Biomolecule transfer method using virus envelope and composition and system therefor

专利摘要:

公开号:AU2003257675A1
申请号:U2003257675
申请日:2003-08-22
公开日:2004-05-04
发明作者:Yasufumi Kaneda；Ryuichi Morishita；Munehisa Shimamura
申请人:AnGes MG Inc；
IPC主号:A61K35-76

专利说明:
VERIFICATION OF TRANSLATION Patent Application No. PCT/aJP0o3/01067 s 5 by AnGes MG, Inc. I, Takeshi Komatani of Fifteenth Floor, Crystal Tower, 1-2-27 Shiromi, Chuo-ku, Osaka 540-6015, Japan am the translator of the documents attached and I state that the following is a true translation to the best of my knowledge and belief of International Patent Application No. PCT/JPavo3/01067soos dated August 22, 2003 DATED this day of f r, 2005 .... (S..gnature of translator) (Signaiure of transiliawr 1 AN010 DESCRIPTION METHOD FOR INTRODUCING A BIOLOGICAL MOLECULE USING A VIRAL ENVELOPE AND COMPOSITION AND SYSTEM THEREFOR 5 TECHNICAL FIELD The present invention relates to a novel therapy method relating to direct administration of a bio 10 molecule. More specifically, the present invention is related to administration of a biomolecule using a vi ral envelope. The present invention also relates to a method for delivering a biomolecule to the brain in an efficient way. 15 BACKGROUND ART A number of virus and non-virus methods have been developed to introduce genes into cultured 20 cells or biological tissues for the purposes of gene function analysis, gene therapy, and similar applica tions (Mulligan, Science, 260, 926 to 932, 1993; and Ledley, Human Gene Therapy, Vol. 6, 1129 to 1144, 1995) Virus methods are most effective for the delivery of 25 genes into cells. However, virus vectors may raise problems due to the co-introduction of gene elements essential for parent genes derived from the parent vi rus, expression of virus genes, an immunogenicity, and the like. On the other hand, a liposome method, which 30 is a non-virus method, has a lower level of cytotoxic ity and immunogenicity than virus methods, but also tends to have a lower level of gene introduction effi ciency into biological tissues that that of virus vec- 2 AN010 tors. Hemagglutinating virus of Japan (HVJ) was first reported as fusing Ehrlich tumor cells (Okada, 5 Biken Journal, 1, 103-110, 1958), then the mechanism of its ability to fuse cell membranes (hereinafter re ferred to as "fusion activity") was clarified and the possible use of it as a gene introduction vector has been studied. It is known that HVJ has a high level of 10 immunogenicity, and particularly induces Cytotoxic T lymphocyte (CTL) when a large amount of NP protein is produced (Cole G.A. et al., Journal of Immunology, 158, 4301 to 4309, 1997). It is also likely that HVJ inhib its protein synthesis in hosts. To avoid these problems, 15 a technique was devised in which a liposome including a gene or protein is fused with HVJ which has been inac tivated by ultraviolet irradiation to prepare a fusion particle (HVJ-liposome). This technique made it possi ble to introduce a gene non-invasively into cultured 20 cells or organisms (US Patent No. 5,631,237; Dzau et al., Proc. Natl. Acad. Sci. USA, 93, 11421 to 11425, 1996; and Kaneda et al., Molecular Medicine Today, 5, 298 to 303, 1999). However, the technique requires preparation of two different vehicles, a liposome and a 25 viral envelope, which complicates the technique. The fusion particle of a liposome and HVJ disadvantageously has an average diameter about 1.3 times that of HVJ and a fusion activity one-tenth that of HVJ. In addition, for conventional HVJ-based vectors, there are some tis 30 sues in which it is not possible to introduce genes, or if it is possible, it is only possible with very low efficiency.
3 AN010 The present inventors have provided vari ous novel inactivated virus envelope vectors for intro ducing a gene or oligonucleotide into cultured cells or organisms (WO01/57204). Specifically, by packaging 5 genes into envelopes of various envelope viruses (e.g., HVJ, etc.), whose genomes are previously inactivated, the resultant viruses can be used as vectors capable of introducing genes into cultured cells or biological tissues with simplicity and high efficiency. These vi 10 ral vectors are also less toxic to cells. The present inventors have developed a method for producing an inactivated viral envelope at an indus trial scale which is inexpensive, effective and secures 15 a good quality product, by employing the use of an al kylating agent. In the present days, although some treatments ex ist for many diseases, diseases and disorders of the 20 brain is a field where few solutions are found. Further, demand for such treatment and prevention is increasing very year. Among encephalopathies, cerebral occlusive dis 25 ease caused by atherosclerosis of the cerebral arteries or Moyamoya disease, often causes chronic hypoperfusion of the brain. Although such a condition leads not only to cerebral ischemic events, but also to neuropa thological changes including dementia ( Kalaria RN, 30 Bhatti SU, Lust WD, Perry G., Ann N Y Acad Sci. 1993; 695: 190-3.; Kudo T, Takeda M, Tanimukai S, Nishimura T., Stroke. 1993;24:259-64; discussion 265.;Kurumatani T, Kudo T, Ikura Y, Takeda M., Stroke. 1998;29:1058- 4 AN010 62.; and Sekhon LH, Morgan MK, Spence I, Weber NC., Stroke. 1994;25:1022-7) , an effective treatment fo hy poperfusion has not yet been established. 5 Recently, preclinical studies have demon strated that angiogenic growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and hepatocyte growth factor (HGF) can stimulate the development of collateral arteries in an 10 animal brain ischemia model (Harrigan MR, Ennis SR, Ma sada T, Keep RF.,Neurosurgery. 2002;50:589-98;Lyons MK, Anderson RE, Meyer FB., Brain Res. 1991;558:315-20.;and Yoshimura S, et al., Hypertension. 2002;39:1028-34.) , a concept called therapeutic angiogenesis. The efficacy 15 of therapeutic angiogenesis using gene transfer of an giogenic growth factors has been reported in human pa tients with critical limb ischemia or myocardial in farction (Baumgartner I, et al., Circulation. 1998;97:1114-23.;Losordo DW, et al. , Circulation. 20 1998;98:2800-4.;Symes JF, et al. ,Ann Thorac Surg. 1999;68:830-6; discussion 836-7.; and Rosengart TK, et al., Circulation. 1999;100:468-74.). Accordingly, pos sible therapeutic angiogenesis should be possible for the treatment of patients with cerebral ischemia, if 25 safe and effective gene transfer methods were developed for human treatment. From this viewpoint, the current gene transfer techniques are not ideal. Gene transfer to the central nervous system (CNS) can be achieved by using various viral vectors including adeno-associated 30 virus (AAV) (Fan D, et al., Neurosci Lett. 1998;248:61 4), retrovirus (Franceschini IA, et al., J Neurosci Res. 2001;65:208-19), adenovirus (Miyaguchi K, Maeda Y, Collin C, Sihag RK., Brain Res Bull. 2000;51:195-202.)
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5 AN010 and herpes simplex virus 1 (Johnson PA, Yoshida K, Gage FH, Friedmann T.,Brain Res Mol Brain Res. 1992;12:95 102.). These vector systems have advantages and disad vantages for human gene therapy. Although these methods 5 are efficient for in vivo gene transfer into CNS, nu merous problems such as safety and production are yet to be resolved toward human gene therapy. In order to solve these problems, we have 10 developed and used HVJ (Hemagglutinating Virus of Japan = Sendai virus) envelope vector, which is a novel non viral vector system, as mentioned above. This vector system was developed based on HVJ-based gene transfer of the first generation using a viral envelope and 15 liposome (Yamada K, et al., Am J Physiol. 1996; 271: R1212-20; Kaneda Y, et al., Exp Cell Res. 1987;173:56 69.) (HVJ liposome methods). The first generation HVJ vector has great potential with regards to transfection to the CNS of rat and primate (Yamada K. et al. ibid ; 20 Hagihara Y, et al.,Gene Ther. 2000; 7:759-63.). However, there are some deficiencies in that it requires compli cated procedure to prepare the vector, and has diffi culties in its storage. A HVJ-envelope (HVJ-E) vector, a novel non-viral vector system, merely uses an enve 25 lope of HVJ for transfer of a foreign gene. (PROBLEM TO BE SOLVED BY THE PRESENT INVENTION) As such, there is a demand in the art for a method for efficient delivery of a biomolecule (such 30 as gene introduction) into the brain and the central nervous system (CNS) using a viral envelope both in vi tro and in vivo.
6 AN010 Further, the efficacy of delivery of a biomolecule using a viral envelope could be improved. Accordingly, there is a demand in the art for develop ing a system and a method for highly efficient delivery 5 of a biomolecule using a viral envelope. The object of the present invention is to provide an improved method for efficient delivery of a biomolecule into the brain and the central nervous sys 10 tem using a viral envelope. DISCLOSURE OF THE INVENTION 15 (MEANS FOR SOLVING THE PROBLEMS) As a result of a number of studies, the present inventors have found that the above mentioned problems may be solved by adding a glycosaminoglycan to a viral envelope system. 20 The present inventors have also found that by us ing a viral envelope system after occluding artery of the brain or cervix, a biomolecule can be effectively delivered into the brain by using a viral envelope. 25 Therefore, the present invention is pro vided below by way of various embodiments. (1) A system for introducing a biomolecule into a cell, 30 comprising: 1) a biomolecule; 2) a viral envelope; and 3) a glycosaminoglycan.
7 AN010 (2) The system according to Item 1, wherein the viral envelope is inactivated. 5 (3) The system according to Item 1, wherein the glyco saminglycan is heparin. (4) The system according to Item 1, wherein the molecu lar weight of the glycosaminoglycan is at least 10,000 10 kDa. (5) The system according to Item 1, wherein the glyco saminoglycan is comprised at the concentration of at least 50 U/ml. 15 (6) The system according to Item 3, wherein the molecu lar weight of said heparin is from 12,000 to 15,000 kDa. (7) The system according to Item 3, wherein the degree 20 of sulfation of said heparin is pharmaceutically ac ceptable. (8) The system according to Item 1, wherein the viral envelope is an envelope of an RNA virus. 25 (9) The system according to Item 1, wherein the viral envelope is an envelope of a virus belonging to the Pa ramixovirus genus. 30 (10) The system according to Item 1, wherein the viral envelope is an envelope of HVJ. (11) The system according to Item 1, wherein the bio- 8 AN010 molecule is selected from the group consisting of a nu cleic acid, a polypeptide, a lipid, a sugar, and a com plex molecule thereof. 5 (12) The system according to Item 1 wherein the bio molecule comprises a nucleic acid. (13) The system according to Item 1 wherein the bio molecule comprises a polypeptide. 10 (14) The system according to Item 1 wherein the bio molecule is a nucleic acid encoding a gene selected from the group consisting of Vascular endothelial growth factor (VEGF), Fibroblast growth factor (FGF), 15 and Hepatocyte growth factor (HGF). (15) The system according to Item 1 wherein the bio molecule is a polypeptide selected from the group con sisting of Vascular endothelial growth factor (VEGF), 20 Fibroblast growth factor (FGF), and Hepatocyte growth factor (HGF). (16) The system according to Item 1, wherein the hepa rin, the biomolecule and the viral envelope are con 25 tained in a same composition. (17) The system according to Item 1, wherein the hepa rin is contained in a different composition from that which comprises the biomolecule and the viral envelope. 30 (18) The system according to Item 1, wherein the bio molecule is contained in the viral envelope.
AN010 (19) A method for introducing a biomolecule into a cell, comprising the steps of: A) administering to a cell a composition compris ing a viral envelope and a biomolecule; and 5 B) administering a glycosaminoglycan to the cell. (20) The method according to Item 19, wherein the step of administering the glycosaminoglycan is performed si multaneously with the step of administering the compo 10 sition. (21) The method according to Item 19, wherein the step of administering the glycosaminoglycan is performed prior to the step of administering the composition. 15 (22) The method according to Item 19, wherein the step of administering the glycosaminoglycan is performed af ter the step of administering the composition. 20 (23) Use of a glycosaminoglycan for manufacturing a medicament for introducing a biomolecule into a cell, wherein the medicament comprises A) a biomolecule; B) a viral envelope; and 25 C) a glycosaminoglycan. (24) A method for delivering a biomolecule to a brain, comprising the steps of: A) transiently occluding an artery of the head 30 portion or cervical portion; and B) introducing a biomolecule into the brain dur ing the occluding of the artery of a head por tion or a cervical portion.
10 AN010 (25) The method according to Item 24, wherein the bio molecule is selected from the group consisting of a nu cleic acid, a polypeptide, a lipid, a sugar, and a com 5 plex molecule thereof. (26) The method according to Item 24, wherein the bio molecule comprises a nucleic acid. 10 (27) The method according to Item 24, wherein the bio molecule is a nucleic acid encoding a gene selected from the group consisting of Vascular endothelial growth factor (VEGF), Fibroblast growth factor (FGF), and Hepatocyte growth factor (HGF). 15 (28) The method according to Item 26, wherein the nu cleic acid is delivered by a vector. (29) The method according to Item 24, wherein the bio 20 molecule is introduced with a viral envelope. (30) The method according to Item 29, wherein the vi ral envelope is inactivated. 25 (31) The method according to Item 29, wherein the vi ral envelope is an envelope of an RNA virus. (32) The method according to Item 29, wherein the vi ral envelope is an envelope of a virus belonging to the 30 Paramixovirus genus. (33) The method according to Item 29, wherein the vi ral envelope is an envelope of HVJ.
