U.S. patent application number 11/630522 was filed with the patent office on 2008-02-07 for anticancer agent containing minus-strand rna virus.
This patent application is currently assigned to DNAVEC RESEARCH INC.. Invention is credited to Mamoru Hasegawa, Haruhiko Kondo, Shinji Okano, Satoko Shibata, Katsuo Sueishi, Yoshikazu Yonemitsu.
Application Number | 20080031855 11/630522 |
Document ID | / |
Family ID | 37866022 |
Filed Date | 2008-02-07 |
United States Patent
Application |
20080031855 |
Kind Code |
A1 |
Okano; Shinji ; et
al. |
February 7, 2008 |
Anticancer Agent Containing Minus-Strand Rna Virus
Abstract
Minus-strand RNA viruses were found to have an effective
tumor-suppressive effect even when they did not carry a
therapeutically effective gene. The present invention provides
anticancer agents comprising a minus-strand RNA virus. Furthermore,
the present invention provides methods for producing the anticancer
agents, which comprise the step of mixing a minus-strand RNA virus
with pharmaceutically acceptable carriers. Furthermore, the present
invention provides methods for suppressing cancer, which comprise
the step of administering a minus-strand RNA virus to cancer
tissues.
Inventors: |
Okano; Shinji; (Fukuoka-shi,
JP) ; Yonemitsu; Yoshikazu; (Chiba-shi, JP) ;
Sueishi; Katsuo; (Fukuoka-shi, JP) ; Shibata;
Satoko; (Fukuoka-shi, JP) ; Hasegawa; Mamoru;
(Tsukuba-shi, JP) ; Kondo; Haruhiko; (Fukuoka-shi,
JP) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Assignee: |
DNAVEC RESEARCH INC.
Tsukuba-shi, Ibaraki
JP
305-0856
|
Family ID: |
37866022 |
Appl. No.: |
11/630522 |
Filed: |
April 28, 2005 |
PCT Filed: |
April 28, 2005 |
PCT NO: |
PCT/JP05/08124 |
371 Date: |
August 24, 2007 |
Current U.S.
Class: |
424/93.6 |
Current CPC
Class: |
C12N 2760/18871
20130101; A61K 2039/5154 20130101; A61K 35/76 20130101; C12N
2760/18832 20130101; A61K 38/179 20130101; A61P 31/00 20180101;
A61K 38/215 20130101; A61P 43/00 20180101; A61P 13/08 20180101;
A61P 35/00 20180101; A61K 35/768 20130101 |
Class at
Publication: |
424/093.6 |
International
Class: |
A61K 35/00 20060101
A61K035/00; A61P 43/00 20060101 A61P043/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 24, 2004 |
JP |
2004-187028 |
Oct 29, 2004 |
JP |
PCT/JP2004/016089 |
Claims
1. An anticancer agent comprising a minus-strand RNA virus.
2. The anticancer agent of claim 1, wherein the minus-strand RNA
virus does not encode a foreign protein.
3. The anticancer agent of claim 1, wherein the minus-strand RNA
virus is an infectious or a non-infectious virion.
4. The anticancer agent of claim 1, wherein the RNA virus is a
genomic RNA-protein complex.
5. The anticancer agent of claim 1, wherein the minus-strand RNA
virus is a paramyxovirus.
6. The anticancer agent of claim 5, wherein the paramyxovirus is a
Sendai virus.
7. A method for producing an anticancer agent, comprising the step
of mixing a minus-strand RNA virus with a pharmaceutically
acceptable carrier or medium.
8. A method for suppressing cancer, comprising the step of
administering a minus-strand RNA virus to a cancer tissue.
9. The anticancer agent of claim 2, wherein the minus-strand RNA
virus is an infectious or a non-infectious virion.
10. The anticancer agent of claim 2, wherein the RNA virus is a
genomic RNA-protein complex.
11. The anticancer agent of claim 3, wherein the RNA virus is a
genomic RNA-protein complex.
12. The anticancer agent of claim 9, wherein the RNA virus is a
genomic RNA-protein complex.
13. The anticancer agent of claim 2, wherein the minus-strand RNA
virus is a paramyxovirus.
14. The anticancer agent of claim 3, wherein the minus-strand RNA
virus is a paramyxovirus.
15. The anticancer agent of claim 4, wherein the minus-strand RNA
virus is a paramyxovirus.
16. The anticancer agent of claim 9, wherein the minus-strand RNA
virus is a paramyxovirus.
17. The anticancer agent of claim 10, wherein the minus-strand RNA
virus is a paramyxovirus.
18. The anticancer agent of claim 11, wherein the minus-strand RNA
virus is a paramyxovirus.
19. The anticancer agent of claim 12, wherein the minus-strand RNA
virus is a paramyxovirus.
20. The anticancer agent of claim 13, wherein the paramyxovirus is
a Sendai virus.
21. The anticancer agent of claim 14, wherein the paramyxovirus is
a Sendai virus.
