- Email: [email protected]
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co-reconstituted with the phage receptor and components or entrapped within the liposomes to favor the gene delivery process or to protect the DNA from degradation. Also, the technique might be improved by using phage lambda, which is used extensively as a cloning vector and in recombinant technology and whose receptor, the LamB protein, has been reconstituted functionally into liposomes. Acknowledgments We acknowledge J. Bo¨hm and W. Baumeister for their fruitful collaboration on electron tomography. We have benefited through this work with the extensive collaboration of L. Letellier and her collaborators. We also thank W. Gelbart and D. Levy for stimulating discussions.
[30] The Hemagglutinating Virus of Japan–Liposome Method for Gene Delivery By Yasufumi Kaneda, Seiji Yamamoto, and Kazuya Hiraoka Introduction
Toward the success of human gene therapy, numerous viral and nonviral (synthetic) methods of gene transfer have been developed,1,2 but each has its own limitations as well as advantages. Therefore, to develop in vivo gene transfer vectors with high efficiency and low toxicity, several groups have attempted to overcome the limitations of one vector by combining them with the strengths of another. Our basic concept is the construction of novel, hybrid-type liposomes with functional molecules inserted into them.3,4 On the basis of this concept, DNA-loaded liposomes were fused with ultraviolet (UV) inactivated hemagglutinating virus of Japan; Sendai virus (HVJ) to form HVJliposomes (approximately 400–500 nm in diameter). Those viral liposomes bind to cell surface sialic acid receptors and fuse with cell membrane to directly introduce DNA into the cytoplasm without degradation. The HVJ–liposomes can encapsulate DNA smaller than 100 kb. RNA, oligodeoxynucleotides (ODN), proteins, and drugs can also be enclosed and 1
R. C. Mulligan, Science 260, 926 (1993). F. D. Ledley, Hum. Gene Ther. 6, 1129 (1995). 3 Y. Kaneda, Biogenic Amines 14, 553 (1998). 4 Y. Kaneda, Y. Saeki, and R. Morishita, Mol. Med. Today 5, 298, (1999). 2
METHODS IN ENZYMOLOGY, VOL. 373
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delivered to cells. Recently, we explained by use of fluorescence resonance energy transfer (FRET) that the degradation of ODN in the process of delivery to the cytoplasm is inhibited by HVJ-liposomes but not with simple cationic liposomes.5 HVJ-liposomes are useful for in vivo gene transfer.6 When HVJ– liposomes containing the LacZ gene were injected directly into one rat liver lobe, approximately 70% of cells expressed LacZ gene activity, and no pathological hepatic changes were observed.7 One advantage of HVJ– liposomes is allowance for repeated injections. Gene transfer to rat liver cells was not inhibited by repeated injections. After repeated injections, anti-HVJ antibody generated in the rat was not sufficient to neutralize HVJ–liposomes. Cytotoxic T cells recognizing HVJ were not detected in rats transfected repeatedly with HVJ-liposomes.7 The safety of HVJ– liposomes has been tested and evaluated in monkeys.8 To increase the efficiency of gene delivery by HVJ–liposomes, we investigated the lipid components of liposomes9 and concluded that the most efficient gene expression occurred with liposomes consisting of a phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, phospatidylserine, and cholesterol at the molar ratio of 13.3:13.3:13.3:10 and 50, respectively. The lipid components of the liposomes are very similar to the HIV envelope and mimic the red blood cell membrane.10 We called the liposomes HVJ–AVE liposomes (i.e., HVJ-artificial viral envelope liposomes). With HVJ–AVE liposomes, gene expression in heart, liver, and muscle was 5–10 times greater than that observed with various nonviral gene transfer methods such as conventional HVJ–liposomes, cationic-lipid–mediated lipofection and naked DNA injection.4 Another improvement was construction of cationic-type HVJ– liposomes using cationic lipids. Of the cationic lipids, positively charged DC-cholesterol (DC)11 has been the most efficient for gene transfer. For luciferase expression, HVJ–cationic DC liposomes were 100 times more 5
N. Nakamura, D. A. Hart, C. B. Frank, L. L. Marchuk, N. G. Shrive, N. Ota, K. Taira, H. Yoshikawa, and Y. Kaneda, J. Biochem. 129, 755 (2001). 6 V. J. Dzau, M. Mann, R. Morishita, and Y. Kaneda, Proc. Natl. Acad. Sci. USA 93, 11421 (1996). 7 T. Hirano, J. Fujimoto, T. Ueki, H. Yamamoto, T. Iwasaki, R. Morishita, Y. Sawa, Y. Kaneda, H. Takahashi, and E. Okamoto, Gene Ther. 5, 459 (1998). 8 N. Tsuboniwa, R. Morishita, T. Hirano, J. Fujimoto, S. Furukawa, M. Kikumori, A. Okuyama, and Y. Kaneda, Hum. Gene Ther. 12, 469 (2001). 9 Y. Saeki, N. Matsumoto, Y. Nakano, M. Mori, K. Awai, and Y. Kaneda, Hum. Gene Ther. 8, 1965 (1997). 10 R. Chander and H. Schreier, Life Science 50, 481 (1992). 11 K. Goyal and L. Huang, J. Liposome Res. 5, 49 (1995).
