[36] HVJ (hemagglutinating virus of Japan; Sendai virus)-liposome method

[36]

HVJ-LIPOSOME METHOD

619

[36] HVJ (Hemagglutinating Virus of Japan; S e n d a i Virus)-Liposome Method By RYUICHIMORISHITAand YASUFUMIKANEDA 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. Especially for the treatment of cardiovascular disease, it is necessary to develop safe and efficient gene delivery methods. The HVJ-liposome method appears to possess many ideal properties for in vivo gene transfer such as (1) efficiency, (2) safety, (3) simplicity of handling, (4) brevity of incubation time, and (5) no limitation of inserted DNA size. In this method, foreign DNA is complexed with liposomes, a nuclear protein, and the viral protein coat of HVJ (Fig. 1). The HVJ method has been successfully employed for gene transfer in vivo to many tissues including liver, kidney, and vascular w a l l . 3-18 1 R. C. Mulligan, Science 260, 926 (1993). 2 E D. Ledley, Hum. Gene Ther 6, 1129 (1995). 3 y. Kaneda, BiogenicAmines 14, 553 (1998). 4 y. Kaneda, Y. Saeki, and R. Morishita, Mol. Med. Today 5, 298 (1999). 5 V. J. Dzau, M. Mann, R. Morishita, and Y. Kaneda, Proc. Natl. Acad. Sci. U.S.A. 93, 11421 (1996). 6 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 0998). 7 y. Saeki, N. Matsumoto, Y. Nakano, M. Mori, K. Awai, and Y. Kaneda, Hum. Gene Ther. 8, 1965 (1997). 8 y. Sawa, Y. Kaneda, H-Z. Bai, K. Suzuki, J. Fujimoto, R. Morishita, and H. Matsuda, Gene Ther. 5, 1472 (1998). 9 G. Yamada, S. Nakamura, R. Haraguchi, M. Sakai, T. Terashi, S. Sakisaka, T. Toyoda, Y. Ogino, H. Hatanaka, and Y. Kaneda, Cell. Mol. Biol. 43, 1165 (1997). 10 y. Yonemitsu, Y. Kaneda, A. Muraishi, T. Yoshizumi, K. Sugimachi, and K. Sueishi, Gene Ther. 4, 631 (1997). 11 E. Mabuchi, K. Shimizu, Y. Miyao, Y. Kaneda, H. Kishima, M. Tamura, K. Ikenaka, and T. Hayakawa, Gene Ther. 4, 768 (1997). 12 y. Kaneda, K. Iwai, and T. Uchida, Science 243, 375 (1989). 13 y. Kaneda, K. Iwai, and T. Uchida, J. Biol. Chem. 264, 12126 (1989). 14 K. Kato, M. Nakanishi, Y. Kaneda, T. Uchida, and Y. Okada, J. Biol. Chem. 266, 3361 (1991). 15 N. Tomita, J. Higaki, R. Morishita, S. Tomita, M. Aoki, T. Ogihara, and Y. Kaneda, Gene Ther 3, 477 (1996). 16 N. Tomita, J. Higaki, R. Morishita, K. Kato, Y. Kaneda, and T. Ogihara, Biochem. Biophys. Res. Comm. 186, 129 (1992).

METHODSIN ENZYMOLOGY,VOL 346

CopyrightO 2002by AcademicPress. All rightsof reproductionin any formreserved. 0076-6879/02$35.00

620

OTHERSTRATEGIES

[36]

HVJ-Liposome Gene Transfer Method

0

0

0

--.

o ck

0

%iOo,,o

PlasmldDNA

-

Vortex Ultraaonlcatlon

O,~ sO Llplds

@

@

@

DNAcontaining Liposomes

A

~

HVJ-Llposomes

Inactivated HVJ

FIG. 1. Gene transfer by HVJ-liposomes. HVJ-liposomes bind to cell surface sialic acid receptors and associate with lipids in the lipid bilayer to induce cell fusion. By the fusion of the envelope of HVJ-liposomes with cell membrane, DNA in the HVJ-liposomes can be directly introduced into the cytoplasm.

