rat Brain. Three Immediately after they received electrical pulsing [group E+P+(ic)] or a weeks after the electro-gene transfer procedure, tumors in rat brain were sham operation on the head (group Eâ€”P+), 10 @.tgplasmid, pCHllO, a stained by the indirect immunoperoxidase method. Cryostat sections were cut mammalian expression vector of the lacZ gene with the SV4Oearly promoter at a thickness of 6 @mandair dried. To inhibit endogenous peroxidase activity, (20) in 0.5 ml PBS was injected by carotid catheter into the rats with a the sections were first immersed in S mM periodate solution for 10 mm, washed microinfusion pump for 5 ruin. In group E+P+(iv), the same amount of with PBS, and then placed in a 3 mt@isodiumborohydrate solution for 30 ruin plasmid DNA was injected into the tail vein. Previous data indicated that (27). They were then incubated with optimally diluted monoclonal IgG anti uptake by electroporated cells through membrane pores is not affected by human MCP-l (28). After being washed with PBS, they were covered with whether the molecules were present during or added after the electroporating peroxidase-conjugated, species-specific goat antimouse immunoglobulin pulse (21, 22). Therefore, we preferred to inject DNA immediately after (Fab'2) diluted 1:100 (Amersham, Arlington Heights, IL) for 60 mm. Perox electroporation to supply a higher concentration of DNA into the electropo idase activity was visualized by incubation of the sections with 3,3'-diamino rated region using a smaller amount of DNA. After injection, the catheter was benzidine [0.5 mg/ml Tris-HCI buffer (pH 7.6) containing 0.01% H202; Sigma evacuated, and the external carotid artery was coagulated and ligated with silk. Chemical Co., St. Louis, MO] for 10 mm. X-GalStaining.Threedaysafterapplicationoftheelectricalpulseswith' Measurement of fi.Galactosidase Activity. For this experiment, each without plasmid injection (intraarterial or iv.), the bodies of three rats in each rat's body was perfused with 200 ml of ice-cold PBS. From the brain, the group were perfused via the heart with 200 ml of PBS, followed by 200 ml of region including the inoculated tumor and the contralateral brain were cut out 4% paraformaldehyde in PBS. Brain, heart, lung, liver, spleen, kidney, and and immediately frozen. The organs (heart, lung, liver, spleen, kidney, and testis were removed and fixed in the fixation buffer overnight. Brains were cut testis) were sampled, and about 100 mg of each tissue were minced, immedi by vibratome into 60-pm thick slices. Other tissues were soaked in sucrose ately frozen in a 1.5-ml Eppendorf tube in dry ice powder, and stored at â€”80Â°C buffer (10% sucrose in PBS) overnight, and the concentration of sucrose was until used. Before sample homogenization, 150 pAof ice-cold 0.25 MTris-HCI gradually elevated to 30%. Tissues were then frozen embedded in OCT (pH 7.8) were added to each tube. After repeating the freezing-thawing Compound (Miles, Inc., Elkhart, IN), cut into l0-p@mthick slices with a procedure three times, the samples were centrifuged for 30 ruin at 4Â°C.The cryotome, and stained with H & E to observe histological change. Sections of protein concentrations of the resulting l50-pi supernatants were determined the rat brain, including electrode tracts, were also stained with H & E for using a BCA kit (Pierce, Rockford, IL) and used for @3-galactosidase assay. The histological observation. assay was carried out on microtiter plates. Ten p3 of supernatant were added Expression of /3-galactosidasewas detected using X-Gal histochemical stain to 290 @tlofZ buffer [60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCI, 1 mM (23). Staining tissues that express @-galactosidase with X-Gal turns cells blue. MgC12, and 50 mM @-mercaptoethanol] in a microtiter plate well and incubated When an indoyl is liberated from X-Gal by the catalytic activity of @3-galac at 37Â°Cfor 10 mm. Then, 30 .tl of prewarmed 15 msi chlorophenol red-(3-D- tosidase, its subsequent oxidization and self-coupling forms an indigo blue galactopyranoside in Z buffer were added to each well and incubated at 37Â°C derivative. Therefore, only cells that accepted the injected plasmids and cells for 60 mm (29). The reaction was stopped by adding 75 @tlof 1 M Na2CO3 to with endogenous @3-galactosidasecanbe stained by this method and thus be each well, and the color reaction was quantitated in an ELISA reader set at 570 discriminated from other cells, using light microscopy. nm. The @-galactosidaseactivity was expressed as: (absorbance at 570 nm)/mg Construction ofthe Expression Vector ofthe Human MCP-1 cDNA. To protein X 10,000. Comparisons of values were analyzed by Student's t test test long-lasting transgene expression by our method, we selected another with P < 0.05 considered to indicate statistical significance. marker gene, human MCP-1 cDNA (15, 16). This cytokine is produced at a low level in normal tissues and at a high level in some kinds of tumors, including brain tumors, and has a significant role in intratumoral monocyte RESULTS infiltration (24, 25). Since the antitumor effect of this cytokine is reported Expression of the Intraarterially Injected lacZ Gene in Rat based on the results of the transfection experiment (26), we selected this gene Brains. We performed in vivo electroporation of inoculated brain tumors in rats, followed by intraarterial injection of plasmid DNA 300V/O.5cm containing the lacZ gene. To evaluate the efficiency of gene transfer, we performed X-Gal staining on slices of the treated rat brains. Fig. 2, A-D, shows the representative results of X-Gal staining of rat brain on inoculated tumor the third day following electroporation and intraarterial plasmid in jection [E+P+(ic) rats]. A restricted region centered along the border between inoculated tumor cells and adjacent brain stained blue, mdi cating expression of lacZ (Fig. 14). The high magnification picture shows dense staining of the border region and vascular wall and light staining of the region inside the tumor tissue (Fig. 2, B and C). The high magnification of X-Gal staining with H & E counterstaining clearly demonstrates that the tumor cells but not the adjacent tissue highly expressed the lacZ protein (Fig. 2D). The density of stained cells is higher in the region close to tumor vessels. No staining was DNA observed outside of the tumor border (Fig. 2D, lower right corner) in Fig. I. Schematic expression of the method for electro-gene therapy. this slice. 1051