22. The anticancer agent of claim 15, wherein the paramyxovirus is
a Sendai virus.
23. The anticancer agent of claim 16, wherein the paramyxovirus is
a Sendai virus.
24. The anticancer agent of claim 17, wherein the paramyxovirus is
a Sendai virus.
25. The anticancer agent of claim 18, wherein the paramyxovirus is
a Sendai virus.
26. The anticancer agent of claim 19, wherein the paramyxovirus is
a Sendai virus.
Description
TECHNICAL FIELD
[0001] The present invention relates to the field of cancer
therapy.
BACKGROUND ART
[0002] In recent years, clinical studies have been carded out on
virotherapy that targets advanced cancer and uses replicative
viruses. Virotherapy is a therapy that infects tumor cells with
replicative viruses, such as HSV-1 or adenovirus, and uses the
viral cytocidal effect that accompanies viral propagation for
treating tumor. In the case of HSV-1 or adenovirus, the replicative
viruses for tumor therapy are mutant viruses whose viral genome has
been genetically engineered, such that while their ability to
replicate in tumors is maintained, their pathogenicity in normal
human tissue is suppressed to a minimum. Therapeutic replicative
viruses infect tumor cells and replicate inside the cells, and the
infected cells die out in this process. The propagated viruses
reinfect the surrounding tumor cells, and spread the antitumor
effect (Alemany R. et al., Replicative adenoviruses for cancer
therapy. Nat. Biotechnol., 2000, 18:723-727; Curiel, D. T., The
development of conditionally replicative adenoviruses for cancer
therapy, Clin. Cancer Res., 2000, 6:3395-9; Kim, D., Virotherapy
for cancer: Current status, hurdles, and future directions, Cancer
Gene Therapy, 9:959-960, 2002; Mineta T. et al., Attenuated
multi-mutated herpes simplex virus-1 for the treatment of malignant
gliomas. Nat. Med. 1:938-943, 1995). Anticancer virotherapy has a
wide range of applications and provides excellent practicality: for
example, it can be used in combination with conventional therapy
such as surgery, radiation therapy, and chemotherapy; it can be
applied to solid cancers in general; it can be administered
repeatedly; and it can be made to directly incorporate into the
viral genome therapeutic genes such as those of cytokines to
enhance antitumor effect. Development of a more effective
virotherapy is expected to contribute greatly to cancer therapy.
[0003] Non-Patent Document 1: Alemany R. et al., Replicative
adenoviruses for cancer therapy. Nat Biotechnol., 2000, 18:723-727.
[0004] Non-Patent Document 2: Curiel, D. T., The development of
conditionally replicative adenoviruses for cancer therapy, Clin
Cancer Res., 2000, 6:3395-9. [0005] Non-Patent Document 3: Kim, D.,
Virotherapy for cancer. Current status, hurdles, and future
directions, Cancer Gene Therapy, 2002, 9:959-960. [0006] Non-Patent
Document 4: Mineta T. et al., Attenuated multi-mutated herpes
simplex virus-1 for the treatment of malignant gliomas. Nat Med,
1995, 1:938-943.
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
[0007] An objective of the present invention is to provide a more
effective anticancer virotherapy and anticancer agents. More
specifically, the present invention provides methods for producing
anticancer agents comprising a minus-strand RNA virus, and cancer
therapeutic methods using a minus-strand RNA virus.
Means to Solve the Problems
[0008] To verify the therapeutic effect of in vivo administration
of RNA virus on tumor, the present inventors performed experiments
using mice subjected to ventral subcutaneous inoculation with
melanoma cell lines. As a result, the present inventors found that
tumor size is significantly reduced when minus-strand RNA virus
that does not carry any therapeutic gene is injected into the tumor
of the animals. Therefore, this revealed that in vivo
administration of a minus-strand RNA virus exhibits antitumor
effects even when the virus does not carry any therapeutic
gene.
[0009] Minus-strand RNA viruses are considered to be effective
anticancer agents themselves. Moreover, direct injection of such
viruses into tumor sites (cancer tissues) is expected to yield
effective antitumor effects, and such viruses are useful for
treating cancer.
[0010] Therefore, the present invention relates to antitumor agents
comprising a minus-strand RNA virus, methods for producing the
antitumor agents, and methods for suppressing cancer using the RNA
virus, and more specifically, it relates to inventions described in
each claim. An invention or a combination of inventions set forth
in a claim(s) citing the same claim is already included in
inventions described in the claims. More specifically, the present
invention relates to: [0011] [1] an anticancer agent composing a
minus-strand RNA virus; [0012] [2] the anticancer agent of [1],
wherein the minus-strand RNA virus does not encode a foreign
protein; [0013] [3] the cancer agent of [1] or [2], wherein the
minus-strand RNA virus is an infectious or a non-infections virion;
[0014] [4] the anticancer agent of any one of [1] to [3], wherein
the RNA virus is a genomic RNA-protein complex; [0015] [5] the
anticancer agent of any one of [1] to [4], wherein the minus-strand
RNA virus is a paramyxovirus; [0016] [6] the anticancer agent of
[5], wherein the paramyxovirus is a Sendai virus; [0017] [7] a
method for producing an anticancer agent, comprising the step of
mixing a minus-strand RNA virus with a pharmaceutically acceptable
carrier or medium; and [0018] [8] a method for suppressing cancer,
comprising the step of administer a minus-strand RNA virus to a
cancer tissue.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 depicts the therapeutic effects of in vivo
administration of a GFP-expressing SeV, a soluble FGF
receptor-expressing SeV, or a soluble PDGFR.alpha.-expressing SeV
on melanoma.