Development of HVJ-Liposomes Our basic concept is the construction of novel, hybrid-type liposomes with functional molecules inserted into them. 3"4 Based on this concept, DNA-loaded liposomes were fused with UV-inactivated HVJ (hemagglutinating virus of Japan; Sendai virus) to form HVJ-liposomes (approximately 400 to 500 nm in diameter). These viral liposomes bind to cell surface sialic acid receptors and fuse with cell membranes to directly introduce DNA into the cytoplasm without degradation (Fig. 1). The HVJ-liposomes can encapsulate DNA smaller than 100 kb. RNA, oligodeoxynucleotides including antisense, decoy, or ribozyme, proteins, and drugs can also be enclosed and delivered to cells. HVJ-liposomes are useful for in vivo gene transfer. 5 When HVJ-liposomes containing the LacZ gene were injected directly into rat blood vessels, approximately 80-90% of cells expressed LacZ gene activity, and no pathological hepatic changes were observed. 6 One advantage of HVJ-liposomes is allowance for repeated injections. Gene transfer to rat liver cells was not inhibited by repeated injections up to 8 times. 6' 17 Of importance, 17 R. Morishita, G. H. Gibbons, Y. Kaneda, T. Ogihara, and V.J. Dzau, Biochem. Biophys. Res. Commun. 273, 666 (2000). 18 R. Morishita, G. Gibbons, Y. Kaneda, T. Ogihara, and V. Dzau, J. Clin. Invest. 91, 2580 (1993).

[36]

HVJ-LIPOSOME METHOD

621

after repeated injections, anti-HVJ antibody generated in the rat was not sufficient to neutralize HVJ-liposomes. In addition, cytotoxic T cells recognizing HVJ were not detected in the rat transfected repeatedly with HVJ-liposomes. 6 The HVJ-liposome method also enhances the efficiency and prolongs the halflife of antisense ODN in vitro and in vivo. 19-21 The HVJ-liposome method is suitable for an intraluminal molecular delivery system that has several advantages over the peri-adventitial polymer delivery approach. ODN transfected by the HVJliposome method substantially increases the efficiency of uptake of ODN and appears to increase the stability of the ODN within intracellular compartments. 19-21 HVJ is well known to cause cell fusion, and, thus, the complex in the viral envelope might enter the cells directly via cell membrane-liposome membrane fusion. However direct transfer of ODN has several unresolved problems such as (1) significant intracellular degradation via the lysosomal pathway and (2) efflux from endosomes after trapping. Modification of ODN pharmacokinetics with the use of HVJ-liposome complex will facilitate the potential clinical utility of the agents by (1) allowing a shorter intraluminal incubation time to preserve organ perfusion, (2) prolonging the duration of biological action, and (3) enhancing efficacy such that the nonspecific effects of high doses of ODN can be avoided. Since the virus is inactivated by ultraviolet light, there is little potential for biological hazard with this method as compared to the retroviral in vivo gene transfer approach. Improvements of HVJ-Liposomes

The HVJ-liposome gene delivery system has several advantages, although further modification is necessary for it to be used in humans. To increase the efficiency of gene delivery, we investigated the lipid components of liposomes. 7 First, the most efficient gene expression was achieved with a phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin molar ratio of 1 : 1 : 1. Second, anionic HVJ-liposomes should be prepared using phospatidylserine (PS) as the anionic lipid. Finally, the ratio of phospholipids to cholesterol should be 1 : 1. Accordingly, we also developed new anionic liposomes called HVJ-AVE liposomes, i.e., HVJ-artificial viral envelope liposomes. The lipid components of AVE liposomes are very similar to the HIV envelope and mimic the red blood cell membrane. 22 HVJ-AVE liposomes have yielded gene expression in liver and muscle 5 to 10 times higher than that observed with conventional HVJ-liposomes. 7 Interestingly, HVJ-AVE liposomes were most effective for gene transfer to mouse skeletal 19 R. Morishita, G. H. Gibbons, M. Horiuchi, M. Nakajima, K. E. Ellison, W. Lee, Y. Kaneda, T. Ogihara, and V. J. Dzau, J. Cardiovasc. Pharmacol. Ther. 2, 213 (1996). 20 R. Morishita, G. H. Gibbons, K. E. Ellison, M. Nakajima, H. V. L. Leyen, L. Zhang, Y. Kaneda, T. Ogihara, and V. J. Dzau, J. Clin. Invest. 93, 1458 (1994). 21 R. Morishita, G. H. Gibbons, Y. Kaneda, T. Ogihara, and V. J. Dzau, Gene 149, 13 (1994). 22 R. Chander and H. Schreier, Life Sci. 50, 481 (1992).