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Fig. 2. Microscopic visualization of rat brains treated with electro-gene transfer. X-gal staining of @@ brains with tumors. Photomicrograph demonstrates F .. @-galactosidase-transduced cells in rat brains 3 days after the electro-gene transfer [E+P+(ic) group: A, B, C, and D] and control procedures [E+P- group: E and F; Eâ€”P+ group: G and H; E+P+(iv): i and J]. A, sagittal section of E+P+(ic) rat brain; X-Gal staining, X20. B, higher magnification of A, XlOO. @@ _Â°@s__ C. coronal section of the another E+P+(ic) rat brain; X-GaI staining, X40. D, high magnification of X-0a1 staining with H & E counterstain of the an other E+P+(ic) rat brain, X200. E. coronal section of E+P- rat brain;X-Galstaining,X40.F, X-Gal stainingwithH&EcounterstainofE+P- rat, X40. G, coronal section of E-P+ rat brain; X-Gal stain ing. X40. H. X-Ga1staining with H & E counterstain of Eâ€”P+rat brain, X40. I, coronal sectionof E+P+(iv) rat brain; X-Gal staining, X40. I, X-Gal staining with H & E counterstain of E+P+(iv) rat G H brain, X40.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1996 American Association for Cancer Research. ELECIRO-GENE THERAPY @ E+Pâ€”(electroporationonly) rat brain shows no X-Gal-positive A. cells, even in the border region of inoculated tumor tissues (Fig. 2, E and F). Only several cells inside vascular structures expressed in Eâ€”P+ (plasmid injection only) rat brain (Fig. 2, G and H). Further more, electroporation followed by i.v. plasmid injection could deliver @@@ the lacZ gene into a few tumor cells, including vascular wall : . .: . ,.,:,@, .â€˜â€”@. .

[E+P+(iv) rats; Fig. 2, 1 and J]. ., I Expression of the Human MCP-1 Gene 3 Weeks after Electro poration and Microscopic Observation of the Electrode Tract. The human MCP-1 cDNA expression plasmid with the EBV episomal replicon system was transferred into the inoculated rat brain tumor by electro-gene transfer. Long-lasting transgene expression was exam med by immunohistochemical study on frozen sections of the rat brains at 3 weeks after the transfer procedure, at which time the tumor cells were shown to be expressing human MCP-l protein (Fig. 3, A B and B). Because the antihuman MCP-l monoclonal antibody used in .. â€˜ @ this study does not cross-react with endogenous rat MCP-1 protein â€œ@i': @@ (28), immunoreactivecells express human MCP.l encoded by the â€¢;@â€˜Z':@Â°',â€˜@i! @@@ . ..t'@.:_. â€¢,,â€˜@ @@â€¢: transferred plasmid. Significantly, large numbers of macrophages and lymphocytes were observed in tumor tissues, especially in the periph eral area (Fig. 3B, lower left corner). The antitumor effects of cx pressed MCP-l and infiltrated host cells are now under investigation. No expression of human MCP-l protein was detected in E+Pâ€” (electroporation only) rats (Fig. 3C). The tracts made by electrode insertions were filled with astrocytes and lymphocytes, but there were no reactive changes in the surround ings (Fig. 3D), consistent with a previous report (17). No adverse @ clinical effects, such as seizure and paresis, were observed in rats in c@.