[0020] FIG. 2 depicts the growth curve of B16 melanoma cells that
were inoculated subcutaneously.
[0021] FIG. 3 depicts the results of .sup.51Cr release assay for
YAC-1 target cells.
[0022] FIG. 4 depicts the results of .sup.51Cr release assay for
TRP2 peptide +EL-4.
BEST MODE FOR CARRYING OUT THE INVENTION
[0023] The present invention provides anticancer agents
(carcinostatic agents) comprising a minus-strand RNA virus as an
active component. In the present invention, an RNA virus refers to
a virus carrying an RNA genome.
[0024] The RNA virus that works as an active component of the
anticancer agents of the present invention is preferably a
minus-strand RNA virus. A minus-strand RNA virus refers to viruses
that include a minus strand (an antisense strand corresponding to a
sense strand encoding viral proteins) RNA as the genome. The
minus-strand RNA is also referred to as negative strand RNA. The
minus-strand RNA virus used in the present invention particularly
includes single-stranded minus-strand RNA viruses (also referred to
as non-segmented minus-strand RNA viruses). The "single-strand
negative strand RNA virus" refers to viruses having a
single-stranded negative strand [i.e., a minus strand] RNA as the
genome. Such viruses include viruses belonging to Paramyxoviridae
(including the genera Paramyxovirus, Morbillivirus, Rubulavirus,
and Pneumovirus), Rhabdoviridae (including the genera
Vesiculovirus, Lyssavirus, and Ephemerovirus), Filoviridae,
Orthomyxoviridae, (including Influenza viruses A, B, and C, and
Thogoto-like viruses), Bunyaviridae (including the genera
Bunyavirus, Hantavirus, Nairovirus, and Phlebovirus), Arenaviridae,
and the like.
[0025] A minus-strand RNA virus preferably used in the context of
the preset invention includes, for example, Sendai virus belonging
to Paramyxoviridae. Other examples include Newcastle disease virus,
mumps virus, measles virus, respiratory syncytial virus (RS virus),
rinderpest virus, distemper virus, simian parainfluenza virus
(SV5), and human parainfluenza viruses 1, 2, and 3; influenza virus
belonging to Orthomyxoviridae; and vesicular stomatitis virus and
rabies virus belonging to Rhabdoviridae.
[0026] Further examples of virus that may be used in the context of
the present invention include: Sendai virus (SeV), human
parainfluenza virus-1 (HPIV-1), human parainfluenza virus-3
(HPIV-3), phocine distemper virus (PDV), canine distemper virus
(CDV), dolphin molbillivirus (DMV), peste-des-petits-ruminants
virus (PDPR), measles virus (MV), rinderpest virus (RPV), Hendra
virus (Hendra), Nipah virus (Nipah), human parainfluenza virus-2
(HPIV-2), simian parainfluenza virus 5 (SV5), human parainfluenza
virus-4a (HPIV-4a), human parainfluenza virus-4b (HPIV-4b), mumps
virus (Mumps), and Newcastle disease virus (NDV). A more preferred
example is a virus selected from the group consisting of Sendai
virus (SeV), human parainfluenza virus-1 (HPIV-1), human
parainfluenza virus-3 (HPIV-3), phocine distemper virus (PDV),
canine distemper virus (CDV), dolphin molbillivirus (DMV),
peste-des-petits-ruminants virus (PDPR), measles virus (MV),
rinderpest virus (RPV), Hendra virus (Hendra), and Nipah virus
(Nipah).
[0027] More preferably, viruses of the present invention are
preferably those belonging to Paramyxoviridae (including
Respirovirus, Rubulavirus, and Morbillivirus) or derivatives
thereof, and more preferably those belonging to the genus
Respirovirus (also referred to as Paramyxovirus) or derivatives
thereof. The derivatives include viruses that are
genetically-modified or chemically-modified in a manner not to
impair their gene-transferring ability. Examples of viruses of the
genus Respirovirus applicable to this invention are human
parainfluenza virus-1 (HPIV-1), human parainfluenza virus-3
(HPIV-3), bovine parainfluenza virus-3 (HPIV-3), Sendai virus (also
referred to as murine parainfluenza virus-1), and simian
parainfluenza virus-10 (HPIV-10).