622

OTHER STRATEGIES

[36]

muscle in various nonviral gene transfer methods. HVJ-AVE liposomes were also very effective for gene delivery to isolated rat heart via the coronary artery. LacZ gene expression was observed in the entire heart, whereas expression was not observed with empty HVJ-AVE liposomes. 8 Safety of HVJ-liposomes has been tested and evaluated in monkeys. There were no significant pathological signs after injection of HVJ-liposomes into skeletal muscle or saphenous vein of cynomolgus monkeys. Messenger RNAs for fusion proteins of HVJ were not detected in monkey tissues after the injection. 23 Another improvement was construction of cationic-type HVJ-liposomes using cationic lipids. Of the cationic lipids, positively charged 3~-[N-(N',N'dimethylaminoethane)-carbamoyl] cholesterol hydroxide (DC) 24 has been the most efficient for gene transfer. For luciferase expression, HVJ-cationic DC liposomes were 100 times more efficient than were conventional HVJ-anionic liposomes. 7 Although it has been very difficult to transfer genes to bone marrow and spleen cells using conventional HVJ-liposomes, HVJ-cationic liposomes have been shown to be effective for gene transfer to both types of cells. However, when introduced into mouse muscle or liver, total luciferase expression after transfection with HVJ-cationic liposomes was shown to be 10 to 150 times lower than that with conventional anionic HVJ-liposomes,7 which were less efficient for in vitro transfection. Furthermore, AVE liposomes were modified further to create AVE+DC 10 (contains 10% PS and 10% DC), AVE+DC20 (containing 10% PS and 20% DC), and AVE-PS (containing neither PS nor DC) liposomes. We examined in vivo gene transfection efficiency with these liposomes after conjugation with the HVJ envelope. AVE yielded the highest luciferase expression in liver. AVE-PS and AVE+DC10 liposomes, which have a net neutral charge, showed intermediate luciferase activities. AVE+DC20 liposomes, which have an excessive amount of cationic lipid, yielded luciferase activities similar to those of HVJ-DC liposomes. Nevertheless, we have found HVJ-cationic liposomes to be more effective in some cases for in vivo gene transfer. High expression of the LacZ gene was obtained in restricted regions of chick embryos after injection of HVJ-cationic liposomes, 9 whereas HVJ-anionic liposomes were ineffective. In addition, when HVJ-cationic liposomes containing the LacZ gene were administered to rat lung with a jet nebulizer, more efficient gene expression in the epithelium of the trachea and bronchus was observed compared to that found with HVJ-anionic liposomes. 1° HVJ-cationic liposomes were also much more effective for gene transfer to tumor masses or disseminated cancers TM in animal models compared to HVJ-AVE liposomes. Therefore, HVJ anionic and cationic liposomes can complement each other, and each liposome should be used for proper targeting. 23M. Kawauchi, J. Suzuki, R. Morishita, Y. Wada, A. Izawa, N. Tomita, J. Amano, Y. Kaneda, T. Ogihara, S. Takamoto,and M. Isobe, Circ. Res. 87, 1063 (2000). 24K. Goyaland L. Huang,J. Liposome Res. 5, 49 (1995).

