Antisense in vivo knockdown of synaptotagmin I by HVJ–liposome mediated gene transfer attenuates ischemic brain damage in neonatal rats

Brain & Development 30 (2008) 313–320 www.elsevier.com/locate/braindev

Original article

Antisense in vivo knockdown of synaptotagmin I by HVJ–liposome mediated gene transfer attenuates ischemic brain damage in neonatal rats Tadaki Omae a,*, Hiroshi Yoshioka a, Taro Tanaka a, Hideyuki Nagai a, Makoto Saji b,c, Kazuko Noda b, Shizuka Kobayashi c, Tohru Sugimoto a a

Department of Pediatrics, Kyoto Prefectural University of Medicine, 465 Kajii-cho Kawaramachi-Hirokoji Kamigyo-ku, Kyoto 602-0841, Japan b Department of Physiology, School of Allied Health Sciences, Kitasato University, 1-15-1 Kitasato, Sagamihara, Kanagawa 225-8555, Japan c Division of Brain Science, Graduate School of Medical Science, Kitasato University, 1-15-1 Kitasato, Sagamihara, Kanagawa 225-8555, Japan Received 31 May 2007; received in revised form 31 July 2007; accepted 2 August 2007

Abstract Synaptic release of the excitatory amino acid glutamate is considered as an important mechanism in the pathogenesis of ischemic brain damage in neonates. Synaptotagmin I is one of exocytosis-related proteins at nerve terminals and considered to accelerate the exocytosis of synaptic vesicles by promoting fusion between the vesicles and plasma membrane. To test the possibility that antisense in vivo knockdown of synaptotagmin I modulates the exocytotic release of glutamate, thus suppressing the excitotoxic intracellular processes leading to neuronal death following ischemia in the neonatal brain, we injected antisense oligodeoxynucleotides (ODNs) targeting synaptotagmin I (0.3 (AS), 0.15 (0.5 AS), or 0.03 lg (0.1 AS), or vehicle) into the lateral ventricles of 7-day-old rats by using a hemagglutinating virus of Japan (HVJ)–liposome mediated gene transfer technique. At 10 days of age, these rats were subjected to an electrical coagulation of the right external and internal carotid arteries, then the insertion of a solid nylon thread into the right common carotid artery toward the ascending aorta up to 10–12 mm from the upper edge of the sternocleidomastoid muscle. Cerebral ischemia was induced by clamping the left external and internal carotid arteries with a clip, and ended by removing the clip 2 h later. Twenty-four hours after the end of ischemia, the extent of ischemic brain damage was neuropathologically and quantitatively evaluated in the neocortex and striatum. While the relative volume of damage in the cerebral cortex and striatum of the vehicle group was extended to 40% and 13.7%, respectively, that in the AS group was significantly reduced to 4.8% and 0.6%. In the 0.5 AS group, the relative volume of ischemic damage in the cerebral cortex and striatum was reduced to 20.5% and 15.4%, respectively, and the difference between the 0.5 AS group and vehicle group was statistically significant in the neocortex, but not in the striatum. These results indicated that antisense in vivo knockdown of synaptotagmin I successfully attenuated ischemic brain damage in neonatal rats and that the effect was dose-dependent. It was also suggested that this treatment was more effective in the neocortex than in the striatum in neonatal rats.  2008 Published by Elsevier B.V. Keywords: Neonate; Brain ischemia; Rat; Synaptotagmin I; Glutamate; Antisense oligonucleotides; HVJ–liposome; Gene transfer

1. Introduction

* Corresponding author. Address: Never Land Gokoumachi-Oike Room No. 1201, 375 Gokoumachi Oike Agaru Nakagyou-ku, Kyoto 604-0941, Japan. Fax: +81 75 253 5271. E-mail address: [email protected] (T. Omae).