11 AN010 (34) The method according to Item 24, wherein the bio molecule is introduced with a glycosaminoglycan. 5 (35) The method according to Item 34, wherein the gly cosaminoglycan is heparin. (36) The method according to Item 34, wherein the mo lecular weight of the glycosaminoglkycan is at least 10 10,000 kDa. (37) The method according to Item 34, wherein the gly cosaminoglycan is comprised at the concentration of at least 50 U/ml. 15 (38) The method according to Item 35, wherein the mo lecular weight of said heparin is from 12,000 to 15,000 kDa. 20 (39) The method according to Item 35, wherein degree of sulfation of said heparin is pharmaceutically ac ceptable. (40) The method according to Item 34, wherein the gly 25 cosaminoglycan is simultaneously administered with the biomolecule. (41) The method according to Item 34, wherein the gly cosaminoglycan is administered prior to the administra 30 tion of the biomolecule. (42) The method according to Item 34, wherein the gly cosaminoglycan is administered after the administration 12 AN010 of the biomolecule. (43) The method according to Item 24, wherein the ar tery of the head portion or the cervical portion is 5 closed for 1 minute to 120 minutes. (44) The method according to Item 24, wherein the ar tery of the head portion or the cervical portion is the middle cerebral artery or the carotid artery. 10 (45) The method according to Item 24, wherein the ar tery of the head portion or the cervical portion is the middle cerebral artery. 15 (46) The method according to Item 24, wherein the bio molecule is administered into a carotid artery, on a thalamus, intracerebroventricularly or intrathecally. (47) The method according to Item 24, wherein the bio 20 molecule is administered into the carotid artery. (48) A kit for delivering a biomolecule into a brain, comprising: A) a biomolecule; and 25 B) an instruction indicating a method for admin istering the biomolecule, wherein the method comprises: a) transiently occluding an artery of a head portion or a cervical portion; and 30 b) introducing the biomolecule into the brain during the occluding of the ar tery of the head portion or the cervi cal portion.
13 AN010 (49) The kit according to Item 48, wherein the bio molecule is selected from the group consisting of a nu cleic acid, a polypeptide, a lipid, a sugar, and a com 5 plex molecule thereof. (50) The kit according to Item 48, wherein the bio molecule comprises a nucleic acid. 10 (51) The kit according to Item 48, wherein the bio molecule is a nucleic acid encoding a gene selected from the group consisting of Vascular endothelial growth factor (VEGF), Fibroblast growth factor (FGF), and Hepatocyte growth factor (HGF). 15 (52) The kit according to Item 48, wherein the nucleic acid is delivered by a vector. (53) The kit according to Item 48, further comprising 20 a viral envelope. (54) The kit according to Item 53, wherein the viral envelope is inactivated. 25 (55) The kit according to Item 53, wherein the viral envelope is an envelope of an RNA virus. (56) The kit according to Item 53, wherein the viral envelope is an envelope of a virus belonging to the Pa 30 ramixovirus genus. (57) The kit according to Item 53, wherein the viral envelope is an envelope of HVJ.
14 AN010 (58) The kit according to Item 48, further comprising a glycosaminoglycan. 5 (59) The kit according to Item 58, wherein the glyco saminoglycan is heparin. (60) The kit according to Item 58, wherein the molecu lar weight of the glycosaminoglycan is at least 10,000 10 kDa. (61) The kit according to Item 58, wherein the glyco saminoglycan is comprised at the concentration of at least 50 U/ml. 15 (62) The kit according to Item 59, wherein the molecu lar weight of the heparin is from 12,000 to 15,000 kDa. (63) The kit according to Item 59, wherein the degree 20 of sulfation of said heparin is pharmaceutically ac ceptable. (64) The kit according to Item 58, wherein the glyco saminoglycan is simultaneously administered with the 25 biomolecule. (65) The kit according to Item 58, wherein the glyco saminoglycan is administered prior to the administra tion of the biomolecule. 30 (66) The kit according to Item 58, wherein the glyco saminoglycan is administered after the administration of the biomolecule.
15 AN010 (67) The kit according to Item 48, wherein the artery of the head portion or the cervical portion is closed for 1 minute to 120 minutes. 5 (68) The kit according to Item 48, wherein the artery of the head portion or the cervical portion is the mid dle cerebral artery or the carotid artery. 10 (69) The kit according to Item 48, wherein the artery of the head portion or the cervical portion is the mid dle cerebral artery. (70) The kit according to Item 48, wherein the bio 15 molecule is administered into the carotid artery, the thalamus, intracerebroventricularly or intrathecally. (71) The kit according to Item 48, wherein the bio molecule is administered into the carotid artery. 20 (72) Use of a biomolecule for manufacturing a kit for delivering the biomolecule into a brain, the kit comprising: A) the biomolecule; and 25 B) an instruction indicating a method for admin istering the biomolecule, wherein the method comprises: a) transiently occluding an artery of a head portion or a cervical portion; and 30 b) introducing the biomolecule into the brain during the occluding of the artery of the head portion or the cervical portion.
16 AN010 It is to be understood that the advantages and effects of the present invention will become appar ent to those skilled in the art upon reading and under standing the description of the present specification. 5 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Typical example of the cultured rat cerebral 10 cortex neurons and glial cells transfected with Venus gene using HVJ-E vector. Laser scanning confocal mi croscopy images of Venus (a, d, g), immunofluorescent staining for MAP 2 (b), NeuN (d), GFAP (h), and merged images (c, f, i). Most of the cells expressing Venus 15 were immuno-positive for MAP 2 and NeuN (a-f). This ex periment was repeated at least five times. Figure 2. In vivo gene transfer of a EGFP plasmid into brain using stereotactic injection. A fluorescent ste 20 reomicroscope image of laser scanning confocal micros copy images. Stereotactic injection into thalamus (a, b) showed the limited expression at the site of injec tion. Stereotactic injection into lateral ventricle (c, d) revealed the expression at the choroid plexus and 25 ependymal cells (e, f). CP, choroid plexus; LV, lateral ventricle; Str, striatum; Ep, ependymal cell layer. This experiment was repeated at least three times. Figure 3. In vivo gene transfer of Venus plasmid into 30 brain by the injection via cisterna magna. OB: olfac tory bulb; FC: frontal cortex; M: meninges. This ex periment was repeated at least three times.
17 AN010 Figure 4. Fluorescence due to Venus gene transfer via carotid artery after transient occlusion of middle cerebral artery. Coronal suture section at 3 days after left transient occlusion of middle cerebral artery for 5 60 minutes and a fluorescent stereomicroscope image. HVJ-E vector having Venus gene was infused in the left carotid artery during the reperfusion. Gene expression was observed only at the injured lesion, while no fluo rescence could be detected at the intact hemisphere. 10 This experiment was repeated at least three times. Figure 5. Luciferase activity in injured hemi sphere and the contra lateral hemisphere after gene transfer into carotid artery after transient occlusion 15 of middle cerebral artery. Luciferase activity was measured at day 1 after left transient occlusion of middle cerebral artery for 60 minutes. HVJ-E vector containing luciferase gene was infused in the left ca rotid artery during the reperfusion. n=5 for each group. 20 Figure 6. Effect of heparin on luciferase activity. Luciferase activity was measured at day 1 after trans fection of pGL3 luciferase gene via cisterna magna. Heparin was added to the vector at the concentration of 25 0, 10, 50, or 100 (U/ml), low molecular weight heparin at 10, 50, or 100 (U/ml). **P < 0:01 vs. 0, 10 and 100. n=3 for each group. 30 DETAILED DESCRIPTION OF THE INVENTION Hereinafter the present invention is described. It should be understood throughout the present specifi cation that the singular forms include plural referents 18 AN010 unless the context clearly dictates otherwise. It should also be understood that the terms as used herein have definitions typically used in the art unless oth erwise mentioned. 5 Terms specifically used herein will be de scribed below. As used herein, the term "virus" refers to 10 a transmissible small structure which has DNA or RNA as its genome and proliferates only within infected cells. Viruses include a virus belonging to a family selected from the group consisting of the family Retroviridae, the family Togaviridae, the family Coronaviridae, the 15 family Flaviviridae, the family Paramyxoviridae, the family Orthomyxoviridae, the family Bunyaviridae, the family Rhabdoviridae, the family Poxviridae, the family Herpesviridae, the family Baculoviridarie, and the fam ily Hepadnaviridae. A virus used herein may be prefera 20 bly influenza virus or Sendai virus of the family Or thomyxoviridae. More preferably, a virus used herein is Sendai virus. As used herein, the terms "Sendai virus" 25 or "HVJ" (Hemagglutinating virus of Japan) are used in terchangeably, referring to a virus capable of cell fu sion of the genus paramyxovirus of the family para myxovirus. M. Kuroya et al. reported Sendai virus (1953). The genome is a minus strand of RNA having a 30 base length of about 15,500. The particle of Sendai vi rus comprises an envelope and has a diameter of 150 nm to 300 nm (polymorphism). Sendai virus has RNA poly merase. The virus is unstable to heat, and causes hemo- 19 AN010 lysis and agglutination of substantially all types of red blood cells. The virus grows in the cytoplasm of developing chicken eggs and/or cultured cells derived from the kidney of various animals. When established 5 cells are infected with Sendai virus, persistent infec tion is likely to occur. The virus has the ability to fuse various cells, and therefore, is widely used in the formation of heterokaryons, preparation of hybrid cells, and the like. 10 As used herein, the term "(virus or viral) envelope" refers to a membrane structure which basi cally comprises a lipid bilayer surrounding a nucleo capsid which exists in specific viruses such as Sendai 15 virus and the like. Envelopes are typically observed in mature viruses budding from cells. An envelope gener ally consists of host-derived lipids and small project ing structures consisting of spike proteins encoded by viral genes. Therefore "(viral) envelope vector" is a 20 designation when using the envelope as a vector, and as used herein it can be interchangeably used with "viral envelope", when appropriate. As used herein the term "glycosaminogly 25 can" refers to a polysaccharide in which the main com ponent is hexosamine. Such a glycosaminoglycan includes but is not limited to hyaluronic acid, chondroitin sul fate, dermatan sulfate, keratan sulfate, heparan sul fate, heparin and a mixture thereof. As used herein 30 "hexosamine" refers to a hexose in which a hydroxy group is replaced with an amino acid group. Glycosami noglycan is usually classified as described above, how ever, it has been reported that there are some mole- 20 AN010 cules which are difficult to be definitively classified, such as a molecule a part of which has a chondroitin sulfate structure, and another part has a dermatin sul fate structure and the like. Except for hyaluronic ac 5 ids, which are synthesized and secreted by Streptococ cus (Group A), almost all glycosaminoglycans are side chain components of a proteoglycan produced by an ani mal cell (mainly cells of connective tissue), and can be obtained by enzymatically processing core protein 10 portions thereof, or by digesting the binding between the protein and the side chain of glycosaminoglycan by alkaline processing, in the case of ester type in which the glycosaminoglycan is bound to a serine or a threonine of the protein. The anionic structure with 15 high molecular weight of glycosaminoglycan is useful for comprising a number of water molecules. As used herein, glycosaminoglycans include both glucosaminogly can and galactosaminoglycan. Galactosaminoglycans refer to glycosaminoglycan including galactosamine, and in 20 clude, for example, chondroitin sulfate, dermatan sul fate, and the like. Glucosaminoglycans refer to a gly cosaminoglycan including glycosamine, and include, for example, heparin, heparan sulfate and the like. 25 As used herein, the term "heparin" refers to a glycosaminoglycan essentially consisting of D glycosamine and D-glucuronic acid (for example, polym erized in an alternate manner). Sulfate is bound to the 2,6-amino group of almost all glucosamine, as well as 30 to the hydroxy group at the position 6 thereof, and the position 2 of uronic acid. Heparin that is synthesized in mast cells of an animal, and has a number of sulfate and carboxyl groups, thus it is a negatively charged 21 AN010 electrolyte with high molecular weight. It has an in hibitory activity of blood coagulation. As used herein the term "sulfation" refers to re 6 placement of substituent (for example, amino group, hy droxy group and the like) with a sulfate group. The de gree of sulfation is an important factor determining the degree of charge of a molecule, and it is believed that change in the strength of the cellular membrane 10 surface can affect the efficiency of delivery of a vi ral envelope in the present invention. Accordingly, in a preferable embodiment, the degree of sulfation used is pharmaceutically acceptable. 15 The term "biomolecule" as used herein re fers to a molecule related to an organism. An "organism (or "bio-")" as used herein refers to a biological or ganic body, including, but being limited to, an animal, a plant, a fungus, a virus, and the like. A biomolecule 20 includes a molecule extracted from an organism, but is not so limited. A biomolecule is any molecule capable of having an influence on an organism. Therefore, a biomolecule also includes a molecule synthesized, for example, by combinatorial chemistry, and a low weight 25 molecule capable of being used as a medicament (e.g., a low molecular weight ligand, etc.) as long as they are intended to have an influence on an organism. Examples of such biomolecules include, but are not limited to, proteins, polypeptides, oligopeptides, peptides, 30 polynucleotides, oligonucleotides, nucleotides, nucleic acids (e.g., including DNA (such as cDNA and genomic DNA) and RNA (such as mRNA)), polysaccharides, oligo saccharides, lipids, low weight molecules (e.g., hor- 22 AN010 mones, ligands, signal transduction substances, low weight organic molecules, etc.), and complex molecules thereof, and the like. A biomolecule also includes a cell itself, and a part or the whole of tissue, and the 5 like as long as they can be coupled to a substrate of the present invention. Preferably, a biomolecule in cludes a nucleic acid or a protein. In a preferable em bodiment, a biomolecule is a nucleic acid (e.g., ge nomic DNA or cDNA, or DNA synthesized by PCR or the 10 like). In another preferable embodiment, a biomolecule may be a protein. As used herein, the term "biological ac tivity" refers to the activity which a certain factor 15 (e.g., virus, polynucleotide or polypeptide) has within an organism, including activity exhibiting various functions. For example, when the certain factor is a transcriptional factor, its biological activity in cludes activity to regulate transcriptional activity. 20 When the certain factor is a virus, its biological ac tivity includes infection activity. As another example, when the certain factor is a ligand, its biological ac tivity includes binding to a receptor to which the ligand corresponds. Such biological activity can be 25 "inactivated". As used herein, the term "inactivation" in relation to a virus (e.g., Sendai virus, etc.) indi cates that the genome of the virus is inactivated. The 30 inactivated virus is incapable of replication. Inacti vation is achieved by a method described herein such as alkylation and the like. Such a method for inactivation includes but is not limited to a method comprising the 23 AN010 steps of: (a) inactivating a virus (e.g., HVJ, etc.) with an alkylating agent; (b) obtaining a condensate solution of the virus or the inactivated virus; and (c) purifying the virus or the inactivated virus by column 5 chromatography and then ultrafiltration, and a method comprising the same steps but the order thereof being rearranged. As used herein, the term "alkylation" re 10 fers to an action which substitutes an alkyl group for a hydrogen atom of an organic compound. The term "alky lating agent" refers to a compound which supplies an alkyl group. Examples of alkylating agents include, but are not limited to, organic metal compounds such as al 15 kyl halide, dialkyl sulfate, alkyl sulfonate, alkyl lead, and the like. Examples of preferable alkylating agents include, but are not limited to, P-propiolactone, butyrolactone, methyl iodide, ethyl iodide, propyl io dide, methyl bromide, ethyl bromide, propyl bromide, 20 dimethyl sulfate, diethyl sulfate, and the like. As used herein, "nucleic acid", "nucleic acid molecule", "polynucleotide", and "oligonucleotide" are herein used interchangeably to refer to macromole 25 cules (polymer) comprising a series of nucleotides, unless otherwise specified. A nucleotide refers to a nucleoside whose base is a phosphoric ester. The base of the nucleotide is a pyrimidine or purine base (pyrimidine nucleotide and purine nucleotide). Polynu 30 cleotides include DNA or RNA. As used herein, "nucleotide" refers to any naturally occurring nucleotide and non-naturally occur- 24 AN010 ring nucleotide. "Derived nucleotide" refers to a nu cleotide which is different from naturally occurring nucleotides but has a function similar to that of its original naturally occurring form. Such derived nucleo 5 tides are well known in the art. As used herein, the term "fragment" in re lation to a nucleic acid molecule refers to a polynu cleotide whose length is shorter than the full length 10 of the reference nucleic acid molecule, but is suffi cient as an agent of the present invention. Therefore, the term "fragment" refers to a polynucleotide which has a sequence length ranging from 1 to n-1 with re spect to the full length of the reference polynucleo 15 tide (of length n). The length of the fragment can be appropriately changed depending on the purpose. For ex ample, the lower limit of the length of the fragment includes 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 or more nucleotides. Lengths represented by inte 20 gers which are not herein specified (e.g., 11 and the like) may be appropriate as a lower limit. Homology may be represented by a score measured by a search program BLAST using an algorithm developed by Altschul et al. (J. Mol. Biol., 215, 403-410 (1990)). 25 As used herein, the terms "protein", "polypeptide", and "peptide" are used interchangeably, referring to a macromolecule which consists of a series of amino acids. The term "amino acid" refers to an or 30 ganic molecule which has a carboxyl group and an amino group bound to a carbon atom. Preferably, amino acids herein include, but are not limited to, 20 naturally occurring amino acids.