[36]

HVJ-LIPOSOME METHOD

623

Materials

Preparation of HVJ 1. Seed of HVJ: One hundred microliter aliquots of the chorioallantoic fluid containing HVJ (Z strain) in 10 % dimethy sulfoxide were stored in liquid nitrogen. 2. Polypeptone solution (1% polypeptone, 0.2% NaC1, pH 7.2) and BSS (137 mM NaC1, 5.4 mM KC1, 10 mM Tris-HC1 pH7.6) were sterilized by autoclaving and stored at 4 ° . 3. Embryonated chick eggs (10 to 14 days after fertlization). 4. Incubator: The temperature and the moisture were set at 36.5 ° and at 30 to 40%, respectively. 5. Centrifuge tubes including 50 ml conical tube (Becton-Dickinson, Lincoln Park, NJ), 35 ml centrifuge tube (Beckman), and 10 ml ultracentrifuge tube (Hitachi, Tokyo, Japan) were sterilized. 6. A photometer (Spectrophotometer DU-68, Beckman Instruments, Tokyo, Japan), low-speed centrifuge (05PR-22, Hitachi, Tokyo, Japan), and centrifuge with JA-20 rotor (J2-HS, Beckman Instruments, Tokyo, Japan) were used.

Preparation of Lipid Mixtures 1. Chromatographically pure bovine brain phosphatidylserine-sodium salt (PS) (Avanti Polar Lipids Inc., Birmingham, AL), dioleoyl-L-~-phosphatidylethanolamine (DOPE) (Sigma, St. Louis, MO), sphingomyelin (Sph) (Sigma), egg yolk phosphatidylcholine (PC) (Sigma), 3fl-[N-(N',N~-dimethylaminoethane) carbamoyl] cholesterol hydroxide (DC) (Sigma), and cholesterol (Chol) (Sigma) were stored at - 2 0 ° . 2. Glass tubes (24 mm caliber and 12 cm long) were custom-made (Fujiston 24/40, Iwaki Glass Co. Ltd., Tokyo, Japan). The fresh tubes were immersed in saturated KOH-ethanol (180 g KOH in 500 ml ethanol) solution for 24 hr, rinsed with distilled water, and heated at 180 ° for 2 hr before use. 3. A rotary evaporator with a water bath (Type SR-650, Tokyo Rikakikai Inc., Tokyo, Japan) and vacuum pump with a pressure gauge (Type Asp- 13, Iwaki Glass Co. Ltd., Tokyo, Japan) were used.

Preparation of HVJ-Liposomes 1. Plasmid DNAs were purified by a column procedure (Qiagen, Germany). The preparations were dissolved in BSS. The final concentration of DNA should be more than 1 mg/ml, and stored at - 2 0 °. 2. Cellulose acetate membrane filters (0.45/zm, 0.20/zm; Iwaki glass) were used for sizing liposomes. 3. BSS and 30% (w/v) sucrose in BSS were sterilized by autoclaving and stored at 4 ° .

624

OTHER STRATEGIES HVJ from chick egg Centrifugation

UV irradiation

[361 Plasmid DNA

Lipids Evaporation Lipid mixtures

Vortex and Sizing

InactivatedHV J

DNA-loaded liposomes

I

I Fusion

Sucrose density centrifugation HVJ-liposomes FIG.2. Flow chart for the preparationof HVJ-liposomes.

4. A water bath (Thermominder Jr 80, TAITEC, Saitama, Japan) for preparing liposomes, a water bath shaker (Thermominder, TAITEC, Saitama, Japan) for fusing liposomes with HVJ, and an ultracentrifuge with RPS-40T rotor (55P-72, Hitachi, Tokyo, Japan) for purifying HVJ-liposomes and an ultraviolet cross-linker (Spectrolinker XL-1000, Spectronics Co.) for inactivating HVJ were used.

Methods A flow chart for the preparation of HVJ-liposomes is shown in Fig. 2.