0387-7604/$ - see front matter  2008 Published by Elsevier B.V. doi:10.1016/j.braindev.2007.08.002

Hypoxic–ischemic brain damage in neonates is a problem of an enormous importance. Synaptic release of the excitatory amino acid glutamate during and after an ischemic event is considered very important to the pathogenesis of brain damage. The extracellular gluta-

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mate concentration in vivo increases many fold with hypoxic–ischemic insults. The increased amount of extracellular glutamate excessively activates glutamatereceptors and calcium overload, and may promote ischemic neuronal death. However, it is not clear whether the synaptic release of glutamate at nerve terminals is most responsible for the hypoxic–ischemic brain damage resulting from neuronal death. Furthermore, although the extracellular overflow of glutamate has been documented in neonatal animal models as well as adults, the increases seem to be less extensive in neonates [1–3]. In a variety of perinatal hypoxic–ischemic models, treatments with glutamate-receptor channel blockers have been tried to protect against neuronal death. Recent studies have shown major problems with glutamate-receptor antagonists [4,5]. Since modulation of presynaptic release would affect all the synapses, the antisense knockdown of proteins involved in the release of transmitters is expected to minimize treatmentinduced disruption of the interaction between multiple chemical transmitter systems. Such an approach seems to be a promising way to develop effective treatments for ischemic brain damage with less side-effects. Among exocytosis-related proteins that are abundant at nerve terminals, synaptotagmin I is considered to regulate the exocytosis of synaptic vesicles as a major Ca2+ sensor by promoting fusion between the vesicles and plasma membrane via the assembly and clustering of the SNARE complex [6–10]. It is postulated that the antisense in vivo knockdown of synaptotagmin I would reduce rates of the exocytotic release of transmitters at the synaptic terminals. We have already demonstrated in adult rats that the knockdown of synaptotagmin I prevented amygdaloid seizure-induced damage in the hippocampus [11] and attenuate ischemic damageto the hippocampus [12] and that the nigral injection of antisense oligonucleotides targeting synaptotagmin I successfully disrupted the release of dopamine in the striatum [13]. However, there is no study demonstrating in neonatal animals that antisense in vivo knockdown of synaptotagmin I modulates the exocytotic release of glutamate, thus suppressing the excitotoxic intracellular processes leading to neuronal death. To test the relevance of the ‘transmitter release strategy’ for neuroprotection against ischemic brain damage in neonates, we injected antisense oligodeoxynucleotides (ODNs) against synaptotagmin I into the lateral ventricles using a hemagglutinating virus of Japan (HVJ)–liposome mediated gene transfer technique prior to a transient forebrain ischemia in neonatal rats [14] and examined whether the knockdown of synaptotagmin I in the whole brain could regulate ischemia-induced neuronal death. To achieve a long-lasting downregulation of synaptotagmin I by a single treatment with the antisense ODNs, we used a novel transfection vector (HVJ–liposome) [15– 17]. It has been demonstrated that using this HVJ–lipo-

some method oligodeoxynucleotides can be efficiently delivered into neurons, predominantly in cell nuclei, both in vitro and in vivo [19]. 2. Materials and methods 2.1. Animal care Neonatal rats (Wistar strain) used in this study were housed in clear plastic cages with their dams, which were allowed free access to food and water throughout the experiment. The animals were maintained in a temperature-, humidity-, and light-controlled environment with a 12 h light/dark cycle. All experiments were performed in accordance with the Japanese and International Guidelines on the ethical use of animals, and every effort was made to minimize the number of animals and their suffering. This study was approved the Animal Care and Utilization Committee of Kyoto Prefectural University of Medicine. 2.2. Antisense oligodeoxynucleotides The phosphorothioated ODNs (45 mers) corresponding to specific sequences in the 5 0 -coding region of synaptotagmin I were designed to selectively block the biosynthesis of synaptotagmin I [18] as antisense ODNs (stm-AS: 5 0 -TGAAGCTATGCTAGATGCAGTGGT AGGAACGCATTGGCTCCTGTT-3 0 ). The antisense sequence complementary to the coding region for synaptotagmin I did not overlap with other mammalian sequences determined by a search of the GeneBank/EMBL database. 2.3. Preparation of HVJ–liposomes containing oligodeoxynucleotides The preparation of the anionic HVJ–liposome vector containing ODNs (ODN–HVJ–liposome) has been described elsewhere [19,20]. Briefly, three kinds of lipid (phosphatidylserine, phosphatidylcholine, and cholesterol) dissolved in chloroform (1 mg/ml) were mixed at a weight ratio of 1:4.8:2. Ten milligrams of the lipid mixture was transferred into a glass tube and dried as a thin lipid film using a rotary evaporator filled with nitrogen gas at 40 C. The thin lipid film layering the bottom of the glass tube was hydrated in a 200 ll balanced salt solution (BBS: 137 mM NaCl, 5.4 mM KCl, and 10 mM Tris–HCl, pH 7.5) containing 100 lg of ODNs (7.1 nmol synaptotagmin I antisense) that were dispersed in the aqueous phase at room temperature. The mixture of the hydrated lipid film and ODNs was vortexed for 30 s and incubated for 30 s at 37 C. This procedure was repeated eight times, making fragments of the thin lipid film into liposomes in which ODNs were packaged. Then, 300 ll of BBS was added to the