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efficient than conventional HVJ–anionic liposomes.9 However, HVJ– cationic liposomes were not appropriate for gene transfer to liver, kidney, heart, and muscle, but they were much more effective for gene transfer to tumor masses or disseminated cancers12,13 in animal models compared with anionic-type HVJ–AVE liposomes. Therefore, HVJ-anionic and cationic liposomes can complement each other, and each liposome should be used for proper targeting. Materials and Instrumentation
Chromatographically, pure bovine brain phosphatidylserine-sodium salt (PS) (No. 83032L) is obtained from Avanti Polar Lipids Inc. (Alabaster, AL), and other lipids such as dioleoyl-l-alpha-phosphatidylethanolamine (DOPE) (P 5078), sphingomyelin (Sph) (S 0756), egg yolk phosphatidylcholine (PC) (P 2772), DC-Cholestrol (DC-chol) (C 2832), and cholesterol (Chol) (C 8667) are from Sigma (St. Louis, MO). All the lipids are stored at 20 . Polypeptone (Pancreatic Digest of Casein) (No. 394-00115) is obtained from Wako (Osaka, Japan). EDTA-3Na (ED3SS), sucrose (S 7903), trizma base (T 1503), chloroform (C 2432), NaCl (S 3171), KCl (P 9333), and dimethylsulfoxide (DMSO) (D 8779) are from Sigma. The procedures require 50-ml conical tubes (Becton-Dickinson, Lincoln Park, NJ), 35-ml centrifuge tubes (Beckman Instruments, Tokyo, Japan), ultracentrifuge tubes (Hitachi, Tokyo, Japan) and cellulose acetate membrane filters (0.45 m, No. 190-2545, and 0.20 m, No. 190-2520 (Nalgene Co., Rochester, NY). For preparing lipid mixtures, glass tubes 24 mm in diameter and 12 cm long were custom-made (Fujiston 24/40, Iwaki Glass Co. Ltd., Tokyo, Japan), but similar sterilized tubes resistant to chloroform are available. The fresh glass tubes are immersed in saturated KOH-ethanol solution for 24 h, rinsed with distilled water, and heated at 180 for 2 h before use. A rotary evaporator (Type SR-650, Tokyo Rikakikai Inc., Tokyo, Japan), vacuum pump with a pressure gauge (Type Asp-13, Iwaki Glass Co. Ltd., Tokyo, Japan), water bath (Thermominder Jr 80, TAITEC, Saitama, Japan), vortex mixer (Scientific Industries, Bohemia, NY), and water bath shaker (Thermominder, TAITEC, Saitama, Japan) are used, but similar instruments are also available from other manufacturers for this purpose. For preparation of plasmid DNA, an endotoxin-free column (Qiagen Inc., Germany) is recommended. 12 13
T. Otomo, S. Yamamoto, R. Morishita, and Y. Kaneda, J. Gene Med. 3, 345 (2001). T. Miyata, S. Yamamoto, K. Sakamoto, R. Morishita, and Y. Kaneda, Cancer Gene Ther. 8, 852 (2001).
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For purifying HVJ, a low-speed centrifuge (05PR-22, Hitachi, Tokyo, Japan), a centrifuge with JA-20 rotor (J2-HS, Beckman Instruments), and a photometer (Spectrophotometer DU-68, Beckman Instruments) are needed. A UV cross-linker (Spectrolinker XL-1000, Spectronics Co.) is used for inactivation of HVJ. For purifying HVJ–liposomes, an ultracentrifuge with RPS-40T rotor (55P-72, Hitachi, Tokyo, Japan) is used. Procedures for Preparation of Hemagglutinating Virus of Japan–Liposomes
Preparation of Hemagglutinating Virus of Japan Preparation of Hemagglutinating Virus of Japan in Eggs Reagents. 1. Polypeptone solution (1% polypeptone, 0.2% NaCl, pH 7.2): To make 500 ml, 5 g of polypeptone and 1 g of NaCl are solubilized in distilled water, the pH is adjusted to 7.2 by adding aliquots of 1 M NaOH, and the total volume is brought to 500 ml with distilled water. The solution is sterilized by autoclaving and stored at 4 . 2. BSS (137 mM NaCl, 5.4 mM KCl, 10 mM TRIS-HCl pH7.6): Eight grams of NaCl, 0.4 g of KCl, and 1.21 g of Trizma base are dissolved in distilled water, the pH is adjusted to 7.6 with aliquots of 1 M HCl and the total volume is brought to 1 l with distilled water. The solution is sterilized by autoclaving and store at 4 . 3. Seed of HVJ: Aliquots (100 l) of the best seed of HVJ (Z strain) in 10% DMSO are stored at 80 . Methods. 1. The seed is thawed rapidly and diluted 1000 times with poly peptone solution. The diluted seed should be kept at 4 before proceeding to the next step. 2. Embryonated eggs are observed under illumination in a dark room, and an injection point is marked at about 0.5 mm above the chorioallantoic membrane. The eggs are disinfected with tincture of iodine and punctured at the point marked. 3. The diluted seed (0.1 ml) is injected into each egg using a 1-ml disposable syringe with a 26-gauge needle. The needle should be inserted vertically so as to stab the chorioallantoic membrane. 4. After inoculation of the seed, the hole punctured on the egg is covered with melted paraffin. Then the eggs are incubated for 4 days at 36.5 in sufficient moisture.