[0028] The minus-strand RNA viruses of the present invention are
most preferably Sendai viruses.
[0029] The minus-strand RNA viruses of the present invention may be
derived from natural stains, wild-type strains, mutant strains,
laboratory-passaged strains, artificially constructed strains, or
such. More specifically, the minus-strand RNA viruses may be
minus-strand RNA viruses isolated from nature, or minus-strand RNA
viruses artificially generated by genetic recombination.
Furthermore, as long as the ability to replicate genomic RNA in
infected cells is maintained, any of the genes carried by wild-type
minus-strand RNA viruses can have mutations or deletions. For
example, minus-strand RNA viruses comprising a mutation or deletion
in at least an envelope protein or coat protein-encoding gene of
the minus-strand RNA viruses can be preferably used. Such minus
strand RNA viruses can replicate the RNA genome in infected cells
but cannot form infectious virions. Since there is no need to be
concerned about spread of infection to the surrounding, the viruses
are very safe. For example, a minus-strand RNA virus that does not
comprise at least one or a combination of the genes encoding spike
proteins or envelope proteins such as F, H, HN, or G can be used
(WO00/70055 and WO00/70070; Li, H. --O. et al., J. Virol. 74(14)
6564-6569 (2000)). A genome can be replicated in the infected cells
if proteins that are necessary for genome replication (for example
N, P, and L proteins) are encoded supplying to virus-producing
cells, products of the deficient genes or proteins which can
complement the deficient genes (WO00/70055 and WO00/70070; Li, H.
--O. et al., J. Virol. 74(14) 6564-6569 (2000)). However, for
example, among minus-strand RNA viruses, viruses carrying the M
protein gene release non-infectious virions (VLP) even if they do
not carry genes encoding spike proteins such as F protein or HN
protein; therefore, VLP can be produced without complementing the
viral proteins (WO00/70070). Furthermore, RNP comprising genomic
RNA and the N, L, and P proteins can be amplified in cells even if
they do not have any envelope protein genes; therefore, RNP can be
collected from cell lysates by centrifugation and such.
[0030] Moreover, the anticancer agents of the present invention can
be produced using mutant RNA viruses. For example, many
temperature-sensitive mutations of envelope proteins and coat
proteins are known. RNA viruses comprising these
temperature-sensitive mutant protein genes can be preferably used
in the present invention. Temperature-sensitive mutations refer to
mutations that significantly decrease protein activity at normal
temperatures (for example, 37.degree. C. to 38.degree. C.) of the
virus' host, as compared with the activity at low temperatures (for
example, 30.degree. C. to 32.degree. C.). Such proteins harboring
temperature-sensitive mutations are useful because they enable
virus production at permissible temperatures (low
temperatures).
[0031] Examples of temperature-sensitive mutations of the M gene of
minis-strand RNA viruses include amino acid substitutions at a site
arbitrarily selected from the group consisting of G69, T116, and
A183 in the Sendai virus M protein (Inoue, M. et al., J. Virol.
2003, 77: 3238-3246). The amino acids of the homologous sites in
the M protein of other minus-strand RNA viruses can be identified
easily, and specifically, examples of homologous sites in M
proteins that correspond to G69 of the SeV M protein include (the
letter and number denote the amino acid and its position;
abbreviation of the names is shown inside the parentheses): G69 in
human parainfluenza virus-1 (HPIV-1); G73 in human parainfluenza
virus-3 (HPIV-3); G70 in phocine distemper virus (PDV) and canine
distemper virus (CDV); G71 in dolphin molbillivirus (DMV); G70 in
peste-des-petits-ruminants virus (PDPR), measles virus (MV), and
rinderpest virus (RPV); G81 in Hendra virus (Hendra) and Nipah
virus (Nipah); G70 in human parainfluenza virus-2 (HPIV-2); E47 in
human parainfluenza virus-4a (HPIV-4a) and human parainfluenza
virus-4b (HPIV-4b); and E72 in mumps virus (Mumps). Examples of M
protein homologous sites that correspond to T116 in the SeV M
protein include: T116 in human parainfluenza virus-1 (HPIV-1); T120
in human parainfluenza virus-3 (HPIV-3); T104 in phocine distemper
virus (PDV) and canine distemper virus (CDV); T105 in dolphin
molbillivirus (DMV); T104 in peste-des-petits-ruminants virus
(PDPR), measles virus(MV), and rinderpest virus (RPV); T120 in
Hendra virus (Hendra) and Nipah virus (Nipah); T117 in human
parainfluenza virus-2 (HPIV-2) and simian parainfluenza virus 5
(SV5); T121 in human parainfluenza virus-4a (HPIV-4a) and human
parainfluenza virus-4b (HPIV-4b); T119 in mumps virus (Mumps); and
S120 in Newcastle disease virus (NDV). Examples of M protein
homologous sites that correspond to A183 in the SeV M protein
include: A183 in human parainfluenza virus-1 (HPIV-1); F187 in
human parainfluenza virus-3 (HPIV-3); Y171 in phocine distemper
virus (PDV) and canine distemper virus (CDV); Y172 in dolphin
molbillivirus (DMV); Y171 in peste-des-petits-ruminants virus
(PDPR), measles virus (MV), and rinderpest virus (RPV); Y187 in
Hendra virus (Hendra) and Nipah virus (Nipah); Y184 in human
parainfluenza virus-2 (HPIV-2); F184 in simian parainfluenza virus
5 (SV5); F188 in human parainfluenza virus-4a (HPIV-4a) and human
parainfluenza virus-4b (HPIV-4b); F186 in mumps virus (Mumps); and
Y187 in Newcastle disease virus (NDV). Among the viruses recited
herein, viruses comprising a genome encoding, as their M protein, a
mutant M protein in which the amino acids of any one of the three
sites, preferably a combination of any two of these sites. More
preferably, all three sites mentioned above are substituted with
other amino acids are used in the present invention.