Preparation of HVJ in Eggs 1. The seed was rapidly thawed, and diluted to 1000 times with polypeptone solution. The seed diluted should be kept at 4 ° before proceeding to the next step. 2. Embryonated eggs were observed under illumination in a dark room, and an injected point was marked at about 0.5 mm above the chorioallantoic membrane. The eggs were disinfected with tincture of iodine and punctured at the point marked. 3. The diluted seed of 0.1 ml was injected into each egg using 1 ml disposable syringe with a 26-gauge needle. The needle should be inserted vertically so as to stab the chorioallantoic membrane.

[361

HVJ-LIPOSOME METHOD

625

4. After inoculation of the seed, the hole punctured on the egg was covered with melted paraffin. Then the eggs were incubated for 3 days at 36.5 ° in 30 to 40% moisture. 5. The eggs were chilled at 4 ° for more than 6 hr before harvesting the virus. 6. The eggshell was partially removed, and the chorioallantoic fluid was removed to an autoclaved bottle using 10 ml syringe with an 18-gauge needle. The virus in the fluid stored at 4 ° is stable for at least 3 months. Steps 2, 3, and 6 can be carried out at room temperature.

Purification of HVJ from Chorioallantoic Fluid 1. Two hundred ml of the chorioallantoic fluid was transferred into four 50 ml disposable conical tubes, and span at 3000 rpm (1000g) for 10 min at 4 ° in a low-speed centrifuge. 2. Then, the supernatant was aliquotted into 6 tubes (Beckman JS-20), and centrifuged at 15,000 rpm (27,000g) for 30 min at 4 °. 3. About 5 ml of BSS was added to the pellet in one of the tubes, and the material was kept at 4 ° overnight. 4. The pellets were gently suspended, collected in two tubes, and centrifuged as described in step 2. The resultant pellet in each tube was kept at 4 ° in 5 ml of BSS for more than 8 hr. 5. The pellets were gently suspended and subjected to rotation at 3000 rpm in a low-speed centrifuge. 6. The supernatant was removed to an aseptic tube and stored at 4 °. 7. Virus titer was indicated by measuring the absorbbance at 540 nm of the 10 times-diluted supernatant using a photometer. An optical density at 540 nm corresponded to 15,000 hemagglutinating units (HALT), which was well correlated with fusion activity. The supernatant prepared as above usually showed 20,000 to 30,000 HAU/ml. A virus solution aseptically prepared maintains the fusion activity for 3 weeks.

Preparation of Lipid Mixture 1. Dry reagents of DOPE (12.2 mg), Sph (11.8 mg), and Chol (24.0 mg) were dissolved in 3870 lzl of chloroform. Then, 130/zl of PC (13.0 mg) chloroform solution was added to the 3870/zl lipids solution. This 4000 /zl lipids solution was added is called a basal mixture for liposomes. The basal mixture is ready to prepare anionic or cationic liposomes described below or can be stored at - 2 0 ° after infusing nitrogen gas. 2. To prepare an anionic lipid mixture, 10 mg of PS was added to the basal mixture. To obtain a cationic lipid mixture, 6 mg DC was added to the mixture.

626

OTHER STRATEGIES

[361

3. The lipid solution of 0.5 ml was aliquotted into 8 glass tubes. The tubes were kept on ice or - 2 0 ° in nitrogen gas before evaporation. The lipid solution should be evaporated as soon as possible. 4. The tubes were connected to a rotary evaporator. The tubes should be immersed in a 40 ° water bath at the tip. 5. The organic solvent was evaporated in a rotary evaporator under vacuum. The lipids were dried usually for about 5 to 10 min. Lipids appropriate for liposome preparation were those in the sides of the tubes in a thin layer. Those accumulated at the bottom of the tubes were inappropriate.