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5. The eggs are chilled at 4 for more than 6 h before harvesting the virus. 6. The egg shell is removed partially, and the chorioallantoic fluid is aspirated with a 10-ml syringe (18-gauge needle) and placed in an autoclaved bottle. The fluid should be kept at 4 to avoid freezing. The virus is stable in the fluid at least for 3 months. Steps 2, 3, and 6 can be carried out at room temperature. Purification of Hemagglutinating Virus of Japan from Chorioallantoic Fluid Reagents. 1. BSS is prepared as described previously. Methods. 1. Two hundred milliliters of the chorioallantoic fluid is transferred into four 50-ml disposable conical tubes and subjected to centrifugation at 3000 rpm (1000g) for 10 min at 4 in a low-speed centrifuge. 2. Then, the supernatant is aliquoted into six tubes (Beckman JS-20) and centrifuged at 15,000 rpm (27,000g) for 30 min at 4 . 3. About 5 ml of BSS is added to the pellet in one of the tubes, and the materials are kept at 4 overnight. 4. The pellets are gently suspended, collected in two tubes, and centrifuged as described in step 2. The resultant pellet in each tube is kept at 4 in 5 ml of BSS for more than 8 h. 5. The pellets are suspended gently and centrifuged at 3000 rpm in a low-speed centrifuge. 6. The supernatant is removed to an aseptic tube and stored at 4 . 7. The virus titer is determined by measuring the absorbance at 540 nm of the 10 times–diluted supernatant using a photometer. An optical density at 540 nm corresponds to 15,000 hemagglutinating units (HAU), which is well correlated with fusion activity. The supernatant as prepared previously usually shows 20,000–30,000 HAU/ml. A virus solution prepared aseptically maintains fusion activity for 3 weeks. Preparation of the Lipid Mixture Methods 1. Dry reagents of DOPE (12.2 mg), Sph (11.5 mg), and Chol (23.8 mg) are dissolved in 3870 l of chloroform. The PC choloform solution (130 l) is added to the 3870 l lipid solution. This 4000 l of lipid solution is called a basal mixture for liposomes. The master mixture is
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Fig. 1. After evaporation, a thin lipid film is formed inside a glass tube.
ready to prepare anionic or cationic liposomes (described later) or can be stored at 20 after infusing nitrogen gas over the solution. 2. For preparation of an anionic or cationic lipid mixture, 10 mg of PS on 6 mg DC–Chol is added to the basal mixture, respectively. 3. The lipid solution of 0.5 ml is aliquoted into eight glass tubes; each tube contains 10 mg of lipids. The tubes are kept on ice or 20 under nitrogen gas before evaporation. The lipid solution should be evaporated as soon as possible. 4. The tube is attached to a rotary evaporator. The tube should be immersed in a 40 water bath at the tip. 5. The organic solvent is evaporated in the rotary evaporator under vacuum. The lipids are dried up for about 10 min. (Fig. 1). Preparation of Hemagglutinating Virus of Japan–Liposomes The procedure for the preparation of HVJ–liposomes is illustrated in Fig. 2. Preparation of Hemagglutinating Virus of Japan–Liposomes Containing DNA Reagents. 1. BSS, prepared as described previously.
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Fig. 2. Preparation of HVJ–liposomes and fusion-mediated gene transfer. DNA-loaded liposomes are fused with UV-inactivated HVJ to form HVJ–liposomes. HVJ–liposomes bind cell surface sialic acid receptors, degrade the receptor, and associate with the lipids in the cell membrane to induce membrane fusion. By fusion of the envelope of HVJ–liposomes with the cell membrane, DNA in the HVJ–liposomes can be introduced directly into the cytoplasm.
2. Plasmid DNA: Plasmid DNAs are purified by a column procedure. The preparations are dissolved in TE solution, BSS, or water. The final concentration of DNA should be more than 1 mg/ml, and stored at 20 . 3. Sucrose solutions: To prepare 30% and 60% (w/v) sucrose solutions, 150 g of sucrose is solubilized in BSS and the solution bright up to a total volume to 500 ml and 250 ml, respectively. The solution is sterilized by autoclaving and stored at 4 . Methods. 1. Plasmid DNA (200 g) in 200 l is added to a lipid mixture in the glass tube prepared as described previously, and agitated intensely by vortexing for 30 s. followed by incubation at 37 for 30 s. This cycle is repeated eight times. By this method, plasmid DNA up to 20 kb is
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encapsulated at an efficiency of 10–30% in anionic liposomes or 50–60% in cationic liposomes. 2. For preparing sized unilamellar liposomes, the liposome suspension is filtered through a 0.45-m pore size cellulose acetate filter and then through a 0.2-m filter. Sizing by an extruder with polycarobanate filters is better for preparing sized liposomes. 3. In the meantime, HVJ is inactivated and keep on ice. HVJ (15,000 HAU) is added to the liposome suspension and the tube is incubated on ice for 5 to 10 min. Then, the sample is incubated at 37 for 1 h, with shaking (120/min), in a water bath. 4. One milliliter of 60% sucrose and 7 ml of 30% sucrose are added to a centrifuge tube, and the HVJ–liposome mixture is overlaid on top (Fig. 3a). The HVJ–liposome complexes are separated from the free HVJ by sucrose density gradient centrifugation at 62,000g for 90 min at 4 . 5. The conjugated liposomes just above the 30% layer are collected gently (Fig. 3b). Free HVJ is concentrated at the layer between 30% and 60% (Fig. 3b). The final volume of the HVJ–liposome suspension should be approximately 1 ml.
Fig. 3. HVJ–liposomes and free HVJ can be separated by sucrose-density gradient centrifugation. HVJ–liposome mixture (white layer) was added to a sucrose layer consisting of 1 ml of 60% and 7 ml of 30% sucrose solution before centrifugation (A). The upper white band is the conjugate of HVJ and liposomes, and the lower is free HVJ (B).