25 AN010 As used herein, the term "gene" refers to an element defining a genetic trait. A gene is typi cally arranged in a given sequence on a chromosome. A 5 gene which defines the primary structure of a protein is called a structural gene. A gene which regulates the expression of a structural gene is called a regulatory gene. As used herein, the term "gene" may refer to "polynucleotide", "oligonucleotide", "nucleic acid", 10 and "nucleic acid molecule" and/or "protein", "polypep tide", "oligopeptide" and "peptide". As used herein the term "foreign gene" refers to a nucleic acid sequence to be included in a gene intro 15 duction vector derived from a source other than viral sequence. In one aspect of the invention, the foreign gene is operably linked to an appropriate sequence (for example, promoter, enhancer, terminator, and poly A signal necessary for transcription, and ribosome bind 20 ing site, start codon, termination codon necessary for translation, and the like) in order to express the gene introduced by a gene introduction vector. In another aspect, the foreign gene does not include a regulatory sequence for expressing the foreign gene. In a further 25 aspect, the foreign gene is an oligonucleotide or decoy nucleic acid. As used herein the term "gene library" re fers to a nucleic acid library, including a naturally 30 isolated nucleic acid sequence, or a synthetic nucleic acid sequence. Sources of naturally isolated nucleic acids include, but are not limited to, genomic se quences from eukaryotic cells, prokaryotic cells or vi- 26 AN010 ruses, or cDNA sequences. A library in which an arbi tral sequence (for example, signal, tag sequences or the like) is added to a naturally isolated sequence is also included in the definition of a gene library of 5 the present invention. In one embodiment, the gene li brary includes sequences rendering transcription and/or translation of the nucleic acid such as the promoter and the like. 10 As used herein, the term "screening" re fers to selection or an assay of members having a de sired activity or function from a library such as a gene library. Such a method of screening is known in the art. 15 As used herein, the term "gene introduc tion" refers to introduction of a desired, natural, synthetic or recombinant gene or gene fragment into a target cell in vivo or in vitro in such a manner that 20 the function of the introduced gene is maintained. The gene or gene fragment to be introduced in the present invention includes DNA, RNA having a specific sequence, or a synthetic analog thereof. Further, as used herein, gene introduction is used interchangeably with the 25 terms "transfection" and "transfect". As used herein "gene introduction activ ity" refers to the activity of "gene introduction" by a vector, and can be detected by the function of the gene 30 introduced (for example, when using an expression vec tor, expression and/or activity of a protein encoded thereby).
27 AN010 As used herein, the term "expression" of a polynucleotide, a polypeptide, or the like, indicates that the gene or the like is affected by a predeter mined action in vivo to be changed into another form. 5 Preferably, the term "expression" indicates that a gene, a polynucleotide, or the like is transcribed and trans lated into a polypeptide. In addition, generation of mRNA via transcription may be an aspect of the "expres sion". More preferably, such a polypeptide may have a 10 form modified by post-translational processing. As used herein, the term "regulation" in relation to the ex pression of a gene refers to, but is not limited to, enhancement, reduction, induction, elimination, decel eration, acceleration, and the like of gene expression. 15 Examples of a gene to be treated include, but are not limited to, genes encoding enzymes, hor mones, lymphokines, receptors, growth factors, regula tory proteins, polypeptides affecting the immune system, 20 immunoregulatory factors, antibodies, and the like. Specifically, these genes include, but are not limited to, genes encoding human growth hormones, insulin, in terleukin-2, tumor necrosis factors, nerve growth fac tors (NGFs), epithelial growth factors, tissue plasmi 25 nogen activators (TPAs), Factor VIII:C, calcitonin, thymidine kinase, interferon, granulocyte-macrophage colony-stimulating factors (GMCSFs), erythropoietin (EPO), hepatocyte growth factors (HGFs), and the like. These genes may be present in the form of a nucleic 30 acid or a polypeptide in a medicament of the present invention. As used herein the term "vector", when re- 28 AN010 ferring to a gene, refers to those capable of transfer ring a polynucleotide sequence of interest to a cell of interest. Examples of such vector include one capable of autonomous replication in a host cell such as in an 5 individual animal, or one having a promoter at an ap propriate site for transcription of the polynucleotide of the present invention which is capable of being in corporated in the chromosome of the host. As used herein, the vector may be a plasmid. 10 As used herein, the term "expression vec tor" refers to a nucleic acid sequence in which a structural gene, a promoter regulating the expression thereof, and a variety of regulatory elements are oper 15 ably linked in the host cell. Such regulatory elements preferably may include terminators, selective marker such as drug resistant genes, and enhancers. It is well known to those skilled in the art that the types of ex pression vectors of organisms such as animals, and spe 20 cies of regulatory elements to be used therein may vary depending on the host cell or organism used. In the case of a human, the expression vector to be used in the present invention, may include pCAGGS (Niwa H. et al., Gene; 108: 193-9(1991)). 25 As used herein the term "recombinant vec tor" refers to vectors capable of transferring a polynucleotide sequence of interest to a cell of inter est. Examples of such vectors include one capable of 30 autonomous replication, or of being incorporated in to chromosome in a host cell such as in an individual ani mal and having a promoter at an appropriate site for transcription of the polynucleotide of the present in- 29 AN010 vention. Examples of "recombinant vectors" for ani mal cells include, but are not limited to, pcDNAI/Amp, 5 pcDNAI, pCDM8 (all commercially available from Funako shi), pAGE107 [Japanese Laid-Open Publication No. 3-229, 79, Cytotechnology, 3, 133 (1990), pREP4 (Invitrogen), pAGE103 [J. Biochem., 101, 1307(1987)], pAMo, pAMoA [J. Biol. Chem., 268, 22782-22787(1993)], pCAGGS (Niwa, H., 10 et al., Gene; 108, 193-199 (1991), and the like. As used herein, the term "terminator" re fers to a sequence which is located downstream of a 15 protein-encoding region in a gene and which is involved in the addition of a poly-A sequence and the termina tion of transcription when DNA is transcribed into mRNA. It is known that a terminator contributes to the sta bility of mRNA and has influence on the amount of gene 20 expression. Terminators include, but are not limited to, those from a mammal, as well as CaMV35S terminator, nopaline synthase gene terminator (Tnos), tobacco PRIa gene terminator, and the like. 25 As used herein, the term "promoter" refers to a base sequence which determines the initiation site of transcription of a gene and is a region located in DNA which directly regulates the frequency of tran scription. Transcription is started by RNA polymerase 30 binding to a promoter. Accordingly, a portion of a gene having promoter function herein refers to "promoter moiety". A promoter region is usually located within about 2 kbp upstream of the first exon of a putative 30 AN010 protein coding region. Therefore, it is possible to es timate a promoter region by predicting a protein coding region in a genomic base sequence using DNA analysis software. A putative promoter region is usually located 5 upstream of a structural gene, but depending on the structural gene, a putative promoter region may be lo cated downstream of a structural gene. Preferably, a putative promoter region is located within about 2 kbp upstream of the translation initiation site of the 10 first exon. As used herein when referring to expression of a gene, the term "site specificity" generally refers to specificity of expression of the gene in a site (for 15 example, in the case of an animal, the heart, myocar diac cell and the like) of an organism (for example, an animal). The term "time specificity" refers to speci ficity of expression of a gene depending on a specific stage (for example, at the time of stroke and the like) 20 of an organism (for example, an animal). Examples of a vaccine which may be herein used as a medicament include, but are not limited to, vaccines for cancer, acquired immunodeficiency syndrome, 25 measles, herpes simplex, and the like. These vaccines may be present in the form of a nucleic acid or a pep tide in a medicament of the present invention. The present invention provides a pharma 30 ceutical composition or a medicament comprising the above-described envelope singly or in combination with a stabilizing compound, a diluent, a carrier, or other ingredients and pharmaceutical agents. Preferably, the 31 AN010 present invention may be in the form of a vaccine or in other forms suitable for gene therapy. A pharmaceutical composition and medica 5 ment of the present invention may be used in a form which allows the envelope thereof to be taken into cells at an affected site or cells of a tissue of in terest. 10 A pharmaceutical composition and medica ment of the present invention may be administered within any aseptic biocompatible pharmaceutical carrier including, but not being limited to, physiological sa line, buffered physiological saline, dextrose, water, 15 and the like. Any of these molecules may be adminis tered into patients within a pharmaceutical composition, which is mixed with an appropriate excipient, adjuvant, and/or pharmaceutically acceptable carrier, singly or in combination with other pharmaceutical agents. In a 20 certain embodiment of the present invention, a pharma ceutically acceptable carrier is pharmaceutically inac tive. A pharmaceutical composition and medica 25 ment of the present invention is administered orally or parenterally. Examples of parenteral delivery methods include, but are not limited to, topical, intraarterial (e.g., via the carotid artery, or the like), intramus clar, subcutaneous, intramedullary, subarachnoideal, 30 intraventicular, intravenous, intraperitoneal, and in tranasal administrations, and the like. In the present invention, any route which allows delivery to a site to be treated may be used.
32 AN010 As used herein, the term "head portion" refers to a portion of the body, including cranial bone, the content thereof, and related structures. As used 5 herein "head portion" includes a brain. As used herein, the term "cervical por tion" refers to regions between the head portion and the upper extremity. As used herein, the head-cervical 10 (head and neck) portion or craniocervical portion may refer to the neck and above portions. As used herein the term "transiently" re fers to that when a treatment has taken place, such a 15 treatment is continued for a certain period of time. Accordingly, after the treatment ceases, effects of the treatment will be lost or diminished. As used herein the term "artery" refers to 20 a blood vessel delivering blood from the heart to each portion of the body. Accordingly, the term "cerebral artery" refers to an artery present in the brain. As used herein the term to "occlude" an 25 artery or a blood vessel refers to treat the same so that a blood stream is significantly reduced or stopped compared to when the treatment is not performed. Pref erably, it is preferable not to cause hemorrhage when an occlusion is performed. Means for occluding an ar 30 tery or a blood vessel include, but are not limited to, balloon catheter, clipping and the like. As used herein "introducing" a biomolecule 33 AN010 into a space (for example, "into a cell (intracellu larly)") refers to the transfer of such a biomolecule from a different space to the space of interest. Such an introduction may use any means, and may be active 5 introduction or passive introduction. As used herein the term "system" refers to a product consisting of a plurality of components, in cluding pharmaceuticals, agricultural chemicals, compo 10 sitions (for example, pharmaceutical compositions), vaccines, kit and the like. In addition to an envelope, these pharma ceutical compositions and pharmaceutical agents may 15 comprise a pharmaceutically acceptable carrier contain ing other compounds for promoting processing of the en velope in order to prepare an excipient or pharmaceuti cally acceptable composition. Further details of pre scription and administration are described in, for ex 20 ample, the latest edition of Japanese Pharmacopeia and its latest supplement, the latest edition of "REMING TON'S PHARMACEUTICAL SCIENCES" (Maack Publishing Co., Easton, PA), or the like. 25 A pharmaceutical composition for oral ad ministration may be prepared using a pharmaceutically acceptable carrier well known in the art in a form suitable for administration. Such a carrier can be pre pared as a tablet, a pill, a sugar-coated agent, a cap 30 sule, a liquid, a gel, a syrup, a slurry, a suspension, or the like, with which it is suited for the patient to take the pharmaceutical composition.