[0032] A preferred amino acid mutation is substitution to another
amino acid whose side chains have different chemical properties.
For example, substitution is carried out with an amino acid whose
BLOSUM62 matrix value (Henikoff, S. and Henikoff, J. G. (1992)
Proc. Natl. Acad. Sci. USA 89: 10915-10919) is 3 or less,
preferably 2 or less, more preferably 1 or less, and even more
preferably 0 or less. Specifically, G69, T116, and A183 of the
Sendai virus M protein or homologous sites of other viral M
proteins can be substituted wit Glu (E), Ala (A), and Ser (S),
respectively. It is also possible to use mutations that are
homologous to the mutations in the M protein of a
temperature-sensitive P253-505 measles virus strain (Morikawa, Y.
et al., Kitasato Arch. Exp. Med. 1991: 64; 15-30). Mutations can be
introduced, for example, by using oligonucleotides and such
according to well known methods for introducing mutations.
[0033] Examples of temperature-sensitive mutations of the HN gene
include amino acid substitutions at any of the sites selected from
the group consisting of A262, G264, and K461 in the Sendai virus HN
protein (Inoue, M. et al., J. Virol. 2003, 77: 3238-3246). In a
preferred example, A262, G264, and K461 in the Sendai virus HN
protein or homologous sites of other viral HN proteins are
substituted with Thr (T), Arg (R), and Gly (G), respectively.
Furthermore, for example, the amino acids at positions 464 and 468
in the HN protein can be mutated by referring to the
temperature-sensitive mumps virus vaccine strain, Urabe AM9
(Wright, K. E. et al., Virus Res. 2000: 67; 49-57).
[0034] Minus-strand RNA viruses may comprise mutations in the P
gene or L gene. Specific examples of such mutations include
mutation of Glu at position 86 (E86) of the SeV P protein,
substitution of Leu at position 511 (L511) of the SeV P protein to
another amino acid, or substitution of homologous sites in the P
protein of a different minus-strand RNA virus. Specific examples
include substitution of the amino acid at position 86 to Lys, and
substitution of the amino acid at position 511 to Phe. Regarding
the L protein, examples include substitution of Asn at position
1197 (N1197) and/or Lys at position 1795 (K1795) in the SeV L
protein to other amino acids, or substitution of homologous sites
in the L protein of another minus-strand RNA virus, and specific
examples include substitution of the amino acid at position 1197 to
Ser, and substitution of the amino acid at 1795 to Glu. Mutations
of the P gene and L gene can significantly increase the effects of
persistent infectivity, suppression of the release of secondary
virions, and suppression of cytotoxicity. Furthermore, by combining
mutation and/or deletion of the envelope protein gene, these
effects can be increased dramatically.
[0035] When using enveloped viruses, it is possible to use viruses
that comprise in their envelope, proteins that are different from
the envelope proteins originally carried by the viruses. For
example, by expressing a desired foreign envelope protein in the
virus-producing cells during virus production, viruses comprising
such protein can be produced. Such protein is not particularly
limited, and desired proteins that confer the ability to infect
mammalian cells are used. A specific example is the vesicular
stomatitis virus (VSV) G protein (VSV-G). The VSV-G protein may be
derived from any VSV strain, and for example, VSV-G protein derived
from the Indiana serotype strain (J. Virology 39: 519-528 (1981))
can be used, but it is not limited thereto. The minus-strand RNA
viruses used in the present invention can comprise an arbitrary
combination of envelope proteins derived from other viruses.
[0036] A characteristic of the minus-strand RNA viruses of the
present invention is that they themselves have anticancer effects.
Specifically, the minus-strand RNA viruses do not need to comprise
foreign proteins or nucleic acids encoding the proteins. Therefore,
the present invention relates to anticancer agents comprising
minus-strand RNA viruses that do not encode foreign proteins. The
minus-strand RNA viruses of the present invention may or may not
encode a foreign protein or a foreign gene in their genomic RNA.