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liposome suspension and incubated at 37 C for 30 min with shaking. The liposome suspension (500 ll) was mixed with a 1 ml suspension of HVJ (more than 10,000 hemagglutinating units), the RNA genome of which was inactivated by ultraviolet irradiation (198 mJ/cm2) just before use. The mixture was incubated at 4 C for 10 min and 37 C for 1 h with shaking to facilitate the fusion between the inactivated HVJ and liposome. The ODN–HVJ–liposome complex was loaded onto a discontinuous sucrose gradient and centrifuged at 25,000 rpm for 1.5 h at 4 C to separate the ODN–HVJ–liposome complex from free HVJ. The purified ODN–HVJ–liposome suspensions were adjusted to OD 1.0 (540 nm) with 200–250 ll of BBS. Since 10– 30% of the ODNs (10–30 lg) are available for packing into the liposomes [15], it is estimated that 1 ll of the purified ODNs–HVJ–liposomes (OD 1.0) contains about 40–120 ng of ODNs. 2.4. Intraventricular injection of ODN–HVJ–liposome complex and transient forebrain ischemia In a previous study [12], we injected intraventricularly 30 ll of HVJ–liposome containing ODNs into adult rats without any trouble. Thus, we believe this to be a critical dose for adult rats to avoid an abnormal increase of intraventricular pressure. Considering the volume of cerebrospinal fluid (300–500 ll) and body weight (about 250 g) of adult rats, we determined that 2 ll of HVJ– liposome containing ODNs is a critical dose for 7-dayold rats (body weight: about 15 g). Seven-day-old rats of the Wistar strain (14–16 g) were anesthetized with an intraperitoneal pentobarbital injection (25 mg/kg), and 1 ll of HVJ–liposome containing ODN (antisense to synaptotagmin I) or not was stereotaxically injected into the lateral ventricles on each side. The coordinates in mm with respect to the Bregma were as follows: anterior 1 mm; lateral ±1 mm. A silicon-coated glass micropipette (tip size: 30–40 lm; volume 2.5 ll/cm) made from disposable micropipette (20 ll, Drummond) that was connected to an air pressure system was used for focal injection of the ODN–HVJ–liposome suspension. To study the dose-dependent effect, we injected HVJ– liposomes containing 0.3 lg of antisense ODN into 12 pups (AS group), 0.15 lg of ODN into 12 pups (0.5 AS group), 0.03 lg of ODN into 12 pups (0.1 AS group), and no ODN into 12 animals (vehicle group). At 10 days of age, the rats were subjected to 2 h of forebrain ischemia by a method described previously [14]. Briefly, they were anesthetized with an intraperitoneal pentobarbital injection (25 mg/kg), and subjected to an electrical coagulation of the right external and internal carotid arteries, then the insertion of a solid nylon thread (0.47 mm in diameter) into the right common carotid artery toward the ascending aorta up to 10–12 mm from the upper edge of the sternocleidomastoid muscle. Cere-