34 AN010 The pharmaceutical composition for oral use may be obtained in the following manner: an active compound is combined with a solid excipient, the resul tant mixture is pulverized if necessary, an appropriate 5 compound is further added if necessary to obtain a tab let or the core of a sugar-coated agent, and the granu lar mixture is processed. The appropriate excipient may be a carbohydrate or protein filler, including, but not being limited to, the following: sugar including lac 10 tose, sucrose, mannitol, or sorbitol; starch derived from maize, wheat, rice, potato, or other plants; cel lulose such as methylcellulose, hydroxypropylmethylcel lulose, or sodium carboxymethylcellulose; and gum in cluding gum Arabic and gum tragacanth; and proteins 15 such as gelatin and collagen. A disintegrant or a solu bilizing agent such as crosslinked polyvinyl pyrroli done, agar, alginic acid or a salt thereof (e.g., so dium alginate) may be used if necessary. 20 The sugar-coated agent core is provided along with an appropriate coating, such as a condensed sugar solution. The sugar-coated agent core may also contain gum arabic, talc, polyvinyl pyrrolidone, car bopolygel, polyethylene glycol, and/or titanium dioxide, 25 a lacquer solution, and an appropriate organic solvent or a mixed solvent solution. To identify a product, or characterize the amount of an active compound (i.e., dose), dye or pigment may be added to tablets or sugar coated agents. 30 The pharmaceutical composition which may be orally used may contain, for example, a soft sealed capsule consisting of a gelatin capsule, gelatin and 35 AN010 coating (e.g., glycerol or sorbitol). The gelatin cap sule may contain an active ingredient mixed with a filler or binder such as lactose or starch, a lubricant such as talc or magnesium stearate, and optionally a 5 stabilizer. In the soft capsule, the decoy compound may be dissolved or suspended in an appropriate liquid, such as fatty oil, liquid paraffin or liquid polyethyl ene glycol, with or without a stabilizer. 10 The pharmaceutical composition for par enteral administration contains an aqueous solution of an active compound. For the purpose of injection, the pharmaceutical composition of the present invention is prepared in an aqueous solution, preferably Hank's so 15 lution, Ringer's solution, or a physiologically suit able buffer such as a buffered physiological saline. The aqueous suspension for injection may contain a sub stance for increasing the viscosity of a suspension (e.g., sodium carboxymethylcellulose, sorbitol, or dex 20 tran). Further, the suspension of the active compound may be prepared as an appropriate oily suspension. Ap propriate lipophilic solvents or vehicles include fatty acids such as sesame oil, synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. 25 The suspension may contain a stabilizer which allows a high-concentration solution preparation, or an appro priate pharmaceutical agent or reagent for increasing the solubility of the compound, if necessary. 30 The pharmaceutical composition of the pre sent invention may be produced using a process similar to processes known in the art (e.g., conventional mix ing, dissolution, rendering to granules, preparation of 36 AN010 a sugar-coated agent, elutriation, emulsification, cap sulation, inclusion, or freeze drying). A pharmaceutical composition of the pre 5 sent invention includes a composition containing an ef fective amount of an envelope of the present invention which can achieve the intended purpose of the decoy compound. "Therapeutically effective amount" and "phar macologically effective amount" are terms which are 10 well recognized by those skilled in the art and which refer to an amount of pharmaceutical agent effective for production of an intended pharmacological effect. Therefore, therapeutically effective amount is an amount sufficient for reducing the manifestation of the 15 disease to be treated. A useful assay for confirming an effective amount (e.g., a therapeutically effective amount) for a predetermined application is to measure the degree of recovery from a target disease. The amount actually administered depends on the individual 20 to be treated. The amount is preferably optimized so as to achieve a desired effect without significant side effects. The determination of therapeutically effective dose is within the ability of those skilled in the art. 25 A therapeutically effective dose of any compound can be initially estimated using either a cell culture assay or any appropriate animal model. The ani mal model is used to achieve a desired concentration range and an administration route. Thereafter, such in 30 formation can be used to determine a dose and route useful for administration into humans. The term "therapeutically effective 37 AN010 amount" in relation to an envelope refers to an amount which results in amelioration of symptoms or conditions of a disease. Therapeutic effect and toxicity of an en velope may be determined by standard pharmaceutical 5 procedures in cell cultures or experimental animals (e.g., ED 50 , a dose therapeutically effective for 50% of a population; and LDs 50 , a dose lethal to 50% of a popu lation). The dose ratio between therapeutic and toxic effects is therapeutic index, and it can be expressed 10 as the ratio of ED50/LDs 5 0. Pharmaceutical compositions which exhibit high therapeutic indices are preferable. The data obtained from cell culture assays and animal studies can be used in formulating a dosage range for use in humans. The dosage of such compounds preferably 15 lies within a range of circulating concentrations that include the ED 50 but have little or no toxicity. Such a dosage may vary within this range depending upon the dosage form employed, the susceptibility of the patient, and the route of administration. As an example, the 20 dose of an envelope is appropriately selected depending on the age and other conditions of a patient, the type of a disease, the type of the envelope employed, and the like. 25 When an envelope vector of the present in vention is administered into a human, from 400 HAU to 400,000 HAU of the envelope vector may be administered per subject, preferably 1,200 HAU to 120,000 HAU, and more preferably 4,000 HAU to 40,000 HAU. The amount of 30 an exogenous gene contained in an envelope to be admin istered may be from 2 pg to 2,000 pg per subject, pref erably from 6 jg to 600 pg per subject, and more pref erably from 20 pg to 200 pg.
38 AN010 As used herein, the term "HAU" refers to an amount of viral activity capable of agglutinating 0.5% of chicken red blood cells. 1 HAU corresponds to 5 24,000,000 virus particles (Okada Y. et al., Biken Journal, 4, 209-213, 1961). The above-described amount can be administered, for example, from once per day to several times per day. 10 An exact dose may be selected by an indi vidual practitioner in consideration of the patient to be treated. Doses and administration are adjusted to provide a sufficient level of activity or to maintain a desired effect. Further factors to be considered in 15 clude severity of a disease state (for example, size and position of a tumor; age, bodily weight and sex of a patient; diet regulation; period of time and fre quency of administration; combination of drugs; re sponse sensitivity; and resistant/response to the 20 treatment). Depending on the half-life of a specific formulation and clearance rate thereof, sustained release pharmaceutical compositions may be administered once per three to four days, weekly or bi-weekly. Guid ance as to a specific dose and mode of delivery are 25 provided in references known in the art. The present invention may also comprise a biocompatible material as a composition and medicament. The biocompatible material may comprise at least one 30 selected from the group consisting of silicone, colla gen, gelatin, glycolic acid/lactic acid copolymer, eth ylene/vinyl acetate copolymer, polyurethane, polyethyl ene, polytetrafluoroethylene, polypropylene, polyacry- 39 AN010 late, and polymethacrylate. Silicone is preferable be cause it is easy to mold. Examples of biodegradable macromolecules include, but are not limited to, poly mers, copolymers or mixtures thereof, which are synthe 5 sized by noncatalyzed hydration of at least one se lected from the group consisting of collagen, gelatin, a-hydroxycarboxylic acids (e.g., glycolic acid, lactic acid, hydroxybutyric acid, etc.), hydroxydicarboxylic acids (e.g., malic acid, etc.), and hydroxytricarbox 10 ylic acids (e.g., citric acid, etc.); polyacid anhy drides (e.g., poly-a-cyanoacrylic ester, polyamino acid (e.g., poly-y-benzyl-L-glutamic acid, etc.); maleic an hydride-based copolymers (e.g., styrene/maleic acid co polymer, etc.); and the like. The manner of polymeriza 15 tion may be any of random, block, and graft polmeriza tion. When a-hydroxycarboxylic acids, hydroxydicarbox ylic acids, or hydroxytricarboxylic acids have an opti cally active center within a molecule, any of D-isomers, L-isomers, and DL-isomers can be used. Preferably, gly 20 colic acid/lactic acid copolymers may be used. The composition and medicament of the pre sent invention may be provided in a sustained-release form. Any sustained-released dosage form may be used in 25 the present invention. Examples of sustained-release dosage forms include, but are not limited to, rod-like formulations (e.g., pellet-like, cylinder-like, needle like formulations, etc.), tablet formulations, disk like formulations, sphere-like formulations, sheet-like 30 formulations, and the like. Methods for preparing sus tained-release dosage forms are well known in the art, as described in, for example, the Japanese Pharmacopeia, the U.S. Pharmacopeia, Pharmacopeias of other countries, 40 AN010 and the like. Examples of a method for producing sus tained-release drugs include, but are not limited to, a method using disaggregation of a drug from a complex, a method for preparing an aqueous suspension of liquid 5 drug, a method for preparing an oil injection solution or oil suspended injection solution, a method for pre paring an emulsified injection solution (o/w or w/o type emulsified injection solution, or the like), and the like. 10 The use of the composition and medicament of the present invention is usually performed under the supervision of a doctor, or without supervision of a 15 doctor if approved by the authorities and laws of a country in which the present invention is used. The present invention may also be provided in the form of a vaccine. A vaccine means an antigen in 20 any of various forms (e.g., protein, DNA, and the like) which is used to prevent (or treat) a certain type of disease (e.g., contagious diseases, infectious diseases, and the like). Attenuated live pathogens (live vaccine), inactive pathogens (or a part thereof), metabolites of 25 a pathogen (toxin, inactivated toxin (i.e., toxoid), or the like), DNA vaccines, or the like are used depending on the type of infection, transmission, epidemic, or the like. Vaccination cause the active development of immunity (humoral immunity, cell-mediated immunity, or 30 both) within the body of organisms (humans, livestock, and vectors) and prevents infection, transmission, epi demic, or the like caused by pathogens.
41 AN010 The vaccines of the present invention are not particularly limited to any dosage form, and are prepared in accordance with methods known in the art. Further, the vaccines of the present invention may be 5 in the form of an emulsion containing various adjuvants. The adjuvants aid sustenance of a high level of immu nity when the above-described HSV gene recombinant is used in a smaller dose than when it is used alone. Ex amples of the adjuvants include Freund's adjuvant (com 10 plete or incomplete), adjuvant 65 (including peanut oil, mannide monooleate and aluminum monostearate), and alu minum hydrate, aluminum phosphate or mineral gel such as alum. For vaccines for humans, or animals used as a food source, adjuvant 65 is preferable. For vaccines 15 for commercial animals, mineral gel is preferable. In addition to the above-described adju vants, the vaccines of the present invention may con tain one or more additives for preparation selected 20 from diluents, aroma chemicals, preservatives, excipi ents, disintegrants, lubricants, binders, surfactants, plasticizers, and the like. The administration routes of the vaccines 25 of the present invention are not particularly limited, but parenteral administration is preferred. For example, the vaccines are administered parenterally (e.g., in travenously, intraarterially, subcutaneously, intrader mal, intramuscularly or intraperitoneally). Preferably, 30 the vaccine of the present invention may be adminis tered via the carotid. The dose of the vaccines of the present 42 AN010 invention can be selected depending on various condi tions: what administration is intended; whether infec tion is primary or recurrent; the age and weight; con ditions of patients; the severity of disease; and the 5 like. When intended to treat diseases caused by recur rent infection, the dose of the vaccines of the present invention is preferably about 0.01 ng to 10 mg per kg body weight, and more preferably about 0.1 ng to 1 mg per kg body weight. 10 The number of administrations of the vac cines of the present invention varies depending on the above-described conditions, and is not necessarily de termined in the same manner. However, preferably, the 15 vaccines are repeatedly administered at intervals of days or weeks. Particularly, administration is con ducted at several times, or preferably about one to two times, at the interval of about 2 to 4 weeks. The num ber of administrations (administration time) is pref 20 erably determined by symptomatology or a fundamental test using antibody titer while monitoring the condi tions of the disease. Compositions (e.g., vaccines) are herein 25 provided for treating or preventing pathogen infections (e.g., viruses (e.g., HIV, influenza virus, rotavirus, and the like), or bacteria). Such compositions comprise at least one gene or protein of the pathogen. The ex ogenous gene preferably is full length but may be a 30 partial sequence as long as it contains at least an epitope capable of triggering immunity. The term "epi tope" as used herein refers to an antigenic determinant whose structure has been revealed. A method for deter- 43 AN010 mining an epitope is known in the art. Once the primary nucleic acid or amino acid sequence of a protein is provided, such epitopes can be determined by such a known routine technique. A useful epitope may have at 5 least a length of three amino acids, preferably, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, or at 10 least 25 amino acids. As used herein, the term "neutralizing an tibody" refers to an antibody which is involved in a reaction which neutralizes the biological activity of 15 an antigen, such as an enzyme, a toxin, a bacterium, a virus, or the like. The term "neutralizing reaction" refers to a reaction in which an antigen is bound to a neutralizing antibody, so that the activity of the an tigen and the antibody is eliminated or lowered. If a 20 vaccine is administered, a neutralizing antibody is produced and serves to get rid of pathogens. As used herein, the term "gene therapy" or "gene therapeutic method" refers to a method for treat 25 ing diseases caused by a damaged (or defective) gene by introducing a healthy or modified nucleic acid (e.g., DNA) to patients. Some gene therapies use the step of injecting a naked nucleic acid, though vectors are of ten used. A virus envelope of the present invention may 30 be used as such a vector. The present invention may be provided in the form of a kit comprising a composition and medica- 44 AN010 ment. The kit comprises a composition and medicament of the present invention; and instructions which provide guidance in administering the composition and medica ment. The instructions describe a statement indicating 5 an appropriate method for administering a composition or a medicament of the present invention. The instruc tions are prepared in accordance with a format defined by an authority of a country in which the present in vention is practiced (e.g., Health, Labor and Welfare 10 Ministry in Japan, Food and Drug Administration (FDA) in the U.S., and the like), explicitly describing that the instructions are approved by the authority. The in structions are a so-called package insert and are typi cally provided in paper media. The instructions are not 15 so limited and may be provided in the form of elec tronic media (e.g., web sites, electronic mails, and the like provided on the Internet). The amount of a composition and medicament 20 used in the process of the present invention can be easily determined by those skilled in the art with ref erence to the purpose of use, a target disease (type, severity, and the like), the patient's age, weight, sex, and case history, the form or type of the cell physio 25 logically active substance, and the like. The frequency of the treatment method of the present invention applied to a subject (or patient) is also determined by those skilled in the art with re 30 spect to the purpose of use, target disease (type, se verity, and the like), the patient's age, weight, sex, and case history, the progression of therapy, and the like. Examples of the frequency include once per day to 45 AN010 several months (e.g., once per week to once per month). Preferably, administration is performed once per week to month with reference to the progression. 