Since minus-strand RNA viruses that do not encode any foreign
proteins also exhibit anticancer (carcinostatic) effects, foreign
genes are not necessarily required. Therefore, the present
invention is advantageous in hat a desired minus-strand RNA virus
such as wild-type minus-strand RNA virus, or minus-strand RNA virus
isolated from nature (including mutant strains) can be used. For
example, RNA viruses used in the present invention may be RNA
viruses that do not encode proteins with anticancer therapeutic
effects. Such viruses include RNA viruses encoding desired foreign
proteins that do not have any anticancer therapeutic effects. For
example, RNA viruses encoding marker proteins such as green
fluorescent protein (GFP), luciferase, or various peptide tags can
be used to detect the introduction of RNA viruses. Alternatively,
by further incorporating into the minus-strand RNA viruses foreign
proteins or foreign genes that assist anticancer (carcinostatic)
effects, the carcinostatic (anticancer) effects can also be further
enhanced.
[0037] Recombinant minus-strand RNA viruses that carry foreign
genes can be reconstituted using well-known methods. Specific
procedures for such production typically include the steps of (a)
transcribing a cDNA encoding the genomic RNA of a minus-strand RNA
virus in a cell expressing viral proteins necessary for virion
formation, and (b) collecting the culture supernatant comprising
the produced virus. The viral proteins can be expressed from the
transcribed viral genomic RNA, or they may be provided in trans
from sources other than the genomic RNA. When viral genes that are
required for virion formation are deficient from the genomic RNA,
these viral genes are separately expressed in the virus-producing
cells to complement virion formation. To express a viral protein or
RNA genome in cells, a vector, in which a DNA encoding the protein
or the genomic RNA is linked downstream of a suitable promoter that
functions in the host cells, is introduced into the host cells.
Transcribed genomic RNAs are replicated in the presence of viral
proteins, and infectious virions are formed. When producing
deficient viruses that lack envelope protein genes and such, the
deficient proteins, viral proteins that can complement their
functions, or such are expressed in the virus-producing cells.
[0038] For example, the minus-strand RNA viruses of the present
invention can be produced using the following known methods
(WO97/16539; WO97/16538; WO00/70055; WO00/70070; WO01/18223; Hasan,
M. K. et al., J. Gen. Virol. 78: 2813-2820, 1997; Kato, A. et al.,
1997, EMBO J. 16: 578-587; Yu, D. et al., 1997, Genes Cells 2:
457-466; Durbin, A. P. et al., 1997, Virology 235: 323-332; Whelan,
S. P. et al., 1995, Proc. Natl. Acad. Sci. USA 92: 8388-8392;
Schnell. M. J. et al., 1994, EMBO J. 13: 4195-4203; Radecke, F. et
al., 1995, EMBO J. 14: 5773-5784; Lawson, N. D. et at., Proc. Natl.
Acad. Sci. USA 92: 4477-4481; Garcin, D. et al., 1995, EMBO J. 14:
6087-6094; Kato, A. et al., 1996, Genes Cells 1: 569-579; Baron, M.
D. and Barrett, T., 1997, J. Virol. 71: 1265-1271; and Bridgen, A.
and Elliott, R. M., 1996, Proc. Natl. Acad. Sci. USA 93:
15400-15404). Using these methods, minus-strand RNA viruses
comprising parainfluenza virus, vesicular stomatitis virus, rabies
virus, measles virus, rinderpest virus, and Sendai virus can be
reconstituted from DNA. In the present invention, it is preferable
to use minus-strand RNA viruses, especially single-stranded
minus-strand RNA viruses, more preferably viruses of the
Paramyxoviridae family, and even more preferably viruses of the
genus Respirovirus.
[0039] There is no particular limitation on the foreign genes to be
carried by the minus-strand RNA viruses, and examples of naturally
occur proteins include hormones, cytokines, growth factors,
receptors, intracellular signaling molecules, enzymes, antibodies
(including full-length antibodies, antibody fragments such as Fab,
single-chain antibodies, etc), and peptides. The proteins may be
secretory proteins, membrane proteins, cytoplasmic proteins,
nuclear proteins, and such. Artificial proteins include, for
example, fusion proteins of chimeric toxins and such, dominant
negative proteins (including soluble receptor molecules or
membrane-bound dominant negative receptors), cell surface molecules
and truncated cell adhesion molecules. The proteins may also be
proteins to which a secretory signal, membrane-localization signal,
nuclear translocation signal, or such has been attached. The
function of a particular gene can be suppressed by expressing
antisense RNA molecules, RNA-cleaving ribozymes, or the like as
transgenes. Carcinostatic effects may be enhanced by preparing
viruses using a therapeutic gene showing carcinostatic effects as a
foreign gene.
[0040] For example, the use of a gene that inhibits vascularization
or angiogenesis can further enhance antitumor effects. Examples of
genes that are known to promote vascularization or angiogenesis
include fibroblast growth factor 2 (FGF2) (Baffour, R. et al., J.