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Gene Transfer by Hemagglutinating Virus of Japan–Liposomes Transfer of DNA into Cultured Cells Methods. 1. HVJ–cationic liposomes should be used for in vitro gene transfer, because HVJ–cationic liposomes are approximately 100 times more efficient in gene transfer to cultured cells than HVJ–anionic liposomes.8 2. One hundred microliters of the HVJ–cationic liposome suspension is added to 2 106 cells (6-well plate) in a serum-containing culture medium.14 3. The cells are incubated with the liposomes at 37 for 2 h. Then the medium is replaced with fresh culture medium, and the culture continued. Gene Transfer In Vivo by Hemagglutinating Virus of Japan HVJ–Liposomes Methods. 1. For gene transfer to tissues, HVJ–anionic liposomes are recommended. The liposomes are useful for gene transfer to liver, skeletal muscle, heart, lung, artery, brain, spleen, eye, and joint space of rodents, rabbits, dogs, sheep, and monkeys. For example, to introduce DNA into rat liver, 2–3 ml of HVJ–anionic liposomes is injected into the portal vein with a 5-ml syringe with a butterfly-shaped needle15,16 or directly into the liver under the perisplanchnic membrane using a 5-ml syringe with a 27gauge needle.17,18 For gene transfer into rat kidney, 1 ml of anionic HVJ– liposome solution is injected into the renal artery.19,20 For gene transfer into the rat carotid artery, a lumen of a segment of the artery is filled with 0.5 ml anionic HVJ–liposome complex for 20 min at room temperature using a cannula.21 14
T. Nishikawa, D. Edelstein, X. L. Du, S. Yamagishi, T. Matsumura, Y. Kaneda, M. A. Yorek, D. Beebe, P. J. Oates, H-P. Hammes, I. Giardino, and M. Brownlee, Nature 404, 787 (2000). 15 Y. Kaneda, K. Iwai, and T. Uchida, Science 243, 375 (1989). 16 Y. Kaneda, K. Iwai, and T. Uchida, J. Biol. Chem. 264, 12126 (1989). 17 K. Kato, M. Nakanishi, Y. Kaneda, T. Uchida, and Y. Okada, J. Biol. Chem. 266, 3361 (1991). 18 N. Tomita, R. Morishita, J. Higaki, S. Tomita, M. Aoki, T. Ogihara, and Y. Kaneda, Gene Ther. 3, 477 (1996). 19 N. Tomita, J. Higaki, R. Morishita, K. Kato, Y. Kaneda, and T. Ogihara, Biochem. Biophys. Res. Comm. 186, 129 (1992). 20 Y. Isaka, Y. Fujiwara, N. Ueda, Y. Kaneda, T. Kamada, and E. Imai, J. Clin. Invest. 92, 2597 (1993). 21 R. Morishita, G. Gibbons, Y. Kaneda, T. Ogihara, and V. J. Dzau, J. Clin. Invest. 91, 2580 (1993).
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2. For gene transfer to tumor masses or disseminated tumors, direct injection of cationic HVJ–liposomes (0.1 ml–0.5 ml) is recommended. 3. Repetitive transfection in vivo is successful in cancer masses (Yamamoto and Kaneda: manuscript in preparation), liver,7 and skeletal muscle (Fig. 4). The Storage of Hemagglutinating Virus of Japan–Liposomes Reagents 1. DMSO Methods 1. DMSO is added to HVJ–liposome suspension at a final concentration of 10%. 2. The mixture is stored immediately in a freezer (below 20 ). 3. Before use, frozen HVJ–liposomes should be thawed rapidly in a water bath at 37 . 4. Once thawed, all the samples should be used up for gene transfer.
Luciferase activity (RLU/g tissue)
3,500,000 3,000,000 2,500,000 2,000,000 1,500,000 1,000,000 500,000 0 Lane 1
Lane 2
Fig. 4. Repetitive transfection of HVJ–AVE liposomes to mouse skeletal muscle. One hundred microliters of saline (lane 1) or empty HVJ–AVE liposomes (lane 2) was injected into the left and right deltoid muscles of C57BL/6 mouse on days 0 and 7, respectively, and HVJ–AVE liposomes containing the luciferase gene were injected into the left pretibial muscle of the same mouse on day 21. Twenty-four hours after the third injection, luciferase activity in the muscle was measured. The mean and SD was shown. There was no significant difference between lane 1 and lane 2.
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liposomes in gene delivery and gene therapy 50,000 Fresh 45,000 −80
Luciferase activity (RLU)
40,000
LN2
35,000 30,000 25,000 20,000 15,000 10,000 5000 0 1
2
3
4 5 6 Months after storage
7
8
Fig. 5. Stability of HVJ–AVE liposomes. After preparation of HVJ–AVE liposomes containing the luciferase gene, aliquots were stored at 80 or 170 in liquid nitrogen after adding DMSO at a final concentration of 10%. At each time point, the aliquots were thawed and transferred to cultured BHK-21 cells (2 106 cells). Twenty-four hours after transfection, luciferase activity was measured. As a control, freshly prepared HVJ–AVE liposomes containing luciferase gene were transferred to BHK-21 cells, and luciferase activity was measured similarly.
Notes 1. UV-inactivated HVJ can be stored for more than 6 months in 10% DMSO at 80 or 170 (in liquid nitrogen). 2. The lipids can be stored, after evaporation, at 20 under nitrogen gas for 1 month. 3. HVJ–liposomes stored as described previously can maintain gene transfer activity for more than 8 months in 10% DMSO at 80 or 170 (in liquid nitrogen) (Fig. 5). 4. The most important component of HVJ–liposomes is the fusion activity of the HVJ envelope. Hemagglutination ability should be checked frequently by hemagglutination of chick red blood cells.22 5. Reconstituted fusogenic liposomes can be prepared using isolated fusion proteins derived from HVJ instead of inactivated whole viral particles.23 22 23
Y. Okada and J. Tadokoro, Exp. Cell Res. 26, 98 (1962). K. Suzuki, H. Nakashima, Y. Sawa, R. Morishita, H. Matsuda, and Y. Kaneda, Gene Ther. Reg. 1, 65 (2000).
liposomes in gene delivery and gene therapy
[30]
co-reconstituted with the phage receptor and components or entrapped within the liposomes to favor the gene delivery process or to protect the DNA from degradation. Also, the technique might be improved by using phage lambda, which is used extensively as a cloning vector and in recombinant technology and whose receptor, the LamB protein, has been reconstituted functionally into liposomes. Acknowledgments We acknowledge J. Bo¨hm and W. Baumeister for their fruitful collaboration on electron tomography. We have benefited through this work with the extensive collaboration of L. Letellier and her collaborators. We also thank W. Gelbart and D. Levy for stimulating discussions.