@@ any groups during the 3-week follow-up period. â€˜ â€” ,.. /@,@ @ Ã˜-Galactosidase Activities in Rat Brain and Other Organs I @ ...... : .@f â€¢.@ @\â€œ@,: â€˜@ @â€˜;â€˜@@ â€˜: _@â€˜@ @, :: @@ Outside Electrodes. To quantify the efficiencies of gene transfer and ...@

expression achieved by the electro-gene transfer, we measured the ,â€˜. @.- I @ lacZ enzyme activities in homogenates of the various tissues. The -. â€˜i_ I region that included inoculated C6 cells expressed statistically signif icant higher activities in E+P+ rats than in E+Pâ€” and Eâ€”P+ rats, whereas the contralateral brain does not have high activities (Fig. 4A).

@. @ A previous study demonstrated that systemic i.v. injection of plasmids . . .. _â€¢t...@ mixed with cationic liposomes allowed systemic expression of the @Â°,- rJ 1 transferred gene (30). This nonspecific delivery of injected plasmid is a potential major problem in our system using intraarterial injection. @ Therefore, we examined whether the organs outside electrodes D. . .. I.. .. showed expression of the transferred gene. We measured the lacZ enzyme activities in homogenates of the major organs such as heart, lung, liver, spleen, kidney, and testis and compared the activities in DNA-injected rats [E+P+ (ic), n = 5; Eâ€”P+, n 4] with those in DNA nomnjected rats (E+Pâ€”, n = 5). Each organ had endogenous enzyme activity at various levels. Although kidneys in electroporation and DNA-injected rats expressed relatively high /3-galactosidase ac @: - . .. ,1 tivities, the difference between these levels and those seen in the 1@ . groups not injected with DNA was trivial and not significant (P = 0.281). The enzyme activities in other organs were at the same levels in both groups (Fig. 4B). .. :1

DISCUSSION Fig. 3. Immunohistochemical detection of the human MCP-1 gene product in tumor cells inoculated in rat brain. The human MCP-l expression vector, pCEP/hMCP-l, was intraarterially injected after electroporation into rats with brain tumors. Rats were sacri This study demonstrates that the novel gene transfer method using ficed 3 weeks after the transfer procedure. The brain slice, including tumor tissue, was in vivo electroporation followed by intraarterial plasmid DNA injec subjected to immunohistochemical staining with antihuman MCP-l. A and B, immuno tion can provide efficient and region-controlled gene transfer. Because histochemical staining of human MCP-l protein of E+P+(ic) rat brain. A, most of the residual tumor cells expressed human MCP-l protein. A large number of monocytes are intraarterial plasmid injection alone could not transfer the gene into observed in tumor tissue, especially in the peripheral area of the tumor; X30. B, higher the tumor, in vivo electroporation was essential for the efficient magnification of A; X 100. C, immunohistochemical staining of E+Pâ€” rat brain (elec troporation only). Human MCP-l protein was not detected; X40. D, H & E staining of the transfer of the naked plasmid DNA. Furthermore, electroporation rat brain where the electrode was inserted 3 weeks before. The tract is filled with followed by i.v. injection could not efficiently transfer the plasmid. astrocytes and lymphocytes; X100. 1053

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1996 American Association for Cancer Research. ELECrR0-GENE THERAPY A electric field. Over 80% of the adherent COS-M6 cells took up plasmid DNA and expressed the lacZ gene, whereas the transfection efficiency was less than 20% when the M6 cells were electroporated in suspension (32). They speculated that the high-efficiency transfer into attached cells is explained by the characteristics of the attached .7 . cells, which have the higher surface:volume ratio compared to trypsin EDTA treatedsuspensioncells, andthe intactsubmembranecytoskel eton system. Because our lacZ gene expression system used in the present study was designed to test for transient expression, we measured the (3-ga lactosidase activity at the third day after the electrotransfer procedure. Long-lasting transgene expression was investigated by using the EBV E+P+ E.P+ E+P- E+P+ E-P+ E+P episomal replicon vector, which has human MCP-1 cDNA. The F 1@ transferred MCP-1 cDNA had been expressed in tumor cells for 3 tumor contralateral cerebrum weeks. Furthermore, the expressed MCP-l protein seemed functional B since a large numbers of monocytes were observed in the tumor tissue. There had previously been some trials of gene transfer using 2.25 plasmid DNA, such as local injection of mammalian expression plasmids, to produce local expression of a target gene and genetic I.75 1.5 immunization (33). Also, muscle tissue has been reported to be able to take up injected pla.smid DNA efficiently (34), and this uptake was a 1.25 enhanced if the muscle regeneration was induced by the myotoxic