Vasc. Surg. 16(2):181-91, 1992), endothelial cell growth factor
(FCGF) (Pu, L. Q. et al., J. Surg. Res. 54(6):575-83, 1993),
vascular endothelial growth factor (VEGF)/vascular permeability
factor (VPF) (Takeshita, S. et al., Circulation 90 (5 Pt
2):II228-34, 1994; Takeshita, S. et al., J. Clin. Invest.
93(2):662-70, 1994), and hepatocyte growth factor/scatter factor
(HGF/SF). Genes that encode secretory proteins which inhibit the
effect of these signal molecules can be used as foreign genes.
Specific examples include antibodies that bind to these signal
molecules or their rectors, or polypeptides comprising
antigen-binding fragments of such antibodies, or soluble forms of
such receptor proteins (secretory receptors that carry a ligand
binding site, but not transmembrane region). In particular,
minus-strand RNA viruses encoding soluble-type FGF receptor (FGF-R)
polypeptides can significantly increase the effects of cancer
growth suppression. Therefore, minus-strand RNA viruses that encode
soluble FGF-R can be preferably used in the present invention. For
the soluble FGF-R, naturally occurring soluble FGF-R may be used,
or fragments comprising an extracellular domain of membrane-bound
FGF-R (FGF-R1 and such) may be used (A. Hanneken and A. Baird,
Investigative Ophthalmology & Visual Science, Vol 36,
1192-1196, 1995; Takaishi, S. et al., Biochem. Biophys. Res.
Commun., 267(2):658-62, 2000; Seno M, et al., Cytokine,
10(4):290-4, 1998; and Hanneken, A., FEBS Lett. 489:176, 2001).
[0041] Furthermore, anticancer immune response is increased when
the immune system is stimulated upon expression of cytokines;
therefore, minus-strand RNA viruses into which cytokine-encoding
genes have been introduced are also useful. A minus-strand RNA
virus carrying a gene encoding an immunostimulatory cytokine will
serve as an effective anticancer agent having the activity to
induce tumor immunity. For example, immunostimulatory cytokines
comprise interleukins (for example, IL-1alpha, IL-1beta IL-2, IL-3,
IL-4, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-15, IL-18, IL-19,
IL-20, IL-21, IL-23, and IL-27), interferons (for example,
IFN-alpha, IFN-beta, and IFN-gamma), tumor necrosis factor (TNF),
transforming growth factor (TGF)-beta, granulocyte colony
stimulating factor (G-CSF), macrophage colony stimulating factor
(M-CSF), granulocyte macrophage colony stimulating factor (GM-CSF),
insulin-like growth factor (IGF)-I, IGF-2, Flt-3 ligand, Fas
ligand, c-kit ligand, and other immunomodulatory proteins (such as
chemokines and costimulatory molecules).
[0042] The amino acid sequences of these cytokines are well known
to those skilled in tie art. One may refer to: for IL-4, for
example, Arai et al. (1989), J. Immunol. 142(1) 274-282; for IL-6,
for example, Yasukawa et al. (1987), EMBO J., 6(10): 2939-2945; for
IL-12, for example, Wolf et al. (1991), J. Immunol. 146(9):
3074-3081; for IFN-alpha, for example, Gren et al. (1984) J.
Interferon Res. 4(4): 609-617, and Weismann et al. (1982) Princess
Takamatsu Symp. 12: 1-22. Examples of IFN-beta sequences include
the sequence from position 139 to 636 of Accession number
NM.sub.--002176 (corresponding to position 22 to 187 of the amino
acid sequence of NP.sub.--002167) (Derynck, R. et al., Nature 285,
542-547 (1980); Higashi, Y. et al., J. Bio. Chem. 258, 9522-9529
(1983); Kuga, T. et al., Nucleic Acid Res. 17, 3291(1989).
Moreover, one may refer to: for TNF, for example, Pennica et al.
(1984) Nature 312: 724-729; for G-CSF, for example, Hirano et al.
(1986) Nature 324:73-76; and for GM-CSF, for example, Cantrell et
al. (1985) Proc. Natl. Acad. Sci. (USA) 82(18): 6250-6254. More
specifically, the nucleic acid sequence encoding GM-CSF includes
sequences containing the sequences from position 84 to 461 of
Accession number NM.sub.--000758 (corresponding to position 18 to
144 of the amino acid sequence of NP.sub.--000749). The nucleic
acid sequence encoding IL-4 includes sequences contra the sequences
from position 443 to 829 of Accession number NM.sub.--000589
(corresponding to position 25 to 153 of the amino acid sequence of
NP.sub.--000580). Signal peptides can be appropriately substituted
with signal peptide sequences of other proteins. Using natural
genes encoding these cytokines or using the degeneracy of the
genetic code, mutant genes encoding functional cytokines can be
constructed and used.