5 A composition and medicament of the pre sent invention comprises a material or medical ingredi ent to be introduced into hosts. Such a material or medical ingredient may be a biological macromolecule. Preferably, such a biological macromolecule is selected 10 from the group consisting of a nucleic acid, a polypep tide, a sugar, a lipid, and a complex molecule thereof. Preferably, such a medical ingredient may be a nucleic acid encoding a polypeptide which is expressed in the host into which the ingredient is introduced. 15 A composition and medicament of the pre sent invention may comprise one or more additional medical ingredients. Such a medical ingredient may be contained in the pharmaceutical composition. Examples 20 of such a medical ingredient include, but are not lim ited to, those described below: central nerve system drugs (e.g., general anesthetics, sedative-hypnotics, anxiolytics, antiepi 25 leptics, anti-inflammatory agents, stimulants, antihyp notics, antiparkinson agents, antipsychotics, combina tion cold remedies, and the like); peripheral nerve agents (e.g., local anes 30 thetics, skeletal muscle relaxants, autonomic nerve agents, antispasmodic agents, and the like); sensory organ drugs (e.g., ophthalmologi- 46 AN010 cal agents, otorhinolaryngological agents, antidinics, and the like); circulatory organ drugs (e.g., cardioton 5 ics, antiarrhythmics, diuretics, antihypertensive agents, vasoconstrictors, vasodilators, antihyperli pemia agents, and the like); respiratory organ drugs (e.g., respiratory 10 stimulants, antitussives, expectorants, antitussive ex pectorants, bronchodilators, collutoriums, and the like); digestive organ drugs (e.g., stegnotics, 15 antiflatuents, peptic ulcer agents, stomachics, antac ids, cathartics, enemas, cholagogues, and the like); hormone agents (e.g., pituitary gland hor mone agents, salivary gland hormone agents, thyroid 20 gland hormone agents, accessory thyroid gland hormone agents, anabolic steroid agents, adrenal gland hormone agents, androgenic hormone agents, estrogen agents, progesterone agents, mixed hormone agents, and the like); 25 urogenital organ and anal drugs (e.g., urinary organ agents, genital organs agents, uteroton ics, hemorrhoid agents, and the like); 30 dermatologic drugs (e.g., dermatologic disinfectants, wound protecting agents, pyogenic dis ease agents, analgesics, antipruritics, astringents, antiphlogistics, parasitic skin disease agents, emol- 47 AN010 lients, hair agents, and the like); dental and oral agents; 5 drugs for other organs; vitamin agents (e.g., vitamin A agents, vitamin D agents, vitamin B agents, vitamin C agents, vitamin E agents, vitamin K agents, mixed vitamin 10 agents, and the like); nutritive agents (e.g., calcium agents, inorganic preparations, saccharide agents, protein amino acid preparations, organ preparations, infant 15 preparations, and the like); blood and body fluid drugs (e.g., blood substitute agents, styptics, anticoagulants, and the like); 20 dialysis drugs (e.g., kidney dialysis agents, peritoneal dialysis agents, and the like); other metabolic drugs (e.g., organ disease 25 agents, antidotes, antabuses, arthrifuges, enzyme preparations, diabetic agents, and others); cell activating agents (e.g., chlorophyll preparations, pigment agents, and the like); 30 tumor agents (e.g., alkylation agents, an timetabolites, antineoplastic antibiotic preparations, antineoplastic plant extract preparations, and the 48 AN010 like); radiopharmaceuticals; 5 allergy drugs (e.g., antihistamic agents, irritation therapy agents, non-specific immunogen preparations, and other allergy drugs, crude drugs and drugs based on Chinese medicine, crude drugs, Chinese medicine preparations, and other preparations based on 10 crude drug and Chinese medicine formulation); antibiotic preparations (e.g., acting on gram-positive bacteria, gram-negative bacteria, gram positive mycoplasmas, gram-negative mycoplasmas, gram 15 positive rickettsia, gram-negative rickettsia, acid fast bacteria, molds, and the like); chemotherapeutic agents (e.g., sulfa drugs, antitubercular agents, synthetic antimicrobial agents, 20 antiviral agents, and the like); biological preparations (e.g., vaccines, toxoids, antitoxins, leptospire antisera, blood prepa rations, biological test preparations, and other bio 25 logical preparations, and antiprotozoal drugs, anthelmintics, and the like). As used herein, molecular biological tech niques, biochemical techniques, and microbiological 30 techniques well known in the art are optionally used. These methods are described in, for example, Ausubel F.A., et al., editors (1988), "Current Protocols in Mo lecular Biology", Wiley, New York, NY; Sambrook J., et 49 AN010 al. (1987), "Molecular Cloning: A Laboratory Manual", 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Bessatsu Jikken Igaku, "Idenshi Donyu & Hatsugen-Kaiseki-Jikkenho" [Experimantal Medi 5 cine, Special Issue, "Experimental Methods for Gene In troduction & Expression Analysis", Yodo-sha, 1997; and the like. Viruses (e.g., HVJ) proliferated in fer 10 tilized chicken eggs by inoculating seed virus there into may be generally used. Alternatively, viruses pro liferated in a persistent infection line of cultured cells or tissues of a monkey or human is used (culture medium supplemented with a hydrolytic enzyme, such as 15 trypsin or the like). Alternatively, viruses prolifer ated in cultured cells which are infected with a cloned viral genome to elicit persistent infection may be used in the present invention. These mutant lines can also be used in the present invention. In addition, viruses 20 (e.g., HVJ, etc.), which can be obtained by other meth ods, can also be used. Recombinant HVJ (Hasan M.K. et al., Journal of General Virology, 78, 2813 to 2830, 1997; or Yonemitsu Y. et al., Nature Biotechnology, 18, 970 to 973, 2000) can be used. Any HVJ may be used. 25 The Z line (e.g., Accession No. ATCC VA 2388 or one commercially available from Charles River SPAFAS) or the Cantell line (e.g., Johnston M.D., J. Gen. Virol., 56, 175-184, 1981 or one commercially available from Charles River SPAFAS) are more desirable. 30 BEST MODE FOR CARRYING OUT THE INVENTION In one aspect, the present invention pro- 50 AN010 vides a system for introducing a biomolecule into a cell. The present system comprises 1) a biomolecule; 2) a viral envelope; and 3) a glycosaminoglycan. The pre sent invention revealed the effects of a glycosami 5 noglycan (for example, heparin) to improve introduction of a biomolecule into a cell by the use of a viral en velope. Although not wishing to be bound by any theory, it is now believed that addition of a glycosaminoglycan attains the effect of changing or weakening the 10 strength of a cell, thereby enhancing introduction of a biomolecule into a cell. Accordingly, a glycosaminogly can molecule used may be any glycosaminoglycan, and preferably a glycosaminoglycan having a glucosamine residue may be used, and more preferably a heparin may 15 be used. Although not wishing to be bound by any theory, it is now believed that a glycosaminoglycan is prefer able since it has a glucosamine as a residue, it has the effect of facilitating passage through blood-brain barrier. Among heparins used, heparins having high mo 20 lecular weight (for example, 10,000 kDa or greater, more preferably, 11,000 kDa or greater, still more preferably, 12,000 kDa or greater) are preferable, and more preferably, heparins having an average molecular weight of 12,000 to 15,000 kDa are used. However, hepa 25 rins having less than those molecular weight may be used. A degree of sulfation appears to have some effect on efficiency of delivery. Accordingly, heparins having a pharmaceutically acceptable degree of sulfation are preferable heparins for use. As described above, mole 30 cules other than heparins may be used. Such a molecule includes but is not limited to for example, hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, and mixtures and polymers 51 AN010 thereof. Although not wishing to be bound by any theory, it is believed that these molecules have sulfate resi dues, and thus facilitate delivery into the body. Such glycosaminoglycan is usually comprised in the system of 5 the present invention at 1U/ml. Preferably, such a gly cosaminoglycan is comprised of at least 5U/ml, more preferably of at least 10U/ml, still more preferably of at least 50 U/ml, most preferably of at least 100 U/ml in the system of the present application. 10 In one embodiment, the viral envelope used in the present invention is inactivated. Such inactiva tion includes but is not limited to inactivation by ul traviolet ray radiation, alkylation and the like. 15 In one embodiment, the viral envelope used in the present invention may be an envelope of an RNA virus. Preferably, such a viral envelope may be an en velope of a virus belonging to the Paramixovirus genus 20 (for example, HVJ, influenza virus), preferably, an HVJ envelope. A biomolecule to be introduced by the sys tem according to the present invention, may be any 25 molecule as described hereinabove, and usually includes a molecule selected from the group consisting of a nu cleic acid, polypeptide, lipid, sugar, and a complex molecule thereof. Preferably, such a biomolecule com prises a nucleic acid and polypeptide. In one embodi 30 ment, such a biomolecule comprises a nucleic acid. In another embodiment, such a biomolecule includes a poly peptide.
52 AN010 In a certain embodiment, a biomolecule to be introduced by the present invention is a nucleic acid encoding a gene selected from the group consisting of Vascular endothelial growth factor (VEGF), Fibro 5 blast growth factor (FGF), and Hepatocyte growth factor (HGF). In another embodiment, a molecule to be introduced by the present invention is a polypeptide 10 selected from the group consisting of Vascular endothe lial growth factor (VEGF), Fibroblast growth factor (FGF), and Hepatocyte growth factor (HGF). In a preferred embodiment, the glycosami 15 noglycan (for example, heparin), the biomolecule and the viral envelope may be comprised in the same compo sition. In this case, homogeneity thereof in the compo sition is not problematic. 20 In another preferred embodiment, the gly cosaminoglycan (for example, heparin), the biomolecule and the viral envelope may be comprised in different compositions. In this case, two compositions may be ad ministered concurrently or separately. Preferably, the 25 biomolecule is comprised in the viral envelope, since the biomolecule is therefore efficiently delivered. In another aspect, the present invention provides a method for introducing a biomolecule into a 30 cell, comprising the steps of: 1) administering to a cell a composition comprising a viral envelope and a biomolecule; and 2) administering a glycosaminoglycan to the cell. Preferable embodiments of biomolecules 53 AN010 (for example, nucleic acids or polypeptides), viral en velopes (for example, HVJ envelopes), and glycosami noglycan (for example, heparins) used in the present invention are described hereinabove. 5 In the present invention, the step of ad ministering a glycosaminoglycan (for example, heparin) can be simultaneous to, prior to, or after the step of administering the composition comprising the viral en 10 velope and the biomolecule. Preferably, the glycosami noglycan is administered simultaneously or just before the administration of the composition. More preferably, the composition and the glycosaminoglycan are adminis tered simultaneously. When the simultaneous administra 15 tion is performed, glycosaminoglycans may or may not be contained in the composition. In another aspect, the present invention provides for the use of glycosaminoglycan for manufac 20 turing a medicament for introducing a biomolecule into a cell. In the present use, the claimed medicament com prises 1) a biomolecule; 2) a viral envelope; and 3) a glycosaminoglycan. Preferable embodiments of the bio molecule (for example, nucleic acid and polypeptide), a 25 viral envelope (for example, HVJ envelope) and a glyco saminoglycan (for example, heparin) are described here inabove. In another aspect, the present invention 30 provides a method for delivering a biomolecule to a brain. This method comprises the steps of: 1) tran siently occluding an artery of the head portion or cer vical portion; and 2) introducing a biomolecule into 54 AN010 the brain during the occluding of the artery of the head portion or the cervical portion. As used herein, it was an unexpected effect that the biomolecules can be introduced with high efficiency (for example, two 5 fold or greater) to the brain by transiently occluding an artery, since it was thought that the biomolecules cannot be introduced to the brain by the clear distinc tion of the blood vessel and the brain, due to the presence of the blood-brain-barrier (BBB). Methods for 10 transient occlusion include balloon cathethers, clip ping, cerebral infarction as a physiological occlusion, and the like. Preferably, balloon cathether and clip ping are used. As used herein, "transient" refers to a period of time sufficient for administering a bio 15 molecule (for example, at least one minute, 5 minutes and the like), and preferably 1-120 minutes. In one embodiment, a biomolecule may be any mole cule as described herein above, and preferably is se 20 lected from the group consisting of a nucleic acid, a polypeptide, a lipid, a sugar, and a complex molecule thereof. In a preferable embodiment, the biomolecule comprises a nucleic acid. 25 In a preferable embodiment, the bio molecule is a nucleic acid molecule encoding a gene se lected from the group consisting of vascularization factors such as vascular endhothelial growth factor (VEGF), fibroblast growth factor (FGF), hepatocyte 30 growth factor (HGF), HIF-1, DEL-1 and the like; an an tiapoptosis agent such as Bcl-2 and the like; a brain protective agent such as BDNF and the like; or antioxi dant agents such as Mn-SOD and the like.
55 AN010 In a preferable embodiment, the nucleic acid to be introduced is delivered by a vector. Such a vector optionally includes a transcription regulation 5 sequence such as a promoter, an enhancer, and the like. Preferably, a vector may be an expression vector. Con struction of a vector is well known in the art. In one embodiment, a biomolecule is intro 10 duced with an viral envelope. Preferably, the viral en velope to be used in the present invention is inacti vated. Inactivation reduces unwanted toxicity or the like. Inactivation may be achieved by any method in cluding but not limited to, UV radiation, use of alky 15 lating agent, and the like. In another embodiment, the viral envelope may be an envelope of an RNA virus. Preferably, the vi ral envelope used is an envelope of a virus belonging 20 to the Paramixovirus genus (for example, HVJ, influenza virus). Most preferably, the viral envelope used is an envelope of an HVJ. In a preferably embodiment, introduction 25 to a brain of a biomolecule may be performed together with a glycosaminoglycan. Glycosaminoglycan may be any molecule, and preferably, a heparin. Any glycosami noglycan may be used, and preferably the molecular weight of the glycosaminoglycan is at least 10,000 kDa, 30 more preferably, at least 11,000kDa, more preferably, at least 12,000 kDa. In a preferable embodiment, the molecular weight of a heparin is 12,000 to 15,000 kDa.