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bral ischemia was induced by clamping the left external and internal carotid arteries with a small clip. Recirculation was started by removing the clip after a 2-hour-ischemia. Our model is based on the following two characteristics of blood flow in the brain of rats. First, the innominate artery gives rise to both the right common carotid and right subclavian arteries, and the latter supplies the right vertebral artery. Insertion of the nylon thread interrupts the blood flow in the right common carotid artery completely and reduces that in the right vertebral artery. Second, the cerebral blood flow of the right hemisphere is maintained even when blood flow in the right common carotid artery is interrupted because the circle of Willis is well developed in the rat, until the left external and internal carotid arteries are clamped. Surviving animals were observed for 3 h after recirculation as to whether they exhibited seizures, dyspnea, or pale skin color. Body temperature was maintained at about 37 C by means of a heating pad throughout the experiment. Twelve littermates, which were subjected to a 2-hourischemia without any intraventricular injection, served as the non-treated (NT) group. 2.5. Quantitative neuropathlogical study Twenty-four hours after the end of ischemia, 60 rat pups were perfused-fixed transcardially with 4% paraformaldehyde in phosphate buffer (4% PFA in PB), and their brains were removed. Seven control littermates were also included in this study. After fixation for a week, brains were embedded in paraffin wax. Coronal slices 6 lm thick were sectioned at every 100 lm and stained with nissle. The extent of ischemic brain injury was quantitatively evaluated in the neocortex and striatum. Infarcted areas and the total area were measured with an image analyzer (NIH image 1.61) at seven representative coronal levels for the neocortex (7.0, 5.6, 4.1, 3.2, 2.0, 0.8 and 0.5 mm anterior to the interauricular line according to the rat brain atlas of Sherwood and Timiras (1970)) and at four levels for the striatum (5.6, 4.1, 3.2 and 2.0 mm anterior to the interauricular line). Percentages of infarcted volumes in the neocortex or striatum were derived by numerical integration of sequential infarcted areas divided by the sequential total area of each structure. In addition, we verified pathologically in each animal that the tip of the micropipette had reached the lateral ventricle. 2.6. Statistical analysis Data are presented as means ± standard error (SE). The extent of the infarcted area and volume in the neocortex or striatum were analyzed with a Kruskal–Wallis test and Steel–Dwass Post-hoc test. A P-value of less than 0.05 was considered to represent a significant difference.

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3. Results No animals died before the ischemic manipulation, even though, except in the non-treated group, they received the intraventricular injection. After the ischemic event, 3 of 15 pups (20%) in the non-treated group died. Similarly, the proportion that died during the recovery period was 5 of 17 (29%) in the vehicle group, 6 of 18 (33.3%) in the 0.1 AS group, 4 of 16 (25%) in the 0.5 AS group, and 4 of 16 (25%) in the AS group. These rates did not differ significantly between groups. As shown in Fig. 1, brain damage in the vehicle group was similar to that in the non-treated group. Although brain infarction was severe and extensive in the cerebral cortex and striatum in the non-treated and vehicle groups, infarction in the AS group was apparently mild. Brain infarction group looked slightly milder in the 0.5 AS than non-treated group, and damage in the 0.1 AS group was similar to that in the non-treated group. Fig. 2 shows areas of ischemic infarction on different coronal planes in the cerebral cortex and striatum in all groups except for the non-treated group whose results were similar to those of the vehicle group. There were significant differences between the AS group and nontreated or vehicle group on all coronal planes in both the cerebral cortex and striatum. In the cerebral cortex, there were also significant differences between the 0.5 AS group and non-treated or vehicle group on most coronal planes. In the striatum, however, there was no significant difference between the 0.5 AS group and non-treated or vehicle group on any coronal plane. Between the 0.1 AS group and non-treated or vehicle group, there was no significant difference on any coronal plane in either the cerebral cortex or the striatum. Fig. 3 demonstrates the relative volume of ischemic brain damage in the cerebral cortex and the striatum. As shown in Fig. 3, in the non-treated and vehicle groups, damage extended to 40.1 ± 4.9% and 33.1 ± 2.3% of the cortex, respectively. On the other hand, infarction in the AS group was significantly reduced to 4.85 ± 1.9% of the cortex and the difference between the AS group and vehicle or non-treated group was significant. In the 0.5 AS group, too, ischemic brain damage affected 20.5 ± 1.4% of the cerebral cortex and was significantly milder than in the vehicle or non-treated group, though the difference between the AS and 0.5 AS groups was also statistically significant. 4. Discussion The release of neurotransmitters from a nerve terminal is mediated by the exocytosis of synaptic vesicles. After the release, synaptic vesicles are endocytosed, refilled, and prepared for subsequent rounds of release at nerve terminals. Synaptotagmin I, a synaptic vesicle protein, is a key regulator of Ca2+-dependent exocytosis.