[30] The Hemagglutinating Virus of Japan–Liposome Method for Gene Delivery By Yasufumi Kaneda, Seiji Yamamoto, and Kazuya Hiraoka Introduction
Toward the success of human gene therapy, numerous viral and nonviral (synthetic) methods of gene transfer have been developed,1,2 but each has its own limitations as well as advantages. Therefore, to develop in vivo gene transfer vectors with high efficiency and low toxicity, several groups have attempted to overcome the limitations of one vector by combining them with the strengths of another. Our basic concept is the construction of novel, hybrid-type liposomes with functional molecules inserted into them.3,4 On the basis of this concept, DNA-loaded liposomes were fused with ultraviolet (UV) inactivated hemagglutinating virus of Japan; Sendai virus (HVJ) to form HVJliposomes (approximately 400–500 nm in diameter). Those viral liposomes bind to cell surface sialic acid receptors and fuse with cell membrane to directly introduce DNA into the cytoplasm without degradation. The HVJ–liposomes can encapsulate DNA smaller than 100 kb. RNA, oligodeoxynucleotides (ODN), proteins, and drugs can also be enclosed and 1
R. C. Mulligan, Science 260, 926 (1993). F. D. Ledley, Hum. Gene Ther. 6, 1129 (1995). 3 Y. Kaneda, Biogenic Amines 14, 553 (1998). 4 Y. Kaneda, Y. Saeki, and R. Morishita, Mol. Med. Today 5, 298, (1999). 2
METHODS IN ENZYMOLOGY, VOL. 373
Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00
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delivered to cells. Recently, we explained by use of fluorescence resonance energy transfer (FRET) that the degradation of ODN in the process of delivery to the cytoplasm is inhibited by HVJ-liposomes but not with simple cationic liposomes.5 HVJ-liposomes are useful for in vivo gene transfer.6 When HVJ– liposomes containing the LacZ gene were injected directly into one rat liver lobe, approximately 70% of cells expressed LacZ gene activity, and no pathological hepatic changes were observed.7 One advantage of HVJ– liposomes is allowance for repeated injections. Gene transfer to rat liver cells was not inhibited by repeated injections. After repeated injections, anti-HVJ antibody generated in the rat was not sufficient to neutralize HVJ–liposomes. Cytotoxic T cells recognizing HVJ were not detected in rats transfected repeatedly with HVJ-liposomes.7 The safety of HVJ– liposomes has been tested and evaluated in monkeys.8 To increase the efficiency of gene delivery by HVJ–liposomes, we investigated the lipid components of liposomes9 and concluded that the most efficient gene expression occurred with liposomes consisting of a phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, phospatidylserine, and cholesterol at the molar ratio of 13.3:13.3:13.3:10 and 50, respectively. The lipid components of the liposomes are very similar to the HIV envelope and mimic the red blood cell membrane.10 We called the liposomes HVJ–AVE liposomes (i.e., HVJ-artificial viral envelope liposomes). With HVJ–AVE liposomes, gene expression in heart, liver, and muscle was 5–10 times greater than that observed with various nonviral gene transfer methods such as conventional HVJ–liposomes, cationic-lipid–mediated lipofection and naked DNA injection.4 Another improvement was construction of cationic-type HVJ– liposomes using cationic lipids. Of the cationic lipids, positively charged DC-cholesterol (DC)11 has been the most efficient for gene transfer. For luciferase expression, HVJ–cationic DC liposomes were 100 times more 5
N. Nakamura, D. A. Hart, C. B. Frank, L. L. Marchuk, N. G. Shrive, N. Ota, K. Taira, H. Yoshikawa, and Y. Kaneda, J. Biochem. 129, 755 (2001). 6 V. J. Dzau, M. Mann, R. Morishita, and Y. Kaneda, Proc. Natl. Acad. Sci. USA 93, 11421 (1996). 7 T. Hirano, J. Fujimoto, T. Ueki, H. Yamamoto, T. Iwasaki, R. Morishita, Y. Sawa, Y. Kaneda, H. Takahashi, and E. Okamoto, Gene Ther. 5, 459 (1998). 8 N. Tsuboniwa, R. Morishita, T. Hirano, J. Fujimoto, S. Furukawa, M. Kikumori, A. Okuyama, and Y. Kaneda, Hum. Gene Ther. 12, 469 (2001). 9 Y. Saeki, N. Matsumoto, Y. Nakano, M. Mori, K. Awai, and Y. Kaneda, Hum. Gene Ther. 8, 1965 (1997). 10 R. Chander and H. Schreier, Life Science 50, 481 (1992). 11 K. Goyal and L. Huang, J. Liposome Res. 5, 49 (1995).