56 AN010 In one embodiment, glycosaminoglycan used is comprised of at least at 50 U/ml. As used herein, it is preferable that the 5 degree of sulfation of the glycosaminoglycan (for exam ple, heparin) is pharmaceutically acceptable. In one embodiment, the glycosaminoglycan may be administered simultaneously with, prior to, or 10 after the administration of the biomolecule. Preferably, the glycosaminoglycan and the biomolecule are adminis tered simultaneously. In a preferable embodiment, the artery of 15 the head portion or cervix portion is occluded for 1 to 120 minutes. In one embodiment, the artery of the head portion or the cervical portion is the middle cerebral 20 artery or the carotid artery. In a preferable embodi ment, the artery of the head portion or the cervical portion is the middle cerebral artery. In one embodiment, the biomolecule is ad 25 ministered into the carotid artery or the thalamus, in tracerebroventricularly or intrathecally. In a preferable embodiment, the bio molecule is administered into the carotid artery. The 30 carotid artery is preferable since it supplies blood to a wide range of the brain parenchyma. In another aspect, the present invention 57 AN010 provides a kit for delivering a biomolecule to a brain. The kit comprises 1) a biomolecule; and 2) an instruc tion indicating a method for administering the bio molecule the method comprising: A) transiently occlud 5 ing an artery of a head portion or a cervical portion; and B) introducing the biomolecule into the brain dur ing the occluding of the artery of the head portion or the cervical portion. A method for transient closure is described hereinabove. 10 The biomolecule to be included in a kit may be any molecule but preferably is selected from the group consisting of a nucleic acid, a polypeptide, a lipid, a sugar, and a complex molecule thereof. Pref 15 erably, the biomolecule comprises a nucleic acid. As used herein preferable biomolecules are a nucleic acid molecule encoding a gene selected from the group consisting of vascularization factors such as 20 vascular endhothelial growth factor (VEGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), HIF-1, DEL-1 and the like; an antiapoptosis agent such as Bcl-2 and the like; a brain protective agent such as BDNF and the like; an antioxidant agent such as Mn-SOD 25 and the like. The nucleic acid included in the kit is delivered by a vector. Vectors as used herein may be any one as long as the vector can express such a nu 30 cleic acid. Preferably, a vector is used which can ef ficiently express the nucleic acid in a mammal (pref erably human).
58 AN010 In a preferable embodiment, the kit of the present invention further comprises a viral envelope. The viral envelope is preferably inacti 5 vated so adverse effects may be avoided. Preferably, the viral envelope may be an envelope of an RNA virus. 10 More preferably, the viral envelope is an envelope of a virus belonging to the Paramixovirus ge nus (for example, HVJ, influenza virus, and the like). Most preferably, the viral envelope is an 15 HVG envelope. The kit of the present invention may fur ther comprise a glycosaminoglycan. 20 As used herein, the glycosaminoglycan used is preferable, but not limited to, heparin. In one embodiment, the molecular weight of the glycosaminoglycan used is at least 10,000 kDa, more 25 preferably at least 11,000 kDa, more preferably at least 12,000 kDa. More preferably, the molecular weight of heparin is 12,000-15,000 kDa. In one embodiment, the glycosaminoglycan 30 to be included in the kit of the present invention is comprised at least at 50U/ml. In a preferable embodiment, the degree of 59 AN010 sulfation of the glycosaminoglycan (for example, hepa rin) is pharmaceutically acceptable. In a preferable embodiment, the glycosami 5 noglycan is administered simultaneously with, prior to, or after the administration of the biomolecule. More preferably, the biomolecule and the glycosaminoglycan are administered simultaneously. 10 In a preferable embodiment of the kit of the present invention, the artery of the head portion or the cervical portion is occluded for 1 minute to 120 minutes. In a preferable embodiment, the artery of the head portion or the cervical portion is the middle 15 cerebral artery or the carotid artery. Further, the ar tery of the head portion or the cervical portion is the middle cerebral artery. In a preferable embodiment of the kit of 20 the present invention, the biomolecule is administered into the carotid artery or the thalamus, intracere broventricularly or intrathecally, but the present in vention is not limited thereto. 25 In a preferable embodiment of the kit of the present invention, the biomolecule is administered into the carotid artery. In another aspect, the present invention 30 provides for the use of a biomolecule for manufacturing a kit for delivering a biomolecule into the brain. The kit comprises A) the biomolecule; and B) an instruc tions for indicating a method for administering the 60 AN010 biomolecule the method comprising: a) transiently oc cluding an artery of a head portion or a cervical por tion; and b) introducing the biomolecule into the brain during the occluding of the artery of the head portion 5 or the cervical portion. As such, the present inventors have stud ied the possibility of gene delivery into the CNS using a viral envelope (for example, HVJ-E vector) both in 10 vitro and in vivo, in the present invention. As demon strated by the following Examples, when using a Venus reporter gene, fluorescence was able to be detected in a rat cerebral cortex neuron and a glial cell. By di rect injection into the thalamus, intracerebdovetricu 15 larly, or intrathecally, reporter genes (Venus or EGFP) were successfully transfected into rat brain without induction of immunological response. Gene expression was not observed after the intraartery injection via total cervical artery, however, when a vector was in 20 jected into the middle cerebral artery after transient closure, EGFP or luciferase activity was only detect able in the damaged hemisphere. Lastly, in order to in crease the transfectino efficiency, the effect of hepa rin was examined. Luciferase activity was significantly 25 increased by the addition of 50U/ml heparin (p<0.0 5 ). In conclusion, the HVJ-E vector is highly efficient for transfection of a gene into the CNS with out any evident toxicity. HVJ-E vector is useful for 30 examining the role of a number of genes and for treat ing cerebrovascular diseases. Hereinafter, the present invention is de- 61 AN010 scribed based on the Examples. The following examples are provided only for exemplary purposes. Accordingly, the scope of the present invention is not limited to the above description and the following examples, and 5 merely limited by the appended claims. EXAMPLES EXAMPLE 1: A method using HVJ 10 MATERIALS AND METHODS HVJ-Envelope vector Induction HVJ (Z strain) (10,000 hemagglutinating units) was mixed with plasmid DNA (200 pg) and 0.3% Triton-X. The mixture was washed with balanced salt so 15 lution (BSS; 137mM NaCl; 5.4mM KCI and 10mM Tris-HCl, pH 7.6). It was then suspended in 10 pl (for intracere bral administration), 100 pl (for intrathecal or in traartery administration), or 400 pl (for culturing cells) phosphate-buffered saline (PBS; pH7.5). For in 20 vitro studies, protamine sulfate (Nakalai tesque, Ja pan) was added to the culture plate 10 pg/well) before treatment with the vector to enhance the transfection efficiency. In the case of co-injection with heparin (Aventis, Japan), low molecular weight heparin (Frag 25 min@®, Kissei, Japan), or argatroban (Slonnon®, Daiichi, Japan), they were mixed with PBS and added to the vec tor. Plasmid DNA 30 pEGFP-C1 was purchased from Clontech (CA, USA). pCMV luciferase-GL3 (pcLuc-GL3: 7.4 kb) was constructed by cloning the luciferase gene from the pGL3-Promoter Vec tor (Promega, Madison, WI, USA) into pcDNA3 (5.4 kb) 62 AN010 (Invitrogen, San Diego, CA, USA) at the HindIII and BamHI sites. Plasmids were purified with the Qiagen plasmid isolation kit (Hilden, Germany). pCMV-LacZ (9.2 kb) was constructed by inserting the Hind III-Bam HI 5 fragment of pSV-p-galactosidase (Promega) into pcDNA3. Venus/pCS2 was kindly provided by Dr. Na gai (Laboratory for Cell Function and Dynamics, Ad vanced Technology Development Center, Brain Science In 10 stitute, RIKEN, Japan). (Determination of fluorescence due to Venus) Expression of Venus was examined under a fluorescent stereomicroscope 96 hours after injection. 15 More precise images were obtained with a confocal laser microscope (Bio-Rad, Hercules, CA, USA). (Assay for luciferase activity) Rats transfected with the luciferase gene 20 were sacrificed under anesthesia at 24 hours after transfection. Organ (brain, lung, spleen and liver) were harvested and placed individually in FALCON 50 ml tubes. The luciferase activity assay was performed as described previously [Brewer GJ, Torricelli JR, Evege 25 EK, Price PJ. , J Neurosci Res. 1993;35:567-76.]. Luciferase levels were normalized by determining the protein concentrations of the tissue extracts [Brewer GJ, Torricelli JR, Evege EK, Price PJ. , J Neurosci Res. 1993;35:567-76.]. Luciferase units were expressed as 30 relative light units (RLU) per gram of tissue protein. (In vitro gene transfer) Rat embryonic cerebral cortex neurons were 63 AN010 obtained from pregnant Wistar rats at 19 days gestation (Charles River Japan, Atsugi, Japan) and cultured [Be layev L, et al. , Stroke. 1996;27:1616-22; discussion 1623FF09]. Briefly, the cerebral cortex was dissected S and individual cells were isolated by treatment with papain and triturated in Leibovitz s L-15 medium (Invi trogen, CA, USA). Cells were cultured in poly-D-lysine coated 24-well plastic culture dishes with DMEM (Invi trogen)/B-27 (Invitrogen) at 37 0 C in a humidified at 10 mosphere of 95% air-5% CO 2 . The medium was changed on the first and fourth days. The rate of immuno-positive cells for MAP 2 (microtubule-associated protein) on the seventh day was 92.9%. Before transfection, the medium was changed to fresh 500 pl DMEM/well. HVJ-E vector 15 (250 HAU) containing the Venus gene was added to each well and left for 10 min at 37 'C. After transfection, the medium was changed to fresh DMEM/B-27 and the dishes were incubated at 37 'C. The expression of Venus was observed at 2 days after transfection using laser 20 scanning confocal microscopic images. Transfection ef ficiency (%) was calculated as (the number of cells ex pressing Venus/the number of NeuN immunoreactive cells) x 100. To average the efficiency, five visual fields were randomly selected and the number of cells was 25 counted. (Immunohistochemistry) In vitro cultured cells were fixed with 4% paraformaldehyde at 37 'C for 15 min and treated with 30 0.5% Triton X-100 for 10 min. The cells were blocked with PBS containing 2% goat serum, bovine serum albumin (5 mg/ml), and glycine (50 mM). Then, the cells were incubated with a mouse monoclonal antibody against MAP 2 64 AN010 (1:1000, Sigma-Aldrich, Saint Louis, MO, USA) or a mouse monoclonal antibody against GFAP (Glial Fibril lary Acidic Protein, 1:1000, Sigma-Aldrich) overnight at 4 OC. After washing with PBS, Alexa Fluor 546 5 conjugated goat anti-mouse IgG (Molecular Probes, Eugene, Oregon, USA) was applied as a secondary anti body and the dishes were incubated for 1 hour at room temperature. The image was analyzed with a confocal la ser microscope. 10 (In vivo gene transfer in normal rats) Wistar male rats (270-300 g; Charles River Japan, Atsugi, Japan) were used in the present inven tion. All procedures were conducted in accordance with 15 Osaka University guideline. Rats were anesthetized with ketamine (Sankyo, Japan) and placed in a stereotactic frame (Narishige Scientific Instrument Laboratory, To kyo, Japan), with the skull exposed. A stainless steel canula (30 gauge; Becton-Dickinson, Franklin Lakes, NJ) 20 with a specially designed Teflon connector (FEP tube, Bioanalytical Systems, West Lafayette, IN) was intro duced into the thalamus (3.8 mm posterior to the bregma, 2.4mm lateral to the midline, and 5.0mm below the skull surface) or lateral ventricle (0.48mm anterior to the 25 bregma, 0.8mm lateral to the midline, and 3.8mm below the skull surface). The HVJ-E vector containing the Ve nus gene or EGFP gene was injected at a speed of 1.0 p1i/min. After infusion, the infusion cannula was re moved. No behavioral change such as convulsion or ab 30 normal movement of extremities was observed in any ani mal. For infusion into the subarachnoid space, 65 AN010 the head of each animal was fixed in the prone position and the atlanto-occipital membrane was exposed through an occipitocerebral midline incision. A stainless steel cannula (27 gauge; Becton-Dickinson) was introduced 5 into cisterna magna (subarachnoid space). HVJ-E vector (100 Ipl) containing luciferase or Venus gene was in fused at a speed of 50 pl/min after removing 100 pl CSF. Then, the animals were placed head down for 30 min. For infusion into the common carotid artery, the left com 10 mon carotid artery, the left external carotid artery, and the left internal carotid artery were isolated via a midline incision under an operating microscope (Konan, Japan). The left common carotid artery and internal ca rotid artery were ligated temporally and a PE-50 cathe 15 ter (Clay Adams, Parsippany, NY, USA) was introduced into the left common carotid artery via a cutdown in the left external carotid artery. HVJ-E vector (100 pl) containing EGFP or luciferase gene was injected at a speed of 25 pl/min. After injection, the cannula was 20 removed and blood flow to the common carotid artery was restored by release of the ligatures. In each procedure, EGFP or Venus was observed at 4 days after transfection and luciferase activity was measured at 1 day after transfection. Luciferase activity in spleen, lung, and 25 liver was also measured at 1 day after intrathecal in jection. All rats showed no weight loss or loss of ac tivity after administration. To clarify the histologi cal change after administration of vector, HE staining of coronal section was performed at 14 days after in 30 traventricle and intrathecal injections. The coronal sections were made at +1.0 mm, -3.30 mm, -5.30 mm, 11.30 mm, and -14.60mm from the bregma.