There is evidence supporting the involvement of synaptotagmin I in Ca2+-dependent transmitter release: a microinjection of synaptotagmin antibodies or synthetic peptides from synaptotagmin I [6] and knockout of synaptotagmin genes [21] impaired synaptic transmission. A recent in vitro study with cortical neurons from synaptotagmin knockout mice revealed that synaptotagmin I controls the kinetic efficiency of endocytosis and plays a key role in the recycling of vesiclesat nerve terminals as well as in regulating exocytosis [22]. Kobayashi et al. [11] examined the antisense-induced reduction of synaptotagmin I protein levels in the hippocampus following the intra-hippocampal injection of an HVJ–liposome suspension containing ODNs into adult rats and found that the amount of synaptotagmin I protein in the hippocampus diminished markedly at 4 days post-injection, and this reduction was sustained days before a partial recovery by 10 days post-injection. Kobayashi et al. [23] also investigated the antisenseinduced changes in the levels of drebrin A, a dendritic spine protein, in the neocortex and hippocampus of adult rats at 4, 8, and 18 days after the intraventricular injection of HVJ–liposome vectors containing antisense ODNs. They demonstrated that the levels of drebrin A protein in the neocortex of the rats treated with antisense ODNs decreased markedly at 4 days after the injection, remained reduced for several days, and returned to the original level by 18 days post-injection. There was no previous study, in which the time course of the antisense-induced reduction of synaptotagmin I protein levels was investigated in neonatal brain following the intraventricular injection of an HVJ–liposome suspension containing ODNs. At first, we tried to investigate the time course of the antisense-induced change of synaptotagmin I protein levels in neonatal rat brain following the intraventricular injection of an HVJ–liposome suspension containing ODNs by synaptotagmin I immunohistochemistry and Western blot analysis, but unsuccessful. Based on our previous findings in adult rats stated above [11,23], therefore, we injected HVJ– liposome 3 days before ischemic manipulation in this study. In the present study, the areas and percent volume of ischemic damage in the cerebral cortex and striatum were significantly reduced in the AS group compared to the non-treated and vehicle groups. This result indicated that antisense in vivo knockdown of synaptotagmin I successfully attenuated ischemic brain damage in neonatal rats. Since synaptotagmin I is considered to promote the exocytosis of synaptic vesicles exocytosis at nerve terminals, it is reasonable to consider that the antisense ODNs blocked mRNA relating to synaptotagmin I’s construction, thus suppressing the exocytosis of glutamate from nerve terminals and recycling of vesicles at nerve terminals, and finally attenuated ischemic brain damage in neonatal

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Fig. 1. Coronal sections of neonatal rat brains in the non-treated (a and b), vehicle (c and d), 0.1 AS (e and f), 0.5 AS (g and h) and AS (i and j) groups. Note the gross infarcts in the neocortex (arrowheads) and striatum (arrows) in the non-treated and vehicle groups (a–d). Antisense in vivo knockdown of synaptotagmin I apparently reduced ischemic brain damage (i and j). (Nissl’s stain, original magnification 4.0·.)

rats. To ascertain our speculation that antisense in vivo knockdown of synaptotagmin I suppressed the exocytosis of glutamate from nerve terminals, a future study, in which changes in glutamate concentra-

tions in the extracellular space following antisense in vivo knockdown of synaptotagmin I and ischemic event will be investigated in neonatal rats by microdialysis method, will be necessary.

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Fig. 2. Effect of antisense in vivo knockdown of synaptotagmin I on the extent of ischemic infarction in the neonate on different coronal planes in the neocortex (a) and striatum (b). There are significant differences between the vehicle and AS groups on all coronal planes examined in both the neocortex and striatum. There are also significant differences between the vehicle and 0.5 AS groups on some coronal planes examined in the neocortex, but not in the striatum. Data represent means ± SE. Vertical lines represent ±SE and are omitted when the SE is smaller than the size of the symbols. The number of animals is 12 in each group. **p < 0.01 compared to 0.5 AS, 0.1 AS, vehicle. *a, p < 0.05 compared to 0.5 AS. **b, p < 0.01 compared to 0.1 AS, vehicle. #p < 0.05 compared to 0.1 AS, vehicle. ##p < 0.01 compared to 0.1 AS, vehicle. #c, p < 0.05 compared to 0.1AS. #d, p < 0.05 compared to vehicle. ##d, p < 0.01 compared to vehicle. $ p < 0.05 compared to vehicle.