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efficient than conventional HVJ–anionic liposomes.9 However, HVJ– cationic liposomes were not appropriate for gene transfer to liver, kidney, heart, and muscle, but they were much more effective for gene transfer to tumor masses or disseminated cancers12,13 in animal models compared with anionic-type HVJ–AVE liposomes. Therefore, HVJ-anionic and cationic liposomes can complement each other, and each liposome should be used for proper targeting. Materials and Instrumentation
Chromatographically, pure bovine brain phosphatidylserine-sodium salt (PS) (No. 83032L) is obtained from Avanti Polar Lipids Inc. (Alabaster, AL), and other lipids such as dioleoyl-l-alpha-phosphatidylethanolamine (DOPE) (P 5078), sphingomyelin (Sph) (S 0756), egg yolk phosphatidylcholine (PC) (P 2772), DC-Cholestrol (DC-chol) (C 2832), and cholesterol (Chol) (C 8667) are from Sigma (St. Louis, MO). All the lipids are stored at 20 . Polypeptone (Pancreatic Digest of Casein) (No. 394-00115) is obtained from Wako (Osaka, Japan). EDTA-3Na (ED3SS), sucrose (S 7903), trizma base (T 1503), chloroform (C 2432), NaCl (S 3171), KCl (P 9333), and dimethylsulfoxide (DMSO) (D 8779) are from Sigma. The procedures require 50-ml conical tubes (Becton-Dickinson, Lincoln Park, NJ), 35-ml centrifuge tubes (Beckman Instruments, Tokyo, Japan), ultracentrifuge tubes (Hitachi, Tokyo, Japan) and cellulose acetate membrane filters (0.45 m, No. 190-2545, and 0.20 m, No. 190-2520 (Nalgene Co., Rochester, NY). For preparing lipid mixtures, glass tubes 24 mm in diameter and 12 cm long were custom-made (Fujiston 24/40, Iwaki Glass Co. Ltd., Tokyo, Japan), but similar sterilized tubes resistant to chloroform are available. The fresh glass tubes are immersed in saturated KOH-ethanol solution for 24 h, rinsed with distilled water, and heated at 180 for 2 h before use. A rotary evaporator (Type SR-650, Tokyo Rikakikai Inc., Tokyo, Japan), vacuum pump with a pressure gauge (Type Asp-13, Iwaki Glass Co. Ltd., Tokyo, Japan), water bath (Thermominder Jr 80, TAITEC, Saitama, Japan), vortex mixer (Scientific Industries, Bohemia, NY), and water bath shaker (Thermominder, TAITEC, Saitama, Japan) are used, but similar instruments are also available from other manufacturers for this purpose. For preparation of plasmid DNA, an endotoxin-free column (Qiagen Inc., Germany) is recommended. 12 13
T. Otomo, S. Yamamoto, R. Morishita, and Y. Kaneda, J. Gene Med. 3, 345 (2001). T. Miyata, S. Yamamoto, K. Sakamoto, R. Morishita, and Y. Kaneda, Cancer Gene Ther. 8, 852 (2001).
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For purifying HVJ, a low-speed centrifuge (05PR-22, Hitachi, Tokyo, Japan), a centrifuge with JA-20 rotor (J2-HS, Beckman Instruments), and a photometer (Spectrophotometer DU-68, Beckman Instruments) are needed. A UV cross-linker (Spectrolinker XL-1000, Spectronics Co.) is used for inactivation of HVJ. For purifying HVJ–liposomes, an ultracentrifuge with RPS-40T rotor (55P-72, Hitachi, Tokyo, Japan) is used. Procedures for Preparation of Hemagglutinating Virus of Japan–Liposomes
Preparation of Hemagglutinating Virus of Japan Preparation of Hemagglutinating Virus of Japan in Eggs Reagents. 1. Polypeptone solution (1% polypeptone, 0.2% NaCl, pH 7.2): To make 500 ml, 5 g of polypeptone and 1 g of NaCl are solubilized in distilled water, the pH is adjusted to 7.2 by adding aliquots of 1 M NaOH, and the total volume is brought to 500 ml with distilled water. The solution is sterilized by autoclaving and stored at 4 . 2. BSS (137 mM NaCl, 5.4 mM KCl, 10 mM TRIS-HCl pH7.6): Eight grams of NaCl, 0.4 g of KCl, and 1.21 g of Trizma base are dissolved in distilled water, the pH is adjusted to 7.6 with aliquots of 1 M HCl and the total volume is brought to 1 l with distilled water. The solution is sterilized by autoclaving and store at 4 . 3. Seed of HVJ: Aliquots (100 l) of the best seed of HVJ (Z strain) in 10% DMSO are stored at 80 . Methods. 1. The seed is thawed rapidly and diluted 1000 times with poly peptone solution. The diluted seed should be kept at 4 before proceeding to the next step. 2. Embryonated eggs are observed under illumination in a dark room, and an injection point is marked at about 0.5 mm above the chorioallantoic membrane. The eggs are disinfected with tincture of iodine and punctured at the point marked. 3. The diluted seed (0.1 ml) is injected into each egg using a 1-ml disposable syringe with a 26-gauge needle. The needle should be inserted vertically so as to stab the chorioallantoic membrane. 4. After inoculation of the seed, the hole punctured on the egg is covered with melted paraffin. Then the eggs are incubated for 4 days at 36.5 in sufficient moisture.