66 AN010 (In vivo gene transfer after transient middle cerebral artery occlusion) To make the middle cerebral artery occlu sion model, the left middle cerebral artery was oc 5 cluded by placement of poly-L-lysine coated 4-0 nylon at the origin of MCA as described before [Belayev L et al., Stroke 27, 1616-1622 (1996)]. Briefly, animals were anesthetized with halothane (1-3.5% in a mixture of 70% N 2 0 and 30% 02) using a face mask. The rectal 10 temperature was maintained at 37±1 OC throughout the surgical procedure using a feedback regulated heating pad. Under the operating microscope the left common ca rotid artery, the left external carotid artery, and the left internal carotid artery were isolated via a mid 15 line incision. After 60 min, common carotid artery and internal carotid artery were transiently ligated and the 4-0 nylon was removed and reperfused for 10 minutes. The common carotid artery and the internal carotid ar tery were then transiently ligated again. The PE-50 20 catheter was placed at the common carotid artery from external carotid artery as described above, and the vector was injected at the speed of 10 pl/min after the release of ligation. After injection, the PE-50 cathe ter was removed and the external carotid artery was 25 ligated by 6-0 nylon. The expression of luciferase or EGFP was observed at 1 day or at 3 days after the infu sion. (RESULTS) 30 (Transfection into the cultured rat cere bral cortex cells) In order to develop an effective method for transferring a gene into the CNS, we transfected 67 AN010 the reporter gene (Venus gene) into cultured rat cere bral cortex cells (E19), since Venus reporter gene has been reported as an easily detected transfection method. It is reported that the use of Venus as an acceptor al 5 lows reliable early detection of fluorescence signals in brain slices [Nagai T, et al., Nat Biotechnol. 2002;20:87-90.]. At 2 days after transfection, the cul tured rat cerebral cortex cells demonstrated readily detectable fluorescence in cultured cells (Figures la, 10 id and 1g). Using immunohistochemical staining, the cells were immuno-positive for MAP 2 (microtubulue asso ciated protein 2; the neuronal marker), NeuN (neuron nuclear antigen; the neuron marker) or GFAP (glial fi bril acidic protein; the glial and astrocytic marker) 15 (Figures ic, if and li). The efficiency of transfection into neuronal cells as calculated from the number of Venus positive cells/the number of NeuN positive cells was 26.7±6.4%, while no positive cells could be de tected in cells transfected with a control vector. No 20 cell death was not observed after transfection using HVJ-E vector. (In vivo gene transfer into the brain) Then, we examined the transfection of Ve 25 nus gene into the brain. Initially, we injected the HVJ-E vector containing enhanced green fluorescence protein (EGFP) gene into the thalamus and lateral ven tricle. Stereotactic injection of EGFP gene into the thalamus showed the limited expression at the site of 30 injection (Figure 2a). Fluorescence could be detected at the choroids plexus and ependymal cells (Figures 2b and 2c) after stereotactic injection into the lateral ventricle. Unexpectedly, no fluorescent signal was de- 68 AN010 tected in neurons. On the other hand, no positive staining for fluorescence could be detected in the brain transfected with the control vector or in the un transfected brain. Then, we did injections into the su 5 barachnoid space. In this experiment, we used the Venus reporter gene instead of EGFP, since the use of the Ve nus reporter gene is an easily detectable transfection method. Injection into cerebral spinal fluid via cis terna magna caused widespread Venus expression in men 10 ingis, but less expression in chorioid plexus or neuron (Figures 3a and 3b). Importantly, HE staining of cor onal section at 3 days after intrathecal injection showed no inflammatory change. 15 (Gene transfer into CNS after transient middle cerebral artery occlusion) Considering the treatment of cerebral ischemic disease in the clinical setting, it seems best to employ infusion into the subarachnoid space rather 20 than injection into the lateral ventricle using a stereotactic frame. To further explore the feasibility of gene therapy using HVJ-E vector in cerebral ischemia, we examined gene transfer into CNS via the carotid ar tery. However, intraarterial infusion into the carotid 25 artery produced little expression of the transgene in the brain and microvascular endothelial cells at 3 and 7 days after injection. To overcome this issue, we hy pothesized that gene transfer after brain ischemia might show different transfection efficiencies into CNS, 30 since brain ischemia caused a change in the blood-brain barrier [Belayev L, Busto R, Zhao W, Ginsberg MD.,Brain Res. 1996;739:88-96. ; Kuroiwa T, Ting P, Martinez H, Klatzo I., Acta Neuropathol. 1985;68:122-9.; and 69 AN010 Neuann-Haefelin T, et al., Stroke. 2000;31:1965-72; discussion 1972-3.]. Thus, we infused HVJ-E vector in cluding the Venus gene into the carotid artery after transient middle cerebral cerebral artery occlusion for 5 60 min. Interestingly, fluorescence due to Venus could be detected at the infarcted cerebral cortex at 3 days after transient occlusion (Figure 4). The feasibility of transfection into CNS was confirmed by experiments using the luciferase gene. Luciferase activity at 24 10 hours after injection was much higher in infarcted hemisphere than that in contra lateral hemisphere (Fig ure 5, P < 0:05). On the other hand no luciferase ac tivity was detected in the spleen, lung and liver. 15 Finally, we investigated whether co administration of heparin would increase the transfec tion efficiency. After the making of HVJ-E vector con taining luciferase gene, heparin was added into the vector at concentrations of 10, 50 or 100 U/ml. The 20 HVJ-E vector was injected into CSF via the cisterna magna. As shown in Figure 6, luciferase activity was dramatically enhanced on the addition of 50 U/ml hepa rin (P < 0:05). To clarify the mechanism of the in crease in transfection efficiency with heparin, we ex 25 amined the effects of low molecular weight heparin (LMWH) at the concentrations of 1, 5, 10, 50, 100 or 200 U/ml, or argatroban at a concentration of 0.1 or 0.2 mg/ml. However, neither low molecular weight hepa rin nor argatroban affected the luciferase activity. 30 (Discussion) Previously, we have reported the efficacy of an HVJ-liposome transfection method for the CNS in 70 AN010 rats and in primates [Yamada K, et al. , Am J Physiol. 1996;271:R1212-20.; Hagihara Y, et al., Gene Ther. 2000;7:759-63.; and Hayashi K, et al., Gene Ther. 2001; 8:1167-73.]. The HVJ-liposome method utilized the com 5 bination of liposomes and fusion activities of proteins derived from the HVJ envelope [Kaneda Y, Saeki Y, Mor ishita R.,Mol Med Today. 1999;5:298-303.] for various cells and tissues. However, the procedure to make HVJ liposome vesicleis time-consuming and complex. In addi 10 tion, long-term storage be impossible. These issues might affect the usage of HVJ-liposome complex for hu man gene therapy. To overcome these problems, we have recently developed the second generation of HVJ vector, so-called HVJ-envelope (HVJ-E) vector. In order to pro 15 duce HVJ-E vector, a transgene was inserted into the envelope of HVJ after the viral genome thereof had been completely disrupted. The advantages of HVJ-E vector as compared to those of HVJ-liposome vector are: (1) it is easy to make, (2) the production does not take much 20 time (less than 90 min), and (3) that HVJ-E can be stored for a long period time (at least six months). In the present invention, we have demonstrated the possi bility of gene transfer into the CNS using the HVJ-E vector both in vivo and in vitro. In the in vitro study, 25 reporter genes were successfully expressed in neuron
(MAP
2 positive cells or NeuN positive cells) and astro glial cells (GFAP positive cells) without inducing cell death. With non-viral vector, mitotic cells are trans fected well, but non-mitotic cells such as quiescent 30 (Go) neurons are transfected poorly [Berry M, et al. , Curr Opin Mol Ther. 2001;3:338-49.]. However, in the present invention, reporter genes were successfully transfected into non-mitotic cells with the HVJ-E vec- 71 AN010 tor. Effective transfection can be attained due to the presence of a virus reporter against sialic acid, which is enriched on the surface of a neuron. 5 Importantly, the distribution of gene ex pression with HVJ-E vector was different from that with HVJ-liposome for in vivo gene transfer. By intrathecal injection, f-galactosidase gene expression was observed in cerebral parenchyma by HVJ-liposome method [Hagihara 10 Y, et al., Gene Ther. 2000; 7: 759-63. ; and Hayashi K, et al. , Gene Ther. 2001; 8: 1167-73.], while gene ex pression was detected only in the meningotheles and ad ventitial cells of artery using HVJ-E vector. Addition ally, intraventicle cationic-liposome-mediated (HVJ 15 cationic liposome mediated) gene transfer showed ex pression in cerebral parenchyma [Zou LL, et al.,Gene Ther. 1999; 6: 994-1005.], whereas HVJ-E vector mediated gene transfection revealed transgene expres sion only at the choroid plexus and ependymal cells. 20 Considering the fact that the direct injection of HVJ-E vector into thalamus resulted in successful transfec tion into cerebral parenchyma, the presence of liposome might be important to cross over the meninx or ependy mal cells. The present inventors and others have re 25 ported some gene transfer methods of high transfection efficiency for use in lateral ventricle administration using a stereotactice frame (Yoshimura S, et al. ,Hypertension. 2002;39:1028-34. ; and Yukawa H, et al. ,Gene Ther. 2000;7:942-9.), however, such technol 30 ogy is quite invasive. Luckily, the present inventors have found that transgene expression was observed on the brain surface after infusion into the cisterna magna. Further, the present invention clearly demon- 72 AN010 strates that gene transfer into the CNS is possible af ter transient middle cerebral artery via intraartery injection of the HVJ-E vector upon re-opening the ar tery. The destruction of the blood-brain barrier occlu 5 sion is controversial [Belayev L, Busto R, Zhao W, Ginsberg MD.,Brain Res. 1996;739:88-96. ;Kuroiwa T, Ting P, Martinez H, Klatzo I., Acta Neuropathol. 1985;68:122-9. ; and Neumann-Haefelin T, et al., Stroke. 2000;31:1965-72; discussion 1972-3.]. Kuroiwa et al. 10 demonstrated a biphasic opening of the blood brain bar rier after 1 hour of transient middle cerebral artery occlusion, occurring first at 15 min after the release of occlusion, then at 5 and 72 h of reperfusion (Kuroiwa T, Ting P, Martinez H, Klatzo I., Acta Neuro 15 pathol. 1985;68: 122-9.). Infusion of HVJ-E vector dur ing this limited time period would allow us to trans fect therapeutic genes into cerebral parenchyma via in traarterial means. It should be noted that co administration of heparin increased efficacy of trans 20 fection with HVJ-E vector. This result is similar to the report in which co-infusion with heparin using AAV vector induced higher and more homogeneous gene expres sion [Mastakov MY, Baer K, Kotin RM, During MJ., Mol Ther. 2002;5:371-80. 29. ; and Nguyen JB, Sanchez 25 Pernaute R, Cunningham J, Bankiewicz KS., Neuroreport. 2001;12:1961-4.]. It was predicted that heparin affects 2,3-linked sialic teoglyacid, a virus binding receptor, to enhance efficiency of tranfection, or heparin binds to the viral surface to limit interaction with HSPG on 30 the cellular surface. To clarify the mechanism, we ex amined the effects of low molecular weight heparin (LMWH) and similar substances such as argatroban. How ever, no increase in transfection efficiency was ob- 73 AN010 served using these molecules. The difference between conventional heparin (12,000-15,000 kDa) and LMWH (5000 kDa) is only the molecular size and the degree of sul fation [Ishai-Michaeli R, et al., Biochemistry. 5 1992;31:2080-8.]. These factors likely influence the interaction between HVJ-E vector and targeted cell sur face. Here, the present invention demonstrated potent 10 transfection efficiency using HVJ-E vector into the CNS in vivo as well as in vitro without any apparent toxic ity, while the site of gene expression was different among various administration routes. Successful gene transfection by intra-arterial injection after tran 15 sient arterial occlusion provides a promising approach for treatment of cerebral ischemia. In addition, to our knowledge, there has been no evidence of side effects. HVJ is not pathogenic to humans [Okada Y., Methods En zymol 1993;2211,18-41. ; and Okada Y, Tadokoro J., Exp 20 Cell Res 1962;26,108-118.) and is completely inacti vated by appropriate chemical modification without los ing fusion activity. In the present invention, in trathecal or intraventricular administration showed no loss of weight, no neurological deficits, and no in 25 flammatory change. Additionally, no luciferase activity was observed at any other organs after intrathecal in jection. Therefore, the HVJ-E vector seems to be safe for transfection into the brain. With respect to immu nogenicity, five rounds of repeated administrations of 30 plasmid DNA and antisense decoy DNA oligonucleotide (ODN) by HVJ-liposome method did not result in any loss of biological effects or production of antibodies against HVJ (Morishita R, Gibbons GH, Kaneda Y, Ogihara 74 AN010 T, Dzau VJ. ,Biochem Biophys Res Commun 2000; 273: 666 674 ; and Hirano T, et al., Gene Ther 1998; 5: 459-464.). For these reasons, HVJ-E vector may be a suitable gene transfer method for the treatment of cerebrovascular 5 disease. (EXAMPLE 2: Delivery using other viral vectors) (Preparation of influenza virus) Influenza virus belonging to the Orthomixovirus 10 family was obtained from fertilized chicken egg and propagated basically in accordance with WO96/05294. Briefly, it was performed as follows: fertilized eggs need to be carefully selected and obtained from spe cially secured healthy farms. The eggs are placed in an 15 incubator at 37.8 0 C (100 0 F) for from 9 days to 12 days. The egg is held to the light of a candle to observe the growth or survival of the embryo before an influenza virus is inoculated into the allantois. 20 Thereafter, in order to infect the egg with the virus under optimal conditions, the egg is cultured for from 2 days to 3 days in a culture incuba tor having controlled temperature and humidity. The conditions vary depending on the line and type of the 25 influenza virus used. The culture is rapidly cooled to 5±3 0 C to arrest the proliferation of the virus. There after, allantois liquid containing a large amount of virus particles is recovered from the infected egg. The thus-obtained allantois liquid containing the influenza 30 virus needs to be rapidly purified to remove impurities, such as proteins (e.g., ovalbumin, etc.), lecithin, bacteria, and the like. To achieve this, the recovered material is centrifuged to remove the supernatant, fol- 75 AN010 lowed by ultrafiltration to condense the material 20 fold before purification of the virus. (Alkylation process) 5 Immediately before use, 0.01% P-propiolactone was prepared in 10 mM KH 2 PO. This procedure was rapidly performed at low temperature. 0-propiolactone was added to the influenza 10 virus condensate solution obtained as mentioned above, followed by incubation on ice for 60 minutes. Thereaf ter, incubation was performed at 37 0 C for 2 hours. The resultant solution was dispensed into Eppendorf tubes, followed by centrifugation at 15,000 rpm for 15 minutes. 15 The precipitate was preserved at -20 0 C. Optionally, this influenza virus was ultrafiltered. Ultrafiltration using 500 KMWCO (A/G Tech nology, Needham, Massachusetts) was used to condense 20 the chorioallantoic fluid about 10-fold. 50 mM NaCl, 1 mM MgCl 2 , 2% mannitol, 20 mM Tris (pH 7.5) was used as buffered solution. The HA assay was used to achieve an influenza virus envelope recovery rate of substantially 100%. This is an excellent result. 25 Column chromatography was performed using Q-SepharoseFF (Amersham Pharmacia Biotech K.K., Tokyo) (buffered solution: 20 mM Tris-HCl (pH 7.5) buffer, from 0.2 M to 1 M NaCl)) to purify influenza virus en 30 velope. As a result, the recovery rate was from 40% to 50%, and the purity was 99% or more. As such, inactivated influenza virus enve- 76 AN010 lope was prepared from influenza virus containing chorioallantois liquid. Delivery into the brain using the influ 5 enza viral envelope was investigated. We have investi gated the possibility of gene transfer into the CNS us ing influenza virus envelope in vitro and in vivo as in Example 1. When using the Venus reporter gene, fluores cence could be detected in rat cerebral cortex neuron 10 and glial cells. In in vivo experiment, reporter genes (Venus or EGFP) were successfully transfected into rat brain by direct injection into the thalamus, intracere broventricular injection, or intrathecal injection without inducing immunological change. Gene expression 15 was not observed after intraartery injection via common carotid artery, however, when the vector was injected into the middle cerebral artery after transient occlu sion, EGFP or luciferase activities were only detected in the damaged hemisphere. Lastly, the effect of hepa 20 rin on the increase of transfection efficiency was ex amined. Luciferase activity was significantly increased by the addition of 50 U/ml. As such, it was demonstrated that not only 25 HVJ-E vector, but also vectors derived from HVJ-E vec tors significantly increased gene delivery efficiency into the brain after transient occlusion of cerebral artery, and the addition of heparin increased effi ciency of biomolecule delivery with influenza virus en 30 velope. (Example 3: a method using adeno-associated vi rus) 77 AN010 Next, adeno-associated virus was used to perform experiments similar to those of Examples 1 and 2. AAV Helper -Free System (Stratagene) was used as an adeno-associated virus. 5 The Alkylation process was performed as described in Example 2. Delivery into the brain using an adeno 10 associated virus envelope was investigated. We have in vestigated the possibility of gene transfer into the CNS using adeno-associated virus envelope in vitro and in vivo as in Example 1. When using the Venus reporter gene, fluorescence could be detected in rat cerebral 15 cortex neuron and glial cells. In in vivo experiment, reporter genes (Venus or EGFP) were successfully trans fected into rat brain by direct injection into the thalamus, intracerebroventricular injection, or in trathecal injection, without inducing immunological 20 change. Gene expression was not observed after intraar tery injection via common carotid artery, however, when the vector was injected into the middle cerebral artery after transient occlusion, EGFP or luciferase activi ties were only detected in the damaged hemisphere. 25 Lastly, the effect of heparin on the increase of trans fection efficiency was examined. Luciferase activity was significantly increased by the addition of 50 U/ml. (Example 4: Heparan sulfate) 30 Instead of heparin as in Examples 1-3, heparan sulfate (available from SEIKAGAKU INDUSTRY INC., and Sigma-Aldrich Japan) is used to demonstrate similar ef fects with the use of HVJ, influenza virus vector and 78 AN010 adeno-associated virus vector. As a result, when using heparan sulfate, it is confirmed that the gene delivery efficiency into the 5 brain is enhanced. Accordingly, it is demonstrated that glucosaminoglycan in general is effective in the pre sent invention. (Example 5: chondroitin sulfate) 10 Instead of heparin as in Examples 1-3, chondro itin sulfate (available from SEIKAGAKU INDUSTRY INC., and Sigma-Aldrich Japan) is used to demonstrate similar effects of the use of HVJ, influenza virus vector and adeno-associated virus vector. 15 As a result, when using chondroitin sul fate, it is confirmed that the gene delivery efficiency into the brain is enhanced. Accordingly, it is demon strated that glycosaminoglycans in general including 20 glucosaminoglycan and galactosaminoglycan are effective in the present invention. Although certain preferred embodiments have been described herein, it is not intended that 25 such embodiments be construed as limitations on the scope of the invention except as set forth in the ap pended claims. It should be understood that all patents, published patent applications and publications cited herein are incorporated by reference as if set forth 30 fully herein. INDUSTRIAL APPLICABILITY 79 AN010 A method was provided for efficient delivery of a biomolecule into the brain and central nervous system (CNS) using a viral envelope both in vitro and in vivo. Further, a system and method were provided for highly 5 efficient delivery of a biomolecule using a viral enve lope. Accordingly, the present invention provides a technology for efficiently administering a biomolecule into the nervous system, which provides industrial ap plicability in that such technology can be applied to 10 the administration of a variety of drugs. The present invention can be produced as a composition by not only a medical doctor but also, for example, pharmaceutical entities and the like. There 15 fore, it is believed that the present invention is fully industrially applicable or has full utility. The present invention is also industrially applicable since it is useful as a clinical trial for business purposes in addition to a therapeutic method for pure medical 20 purposes. Further, working of the method for treatment of the present invention in an indirect or direct man ner is believed to have possibilities for use in the businesses medical service-related, and thus the pre sent invention is industrially applicable or useful. 25

权利要求:
Claims (58)
[1] 2. The system according to Claim 1, wherein the vi ral envelope is inactivated.