In a previous study with adult rats [12], we investigated effects of antisense in vivo knockdown of synaptotagmin I on ischemic injury to the hippocampus. When HVJ–liposomes containing antisense ODNs were intraventricularly injected into adult rats 4 days prior to a ligation of the bilateral common carotid arteries for 20 min with permanent coagulation of vertebral arteries, the survival rate of CA1 neurons of the hippocampus increased from 45% to 91%. In the same study, the amount of synaptotagmin I protein in the hippocampus determined with a Western blot assay, decreased by 29% within 4 days after the injection of HVJ–liposomes, while no reduction was observed in the neocortex. The results of the present study may indirectly support stud-

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Fig. 3. Effect of antisense in vivo knockdown of synaptotagmin I on the percent volume of ischemic damage in the neocortex (a) and striatum (b). The extent of infarction was significantly reduced in both the neocortex and striatum in the AS group than in the vehicle, 0.1 AS, or 0.5 AS group. In the 0.5 AS group, only the infarcted volume in the neocortex was significantly less than that in the vehicle group. Data represent means ± SE. The number of animals is 12 in each group. ** p < 0.01 compared to 0.5 AS, 0.1 AS, vehicle, NT. ##p < 0.01 compared to 0.1 AS, vehicle, NT.

ies by Berton F. et al. and by Tocco G. et al. who investigated developmental changes in the expression of synaptotagmin I mRNA in rats using in situ hybridization and found that the mRNA is expressed in the cerebral cortex at birth at levels no less than those in adult brain [24,25]. In the present study, we also investigated whether antisense ODNs for synaptotagmin I have a dose-dependent effect on ischemic brain damage. As shown in Fig. 3, brain infarction in the cerebral cortex was significantly attenuated in the AS and 0.5 AS groups compared to the non-treated and vehicle groups. However, brain damage in the 0.1 AS group was similar to that in the non-treated or vehicle group. In addition, the relative volume of ischemic damage in the cerebral cortex also differed significantly between the AS and 0.5 AS groups. Thus, it was indicated that at least 0.15 lg of antisense ODN targeting synaptotagmin I was necessary to effectively attenuate ischemic damage in the cerebral cortex of neonatal rats. In the striatum, however, the relative volume of damage in the 0.5 AS group was not significantly different from that in the non-treated or vehicle group, though

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the difference between the AS group and vehicle or nontreated group was significant. Therefore, it was suggested that a much higher dose of antisense ODNs was necessary to effectively attenuate ischemic injury in the striatum than cerebral cortex of neonatal rats. The reason why a relatively large dose of antisense ODNs was needed to have a significant effect in the striatum is not clear at present. Berton et al. reported that although synaptotagmin I mRNA was expressed at birth in the rat caudate-putamen, the level was very low and about one half of that in adults [25]. Therefore, 0.3 lg of antisense ODNs targeting synaptotagmin I may not have been enough to effectively attenuate ischemic brain damage in the striatum of neonatal rats. We observed body growth and gross neurological and behavioral development in six neonatal rats after the intraventricular injection of HVJ–liposomes containing antisense ODNs for synaptotagmin I to adulthood. None of the animals showed abnormal behavior like convulsions, hyper- or hypoactive locomotion, defective adaptations, or lack of eating and drinking (unpublished data). Body weight gain was slightly affected for a few days after the injection, but soon recovered. However, the antisense-induced knockdown of synaptotagmin I should modulate the release of neurotransmitters in the whole brain and may result in some neurological aberration in developing rats. Therefore, there is a need for further detailed study on neurological and behavior effects of antisense in vivo knockdown of synaptotagmin I.

[7]

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[16]

Acknowledgement We are grateful to Dr. Yasufumi Kaneda at Osaka University Medical School for kindly teaching the HVJ–liposome mediated gene transfer technique.

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