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5. The eggs are chilled at 4 for more than 6 h before harvesting the virus. 6. The egg shell is removed partially, and the chorioallantoic fluid is aspirated with a 10-ml syringe (18-gauge needle) and placed in an autoclaved bottle. The fluid should be kept at 4 to avoid freezing. The virus is stable in the fluid at least for 3 months. Steps 2, 3, and 6 can be carried out at room temperature. Purification of Hemagglutinating Virus of Japan from Chorioallantoic Fluid Reagents. 1. BSS is prepared as described previously. Methods. 1. Two hundred milliliters of the chorioallantoic fluid is transferred into four 50-ml disposable conical tubes and subjected to centrifugation at 3000 rpm (1000g) for 10 min at 4 in a low-speed centrifuge. 2. Then, the supernatant is aliquoted into six tubes (Beckman JS-20) and centrifuged at 15,000 rpm (27,000g) for 30 min at 4 . 3. About 5 ml of BSS is added to the pellet in one of the tubes, and the materials are kept at 4 overnight. 4. The pellets are gently suspended, collected in two tubes, and centrifuged as described in step 2. The resultant pellet in each tube is kept at 4 in 5 ml of BSS for more than 8 h. 5. The pellets are suspended gently and centrifuged at 3000 rpm in a low-speed centrifuge. 6. The supernatant is removed to an aseptic tube and stored at 4 . 7. The virus titer is determined by measuring the absorbance at 540 nm of the 10 times–diluted supernatant using a photometer. An optical density at 540 nm corresponds to 15,000 hemagglutinating units (HAU), which is well correlated with fusion activity. The supernatant as prepared previously usually shows 20,000–30,000 HAU/ml. A virus solution prepared aseptically maintains fusion activity for 3 weeks. Preparation of the Lipid Mixture Methods 1. Dry reagents of DOPE (12.2 mg), Sph (11.5 mg), and Chol (23.8 mg) are dissolved in 3870 l of chloroform. The PC choloform solution (130 l) is added to the 3870 l lipid solution. This 4000 l of lipid solution is called a basal mixture for liposomes. The master mixture is
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Fig. 1. After evaporation, a thin lipid film is formed inside a glass tube.
ready to prepare anionic or cationic liposomes (described later) or can be stored at 20 after infusing nitrogen gas over the solution. 2. For preparation of an anionic or cationic lipid mixture, 10 mg of PS on 6 mg DC–Chol is added to the basal mixture, respectively. 3. The lipid solution of 0.5 ml is aliquoted into eight glass tubes; each tube contains 10 mg of lipids. The tubes are kept on ice or 20 under nitrogen gas before evaporation. The lipid solution should be evaporated as soon as possible. 4. The tube is attached to a rotary evaporator. The tube should be immersed in a 40 water bath at the tip. 5. The organic solvent is evaporated in the rotary evaporator under vacuum. The lipids are dried up for about 10 min. (Fig. 1). Preparation of Hemagglutinating Virus of Japan–Liposomes The procedure for the preparation of HVJ–liposomes is illustrated in Fig. 2. Preparation of Hemagglutinating Virus of Japan–Liposomes Containing DNA Reagents. 1. BSS, prepared as described previously.
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Fig. 2. Preparation of HVJ–liposomes and fusion-mediated gene transfer. DNA-loaded liposomes are fused with UV-inactivated HVJ to form HVJ–liposomes. HVJ–liposomes bind cell surface sialic acid receptors, degrade the receptor, and associate with the lipids in the cell membrane to induce membrane fusion. By fusion of the envelope of HVJ–liposomes with the cell membrane, DNA in the HVJ–liposomes can be introduced directly into the cytoplasm.
2. Plasmid DNA: Plasmid DNAs are purified by a column procedure. The preparations are dissolved in TE solution, BSS, or water. The final concentration of DNA should be more than 1 mg/ml, and stored at 20 . 3. Sucrose solutions: To prepare 30% and 60% (w/v) sucrose solutions, 150 g of sucrose is solubilized in BSS and the solution bright up to a total volume to 500 ml and 250 ml, respectively. The solution is sterilized by autoclaving and stored at 4 . Methods. 1. Plasmid DNA (200 g) in 200 l is added to a lipid mixture in the glass tube prepared as described previously, and agitated intensely by vortexing for 30 s. followed by incubation at 37 for 30 s. This cycle is repeated eight times. By this method, plasmid DNA up to 20 kb is
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encapsulated at an efficiency of 10–30% in anionic liposomes or 50–60% in cationic liposomes. 2. For preparing sized unilamellar liposomes, the liposome suspension is filtered through a 0.45-m pore size cellulose acetate filter and then through a 0.2-m filter. Sizing by an extruder with polycarobanate filters is better for preparing sized liposomes. 3. In the meantime, HVJ is inactivated and keep on ice. HVJ (15,000 HAU) is added to the liposome suspension and the tube is incubated on ice for 5 to 10 min. Then, the sample is incubated at 37 for 1 h, with shaking (120/min), in a water bath. 4. One milliliter of 60% sucrose and 7 ml of 30% sucrose are added to a centrifuge tube, and the HVJ–liposome mixture is overlaid on top (Fig. 3a). The HVJ–liposome complexes are separated from the free HVJ by sucrose density gradient centrifugation at 62,000g for 90 min at 4 . 5. The conjugated liposomes just above the 30% layer are collected gently (Fig. 3b). Free HVJ is concentrated at the layer between 30% and 60% (Fig. 3b). The final volume of the HVJ–liposome suspension should be approximately 1 ml.
Fig. 3. HVJ–liposomes and free HVJ can be separated by sucrose-density gradient centrifugation. HVJ–liposome mixture (white layer) was added to a sucrose layer consisting of 1 ml of 60% and 7 ml of 30% sucrose solution before centrifugation (A). The upper white band is the conjugate of HVJ and liposomes, and the lower is free HVJ (B).