.75 local anesthetic bupivacaine (35). Thu et a!. (30) reported that sys .5 temic i.v. injection of plasmids mixed with cationic liposomes was .25 nontoxic, which is consistent with other reports (36, 37). However, the @InEnlnin@ftdistribution of the injected DNA by this system was very wide and DNA injection (+) (.) (+) (.) (+) (.) (+) (.) (+) (.) (+) (.) nonspecific, and the amount of plasmid DNA injected for tissue I ii _lI I I I I 1 heart lung liver spleen kidney testis expression in their study was extremely high. To obtain high levels of chloramphenicol acetyltransferase gene expression, more than 100 pg Fig. 4. fi-Galactosidase activities in rat brain and other organs. Rats were sacrificed 3 days after the IacZ gene transfer. Brain. including inoculated tumor and contralateral DNA/20 g mouse tissue (5 pg/g body weight) were injected. In our brain, were cut out and immediately frozen. Major organs (heart, lung. liver, spleen, method, the amount of DNA for injection could be reduced to 10 pg kidney, and testis) were excised. and about 100 mg of each tissue were minced and plasmid/200 g rat tissue (0.05 pg/g body weight) by combining in vivo immediately frozen. @-Galactosidaseactivity was measured by using lysates of homog enized tissues. The @-galactosidaseactivity was expressed as: (absorbance at 570 nm)/mg electroporation and intraarterial plasmid DNA injection. Although we proteinX 10,000.A: @-galactosidaseactivitiesinbrains. @,E+P+rats(electroporation observed very specific and efficient transfer of injected plasmid using and intraarterial plasmid injection), n = 5; @,Eâ€”P+rats (intraarterial plasmid injection only),n = 5; 0, E+Pâ€”rats(electroporationonly),n = 4. Bars,SD.B: @-galactosidaseX-Gal staining,potentialmajorlimitationsof this methodcould have activities in other organs. U, plasmid injection + lE+P+(ic) and Eâ€”P+ rats], n 9; 0, been nonspecific delivery of injected plasmid outside the field be plasmid injection â€”(E+Pâ€” rats), n = 5. Bars, SD. tween the two electrodes, as Zhu et a!. (30) observed for their DNA:liposome complex injection, injury to brain tissues by insertion of the electrodes, and application of high voltage lasting only a This suggests that regionally high DNA concentration in electrically pulsed tissue is a crucial factor for efficient gene transfer. fraction of a millisecond. However, our data on the measurement of Injected DNA was distributed in the whole tumor tissue, while the the enzyme activities in major rat organs with plasmid injection transfer efficiency was uneven within tumor tissue. This efficient proved that the gene was not transferred outside of the vicinity of the transfer could be achieved only by intravascular, especially intraarte electrodes. Consistent with our data, Nabel et a!. (36) observed no rial, delivery of the gene. The fact that the transgene was highly abnormal pathology in histological examinations after systemic (i.v. expressed in the border region between tumor and adjacent brain, and intraarterial) injection of plasmids encoding several different where tumor neovascularization is abundant, may support this hypoth genes, even with plasmid-liposome complexes, into rabbit and pig. esis. The lacZ gene was not well transferred into normal brain cells They also showed that plasmid was not detected in testis or ovary except for cells in vascular walls, even inside the field between the (36). Recently developed materials and techniques for endovascular electrodes. It may be partly explained by the differences in vascular procedures allow us to catheterize tumor-feeding arteries super-selec structure between normal brain and tumor tissue. Capillaries in tumor tively, leading to a high concentration of injected plasmid DNA to the tissue may demonstrate cellular fenestrations, wide intercellular junc electrically pulsed region. This technique drastically cuts down tions, pinocytotic vesicles, and infolding of luminal surfaces (31). By the total amount of plasmid necessary for injection, greatly reducing these significant anatomical changes, molecules normally excluded by the probability of nonspecific gene transfer. the blood-brain barrier can penetrate the structure (31). Furthermore, Bleomycin administration by electrochemotherapy using in vivo the expression efficiency of transgene in rapidly proliferating tumor electroporation was successful for treatment of both inoculated and cells may be better than in normal brain cells by its demethylation of spontaneous tumors in animals (13, 17). Salford et a!. (17) applied this plasmid DNA. When tumor size is big enough, by inserting electrodes method for treatment of brain tumor in an animal model system. No inside tumor tissue, the gene transfer into vascular structure in the adverse effects were detected during the month following electropo normal brain could be avoided. ration, which was performed with 8 to 12 exponential 400-V pulses Theng and Chang (32) demonstrated that high-efficiency gene with a time constant at 325 p.s. Likewise, we did not observe any transfection can be obtained by directly electroporating cultured mam clinical side effects, such as seizure or abnormal behavior, during the malian cells in their attached state using a pulsed radiofrequency 3-week follow-up period under our experimental conditions. Histo 1054