Preparation of HVJ-Liposomes Containing DNA 1. Plasmid DNA (200/zg) in 200/~1 was added to a lipid mixture in the glass tube prepared as above, and agitated intensely by vortexing for 30 sec followed by an incubation at 37 ° for 30 sec. This cycle was repeated 8 times. By this method, plasmid DNA was enclosed at the ratio of 10-30% in anionic liposomes or 50-60% in cationic liposomes. 2. For preparing sized unilamellar liposomes, the liposome suspension is filtered with 0.45/zm pore size cellulose acetate filter and then with 0.2/xm filter. Sizing by an extruder with polycarbanate filters is better for preparing sized liposomes. 3. In the meantime, inactivate the HVJ virus and keep on ice. Add 15,000 HAU of the HVJ virus to the liposome suspension and leave the tube on ice for 5 to 10 min. Then, incubate the sample at 37 ° for 1 hr with shaking (120/min) in a water bath. 4. Add 7 ml 30% sucrose solution to a centrifuge tube and overlay the HVJliposome mixture on it. Separate the HVJ-liposome complexes from the free HVJ by sucrose gradient density centrifugation at 62,000g for 90 min at 4 °. 5. Stop the centrifuge and gently remove only the conjugated liposomes. The final volume of the HVJ-liposome suspension should be approximately 1 ml. Only in the case of HVJ-anionic liposomes, after centrifugation, collect the complexes, add 4 volumes of chilled BSS, spin at 27,000g for 30 min, and suspend the pellet in 0.5 to 1.0 ml of appropriate buffer.

Applications

Transfer of DNA into Cultured Cells 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

[36]

HVJ-LIPOSOME METHOD

627

to cultured cells than HVJ-anionic liposomes. 7 Ten/zl of lml HVJ-cationic liposome suspension was added to 105 cells in serum-containing culture medium. 25 2. Incubate the cells with the liposomes at 37 ° for 2 hr. Then change the medium and continue the culture.

Gene Transfer in Vivo by HVJ-Liposomes 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 rodent, rabbit, dog, lamb, and monkey. For example, for gene transfer to rat carotid artery, a lumen of a segment of the artery was filled with 0.5 ml anionic HVJ-liposome complex for 20 min at room temperature using a cannula.18 For gene transfer into rat kidney, 1 ml of anionic HVJ-liposome suspension was injected into the r e n a l artery. 16'17 To introduce DNA into rat liver, 2 to 3 ml of HVJ-anionic liposomes was injected into portal vein using a 5 ml syringe with a butterfly needle 12'13 or directly into liver under the perisplanchnic membrane using a 5 ml syringe with a 27-gauge needle. 14,15 2. For gene transfer to tumor masses or disseminated tumors, direct injection of cationic HVJ-liposomes (0.1 ml to 0.5 ml) is recommended.

Notes 1. UV-inactivated HVJ can be stored for more than 6 months in 10% dimethyl sulfoxide at - 8 0 °. Do not store it at 4 ° for more than 1 day. 2. Lipid vials should be left at room temperature for about 30 min before opening the lids. Many lipids are highly hygroscopic. 3. The lipid mixture in the glass tube can be stored at - 2 0 ° in nitrogen gas for 1 month after evaporation. 4. HVJ-liposomes can be stored for 3 weeks at 4 °, and for more than 3 months with 10% dimethyl sulfoxide at final concentration in a freezer (below -20°). 5. Gene transfer efficiency of HVJ-liposomes is greatly affected by the fusion activity of HVJ envelope. Hemagglutinating ability should be frequently checked by hemagglutination of chick red blood cells. 26 6. Reconstituted fusion liposomes can be prepared using isolated fusion proteins derived from HVJ instead of from inactivated whole viral particles. 27

25 Z. Nishikawa, D. Du. X. L. Edelstein, S. Yamagishi, T. Matsumura, Y. Kaneda, M. A. Yorek, D. Beebe, E J. Oates, H. E Hammes, I. Giardino, and M. Brownlee, Nature 4114, 787 (2000). 26 y. Okada and J. Tadokoro, Exp. Cell Res. 26, 98 (1962). 27 K. Suzuki, H. Nakashima, Y. Sawa, R. Morishita, H. Matsuda, and Y. Kaneda, Gene Ther. Reg. 1,

65 (20oo).