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Gene Transfer by Hemagglutinating Virus of Japan–Liposomes Transfer of DNA into Cultured Cells Methods. 1. HVJ–cationic liposomes should be used for in vitro gene transfer, because HVJ–cationic liposomes are approximately 100 times more efficient in gene transfer to cultured cells than HVJ–anionic liposomes.8 2. One hundred microliters of the HVJ–cationic liposome suspension is added to 2 106 cells (6-well plate) in a serum-containing culture medium.14 3. The cells are incubated with the liposomes at 37 for 2 h. Then the medium is replaced with fresh culture medium, and the culture continued. Gene Transfer In Vivo by Hemagglutinating Virus of Japan HVJ–Liposomes Methods. 1. For gene transfer to tissues, HVJ–anionic liposomes are recommended. The liposomes are useful for gene transfer to liver, skeletal muscle, heart, lung, artery, brain, spleen, eye, and joint space of rodents, rabbits, dogs, sheep, and monkeys. For example, to introduce DNA into rat liver, 2–3 ml of HVJ–anionic liposomes is injected into the portal vein with a 5-ml syringe with a butterfly-shaped needle15,16 or directly into the liver under the perisplanchnic membrane using a 5-ml syringe with a 27gauge needle.17,18 For gene transfer into rat kidney, 1 ml of anionic HVJ– liposome solution is injected into the renal artery.19,20 For gene transfer into the rat carotid artery, a lumen of a segment of the artery is filled with 0.5 ml anionic HVJ–liposome complex for 20 min at room temperature using a cannula.21 14
T. Nishikawa, D. Edelstein, X. L. Du, S. Yamagishi, T. Matsumura, Y. Kaneda, M. A. Yorek, D. Beebe, P. J. Oates, H-P. Hammes, I. Giardino, and M. Brownlee, Nature 404, 787 (2000). 15 Y. Kaneda, K. Iwai, and T. Uchida, Science 243, 375 (1989). 16 Y. Kaneda, K. Iwai, and T. Uchida, J. Biol. Chem. 264, 12126 (1989). 17 K. Kato, M. Nakanishi, Y. Kaneda, T. Uchida, and Y. Okada, J. Biol. Chem. 266, 3361 (1991). 18 N. Tomita, R. Morishita, J. Higaki, S. Tomita, M. Aoki, T. Ogihara, and Y. Kaneda, Gene Ther. 3, 477 (1996). 19 N. Tomita, J. Higaki, R. Morishita, K. Kato, Y. Kaneda, and T. Ogihara, Biochem. Biophys. Res. Comm. 186, 129 (1992). 20 Y. Isaka, Y. Fujiwara, N. Ueda, Y. Kaneda, T. Kamada, and E. Imai, J. Clin. Invest. 92, 2597 (1993). 21 R. Morishita, G. Gibbons, Y. Kaneda, T. Ogihara, and V. J. Dzau, J. Clin. Invest. 91, 2580 (1993).
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2. For gene transfer to tumor masses or disseminated tumors, direct injection of cationic HVJ–liposomes (0.1 ml–0.5 ml) is recommended. 3. Repetitive transfection in vivo is successful in cancer masses (Yamamoto and Kaneda: manuscript in preparation), liver,7 and skeletal muscle (Fig. 4). The Storage of Hemagglutinating Virus of Japan–Liposomes Reagents 1. DMSO Methods 1. DMSO is added to HVJ–liposome suspension at a final concentration of 10%. 2. The mixture is stored immediately in a freezer (below 20 ). 3. Before use, frozen HVJ–liposomes should be thawed rapidly in a water bath at 37 . 4. Once thawed, all the samples should be used up for gene transfer.
Luciferase activity (RLU/g tissue)
3,500,000 3,000,000 2,500,000 2,000,000 1,500,000 1,000,000 500,000 0 Lane 1
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Fig. 4. Repetitive transfection of HVJ–AVE liposomes to mouse skeletal muscle. One hundred microliters of saline (lane 1) or empty HVJ–AVE liposomes (lane 2) was injected into the left and right deltoid muscles of C57BL/6 mouse on days 0 and 7, respectively, and HVJ–AVE liposomes containing the luciferase gene were injected into the left pretibial muscle of the same mouse on day 21. Twenty-four hours after the third injection, luciferase activity in the muscle was measured. The mean and SD was shown. There was no significant difference between lane 1 and lane 2.
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LN2
35,000 30,000 25,000 20,000 15,000 10,000 5000 0 1
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4 5 6 Months after storage
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Fig. 5. Stability of HVJ–AVE liposomes. After preparation of HVJ–AVE liposomes containing the luciferase gene, aliquots were stored at 80 or 170 in liquid nitrogen after adding DMSO at a final concentration of 10%. At each time point, the aliquots were thawed and transferred to cultured BHK-21 cells (2 106 cells). Twenty-four hours after transfection, luciferase activity was measured. As a control, freshly prepared HVJ–AVE liposomes containing luciferase gene were transferred to BHK-21 cells, and luciferase activity was measured similarly.
Notes 1. UV-inactivated HVJ can be stored for more than 6 months in 10% DMSO at 80 or 170 (in liquid nitrogen). 2. The lipids can be stored, after evaporation, at 20 under nitrogen gas for 1 month. 3. HVJ–liposomes stored as described previously can maintain gene transfer activity for more than 8 months in 10% DMSO at 80 or 170 (in liquid nitrogen) (Fig. 5). 4. The most important component of HVJ–liposomes is the fusion activity of the HVJ envelope. Hemagglutination ability should be checked frequently by hemagglutination of chick red blood cells.22 5. Reconstituted fusogenic liposomes can be prepared using isolated fusion proteins derived from HVJ instead of inactivated whole viral particles.23 22 23
Y. Okada and J. Tadokoro, Exp. Cell Res. 26, 98 (1962). K. Suzuki, H. Nakashima, Y. Sawa, R. Morishita, H. Matsuda, and Y. Kaneda, Gene Ther. Reg. 1, 65 (2000).