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1996 American Association for Cancer Research. ELECTRO-CENE THERAPY logical examination also revealed no significant pathological changes, 14. Kuratsu, J., Leonard, E. J., and Yoshimura, T. Production and characterization of even in the regions of electrode insertion. human glioma cell-derived monocyte chemotactic factor. J. Nail. Cancer Inst., 81: 347â€”351,1989. Further study is essential to determine optimal conditions for gene 15. Yoshimura, T., Yuhki, N., and Moore, S. K. Human monocyte chemoattractant transfer, such as strength of electric current in electroporation, dosage protein-l (MCP-l): full-length cDNA cloning, expression in mitogen-stimulated blood mononuclear leukocytes, and sequence similarity to mouse competence gene of plasmid DNA for injection, timing of DNA injection, and proper JE. FEBS Lett., 244: 487â€”493,1989. vector systems. Our preliminary data indicate that lacZ activities 16. Leonard, E. 3., and Yoshimura, T. Human monocyte chemoattractant protein-I expressed inside tumor tissue correlates with the strength of the (MCP-l). Immunol. Today, 11: 97â€”101,1990. 17. Salford, L. G., Persson, B. R. R., Brun, A., Ceberg, C. P., Kongstad, P. C., and Mir, current used for electroporations (data not shown). To avoid tissue L. M. A new brain tumour therapy combining bleomycin with in viva electroperme injury by electric impulse, optimum electrical parameters for adequate abilization. Biochem. Biophys. Res. Commun., /94: 938â€”943.1993. gene transfer should be determined. Under our conditions of expen 18. Yamaguchi, 0., Irisawa, C., Babe, K., Ogihara, M., Yokota, T., and Shiraiwa, Y. Potentiationof antitumoreffectof bleomycinby local electricpulsesin mouse mentation, the current was kept very low and pulse length was bladder tumor. Tohoku J. Exp. Med., 172: 291â€”293,1994. relatively short (less than 0.1 ms); yet, we achieved a significant 19. Belehradek, J. 3., Orlowski, S., Poddevin, B., Paoletti, C., and Mir, L. M. Electro chemotherapy of spontaneous mammary tumours in mice. Eur. J. Cancer, 27: 73â€”76, amount of gene transfer and transgene expression. Our novel in vivo 1991. gene transfer method is a simple procedure and can solve some of the 20. Hall, C., Jacobb, E., Ringold, G., and Lee, F. Expression and regulation of Esche critical drawbacks of the present gene transfer techniques, thus pro richia coli Inc Z gene fusion in mammalian cells. J. Mol. AppI. Genet., 2: 101â€”109, 1983. viding a new strategy for gene therapy. 21. Sukharev, S. I., Kienchin, V. A., Serov, S. M., Chernommordik, L. V., and Chiz madzhev, Y. A. Electroporation and electrophoretic DNA transfer into cells: the effect of DNA interaction with electropores. Biophys. J., 63: 1320â€”1327,1992. ACKNOWLEDGMENTS 22. Tekle, E., Astumian, R. D., and Chock, P. B. Selective and asymmetric molecular transportacrosselectroporatedcellmembranes.Proc.Natl. 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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1996 American Association for Cancer Research. High-Efficiency in Vivo Gene Transfer Using Intraarterial Plasmid DNA Injection following in Vivo Electroporation

Toru Nishi, Kimio Yoshizato, Shigeo Yamashiro, et al.

Cancer Res 1996;56:1050-1055.

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