[2] 3. The system according to Claim 1, wherein the gly 15 cosaminglycan is heparin.
[3] 4. The system according to Claim 1, wherein the mo lecular weight of the glycosaminoglycan is at least 10,000 kDa. 20
[4] 5. The system according to Claim 1, wherein the gly cosaminoglycan is comprised of at least 50 U/ml.
[5] 6. The system according to Claim 3, wherein the mo 25 lecular weight of said heparin is from 12,000 to 15,000 kDa.
[6] 7. The system according to Claim 3, wherein the de gree of sulfation of said heparin is pharmaceutically 30 acceptable.
[7] 8. The system according to Claim 1, wherein the vi ral envelope is an envelope of an RNA virus. 81 AN010
[8] 9. The system according to Claim 1, wherein the vi ral envelope is an envelope of a virus belonging to the Paramixovirus genus. 5
[9] 10. The system according to Claim 1, wherein the vi ral envelope is an envelope of HVJ.
[10] 11. The system according to Claim 1, wherein the bio 10 molecule is selected from the group consisting of a nu cleic acid, polypeptide, lipid, sugar, and a complex molecule thereof.
[11] 12. The system according to Claim 1 wherein the bio 15 molecule comprises a nucleic acid.
[12] 13. The system according to Claim 1 wherein the bio molecule comprises a polypeptide. 20 14. The system according to Claim 1 wherein the bio molecule is a nucleic acid encoding a gene selected from the group consisting of Vascular endothelial growth factor (VEGF), Fibroblast growth factor (FGF), and Hepatocyte growth factor (HGF). 25
[13] 15. The system according to Claim 1 wherein the bio molecule is a polypeptide selected from the group con sisting of Vascular endothelial growth factor (VEGF), Fibroblast growth factor (FGF), and Hepatocyte growth 30 factor (HGF).
[14] 16. The system according to Claim 1, wherein the heparin and the biomolecule and the viral envelope are 82 AN010 contained in the same composition.
[15] 17. The system according to Claim 1, wherein the heparin is contained in a different composition from 5 that which comprises the biomolecule and the viral en velope.
[16] 18. The system according to Claim 1 wherein the bio molecule is contained in the viral envelope. 10
[17] 19. A method for introducing a biomolecule into a cell, comprising the steps of: A) administering to a cell a composition compris ing a viral envelope and a biomolecule; and 15 B) administering a glycosaminoglycan to the cell.
[18] 20. The method according to Claim 19, wherein the step of administering the glycosaminoglycan is per formed simultaneously with the step of administering 20 the composition.
[19] 21. The method according to Claim 19, wherein the step of administering the glycosaminoglycan is per formed before the step of administering the composition. 25
[20] 22. The method according to Claim 19, wherein the step of administering the glycosaminoglycan is per formed after the step of administering the composition. 30 23. Use of a glycosaminoglycan for manufacturing a medicament for introducing a biomolecule into a cell, wherein the medicament comprises A) a biomolecule; 83 AN010 B) a viral envelope; and C) a glycosaminoglycan.
[21] 24. A method for delivering a biomolecule to a brain, 5 comprising the steps of: A) transiently closing an artery of the head portion or cervical portion; and B) introducing a biomolecule into the brain during the closing of the artery of the head por 10 tion or the cervical portion.
[22] 25. The method according to Claim 24, wherein the biomolecule is selected from the group consisting of a nucleic acid, polypeptide, lipid, sugar, and a complex 15 molecule thereof.
[23] 26. The method according to Claim 24, wherein the biomolecule is a nucleic acid. 20 27. The method according to Claim 24, wherein the biomolecule is a nucleic acid encoding a gene selected from the group consisting of Vascular endothelial growth factor (VEGF), Fibroblast growth factor (FGF), and Hepatocyte growth factor (HGF). 25
[24] 28. The method according to Claim 26, wherein the nu cleic acid is delivered by a vector.
[25] 29. The method according to Claim 26, wherein the 30 biomolecule is introduced with a viral envelope.
[26] 30. The method according to Claim 29, wherein the vi ral envelope is inactivated. 84 AN010
[27] 31. The method according to Claim 29, wherein the vi ral envelope is an envelope of a RNA virus. 5 32. The method according to Claim 29, wherein the vi ral envelope is an envelope of a virus belonging to the Paramixovirus genus.
[28] 33. The method according to Claim 29, wherein the vi 10 ral envelope is an envelope of HVJ.
[29] 34. The method according to Claim 24, wherein the biomolecule is introduced with a glycosaminoglycan. 15 35. The method according to Claim 34, wherein the glycosaminoglycan is heparin.
[30] 36. The method according to Claim 34, wherein the mo lecular weight of the glycosaminoglkycan is at least 20 10,000 kDa.
[31] 37. The method according to Claim 34, wherein the glycosaminoglycan is comprised of at least 50 U/ml. 25 38. The method according to Claim 35, wherein the mo lecular weight of said heparin is from 12,000 to 15,000 kDa.
[32] 39. The method according to Claim 35, wherein the de 30 gree of sulfation of said heparin is pharmaceutically acceptable.
[33] 40. The method according to Claim 34, wherein the 85 AN010 glycosaminoglycan is simultaneously administered with the biomolecule.
[34] 41. The method according to Claim 34, wherein the 5 glycosaminoglycan is administered before the admini stration of the biomolecule.
[35] 42. The method according to Claim 34, wherein the glycosaminoglycan is administered after the administra 10 tion of the biomolecule.
[36] 43. The method according to Claim 24, wherein the ar tery of the head portion or the cervical portion is closed for 1 minute to 120 minutes. 15
[37] 44. The method according to Claim 24, wherein the ar tery of the head portion or the cervical portion is the middle cerebral artery or the carotid artery. 20 45. The method according to Claim 24, wherein the ar tery of the head portion or the cervical portion is the middle cerebral artery.
[38] 46. The method according to Claim 24, wherein the 25 biomolecule is administered into the carotid artery or the thalamus, intracerebroventricularly or intrathe cally.
[39] 47. The method according to Claim 24, wherein the 30 biomolecule is administered into the carotid artery.
[40] 48. A kit for delivering a biomolecule into a brain, comprising: 86 AN010 A) a biomolecule; and B) an instruction indicatinga method for adminis tering the biomolecule wherein the method comprises: 1) transiently closing an artery of the 5 head portion or cervical portion; and 2) introducing the biomolecule into the brain during the closing of the artery of the head portion or the cervical portion. 10 49. The kit according to Claim 48, wherein the bio molecule is selected from the group consisting of a nu cleic acid, polypeptide, lipid, sugar, and a complex molecule thereof. 15 50. The kit according to Claim 48, wherein the bio molecule comprises a nucleic acid.
[41] 51. The kit according to Claim 48, wherein the bio molecule is a nucleic acid encoding a gene selected 20 from the group consisting of Vascular endothelial growth factor (VEGF), Fibroblast growth factor (FGF), and Hepatocyte growth factor (HGF).
[42] 52. The kit according to Claim 48, wherein the nu 25 cleic acid is delivered by a vector.
[43] 53. The kit according to Claim 48, further comprising a viral envelope. 30 54. The kit according to Claim 53, wherein the viral envelope is inactivated.
[44] 55. The kit according to Claim 53, wherein the viral 87 AN010 envelope is an envelope of an RNA virus.
[45] 56. The kit according to Claim 53, wherein the viral envelope is an envelope of a virus belonging to the Pa 5 ramixovirus genus.
[46] 57. The kit according to Claim 53, wherein the viral envelope is an envelope of HVJ. 10 58. The kit according to Claim 48, further comprising a glycosaminoglycan.
[47] 59. The kit according to Claim 58, wherein the glyco saminoglycan is heparin. 15
[48] 60. The kit according to Claim 58, wherein the mo lecular weight of the glycosaminoglycan is at least 10,000 kDa. 20 61. The kit according to Claim 58, wherein the glyco saminoglycan is comprised of at least 50 U/ml.
[49] 62. The kit according to Claim 59, wherein the mo lecular weight of the heparin is from 12,000 to 15,000 25 kDa.
[50] 63. The kit according to Claim 59, wherein the degree of sulfation of said heparin is pharmaceutically ac ceptable. 30
[51] 64. The kit according to Claim 58, wherein the glyco saminoglycan is simultaneously administered with the biomolecule. 88 AN010
[52] 65. The kit according to Claim 58, wherein the glyco saminoglycan is administered before the administration of the biomolecule. 5
[53] 66. The kit according to Claim 58, wherein the glyco saminoglycan is administered after the administration of the biomolecule. 10 67. The kit according to Claim 48, wherein the artery of the head portion or the cervical portion is closed for 1 minute to 120 minutes.
[54] 68. The kit according to Claim 48, wherein the artery 15 of the head portion or the cervical portion is the mid dle cerebral artery or the carotid artery.
[55] 69. The kit according to Claim 48, wherein the artery of the head portion or the cervical portion is the mid 20 dle cerebral artery.
[56] 70. The kit according to Claim 48, wherein the bio molecule is administered into the carotid artery or the thalamus, intracerebroventricularly or intrathecally. 25
[57] 71. The kit according to Claim 48, wherein the bio molecule is administered into the carotid artery.
[58] 73. Use of a biomolecule for manufacturing a kit for 30 delivering the biomolecule into a brain, the kit comprising: A) the biomolecule; and B) an instruction indicating a method for admin- 89 AN010 istering the biomolecule wherein the method comprises: a) transiently closing an artery of the head portion or cervical portion; and 5 b) introducing the biomolecule into the brain during the closing of the artery of the head portion or the cervical portion.

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法律状态:
2005-06-09| DA3| Amendments made section 104|Free format text: THE NATURE OF THE AMENDMENT IS AS SHOWN IN THE STATEMENT(S) FILED 02 MAR 2005 |

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2009-03-19| MK4| Application lapsed section 142(2)(d) - no continuation fee paid for the application|

优先权:

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申请号 | 申请日 | 专利标题

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JP2002247812||2002-08-27||

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JP2002-247812||2002-08-27||

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PCT/JP2003/010675|WO2004035779A1|2002-08-27|2003-08-22|Biomolecule transfer method using virus envelope and composition and system therefor|

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