[0043] Moreover, genes may be modified to express modified forms of
the cytokines. For example, a cytokine that has two forms, the
precursor form and mature form (for example, cytokines producing
active fragments by cleavage of their signal peptides or by limited
proteolysis) may be genetically modified to express either one of
the precursor form and the mature form. Other modified forms, for
example, fusion proteins formed between an active fragment of a
cytokine and a heterologous sequence (for example, a heterologous
signal peptide) cam also be used.
[0044] In the present invention, "recombinant virus" refers to a
virus produced via a recombinant polynucleotide or to an
amplification product of a virus. "Recombinant polynucleotide"
refers to a polynucleotide in which one or both ends are not linked
as in the natural condition. Specifically, a recombinant
polynucleotide is a polynucleotide in which the linkage of the
polynucleotide chain has been artificially modified (cleaved and/or
liked). Recombinant polynucleotides can be produced using well
known gene recombination methods by combining polynucleotide
synthesis, nuclease treatment, ligase treatment, and such.
Recombinant viruses can be produced by expressing a genetically
engineered polynucleotide encoding a viral genome, and then
reconstituting the virus. For example, methods for reconstructing a
virus from cDNA that encodes the viral genome are known (Y. Nagai,
A. Kato, Microbiol. Immunol., 43, 613-624 (1999)).
[0045] In the present invention, "gene" refers to a genetic
substance, a nucleic acid having a sequence to be transcribed in a
sense or antisense strand. Genes may be RNAs or DNAs. In this
invention, a nucleic acid encoding a protein is referred to as a
gene of that protein. Further, a gene may not encode a protein. For
example, a gene may encode a functional RNA, such as a ribozyme or
antisense RNA. A gene may be a naturally-occurring or artificially
designed sequence. Furthermore, in the present invention, "DNA"
includes both single-stranded and double-stranded DNAs. Moreover,
"encoding a protein" means that a polynucleotide includes an ORF
that encodes an amino acid sequence of the protein in a sense or
antisense strand, so that the protein can be expressed under
appropriate conditions.
[0046] Minus-strand RNA viruses may encode an antisense strand for
a foreign gene in the genomic RNA as required. Genomic RNA refers
to RNA that has the function to form a ribonucleoprotein (RNP) with
the viral proteins of a minus-strand RNA virus. Genes contained in
the genome are expressed by the RNP, genomic RNA is replicated, and
daughter RNPs are formed. In general, the genome of a minus-strand
RNA virus is constituted so that the viral genes are situated in an
antisense orientation between the 3'-leader region and 5'-trailer
region. Between the ORFs of individual genes exists a transcription
ending sequence (E sequence)--intervening sequence (I
sequence)--transcription starting sequence (S sequence) that allows
the RNA encoding each ORE to be transcribed as a separate
cistron.
[0047] Genes encoding the viral proteins of a minus-strand RNA
virus include NP, P, M, F, HN, and L genes. "NP, P, M, F, HN, and L
genes" refer to genes encoding nucleocapside-, phospho-, matrix-,
fusion-, hemagglutinin-neuraminidase-, and large-proteins
respectively. Genes in each virus belonging to Paramyxovirinae are
commonly listed as follows. In general, NP gene is also listed as
"N gene." TABLE-US-00001 Respirovirus NP P/C/V M F HN -- L
Rubulavirus NP P/V M F HN (SH) L Morbillivirus NP P/C/V M F H --
L
[0048] For example, the database accession numbers for the
nucleotide sequences of each of the Sendai virus genes are: M29343,
M30202, M30203, M30204, M51331, M55565, M69046, and X17218 for NP
gene; M30202, M30203, M30204, M55565, M69046, X00583, X17007, and
X17008 for P gene; D11446, K02742, M30202, M30203, M30204, M69046,
U31956, X00584, and X53056 for M gene; D00152, D11446, D17334,
D17335, M30202, M30203, M30204, M69046, X00152, and X02131 for F
gene; D26475, M12397, M30202, M30203, M30204, M69046, X00586,
X02808, and X56131 for HN gene; and D00053, M30202, M30203, M30204,
M69040, X00587, and X58886 for L gene. Examples of viral genes
encoded by other viruses are: CDV, AF014953; DMV, X75961; HPIV-1,
D01070, HPIV-2, M55320; HPIV-3, D10025; Mapuera, X85128; Mumps,
D86172; MV, K01711; NDV, AF064091; PDPR, X74443; PDV, X75717; RPV,
X68311; SeV, X00087; SV5, M81442; and Tupaia, AF079780 for N gene;
CDV, X51869; DMV, Z47758; HPIV-1, M74081; HPIV-3, X04721; HPIV-4a,
M55975; HPIV-4b, M55976; Mumps, D86173; MV, M89920; NDV, M20302;
PDV, X75960; RPV, X68311; SeV, M30202; SV5, AF052755; and Tupaia,
AF079780 for P gene; CDV, AF014953; DMV, Z47758; HPIV-1, M74081;
HPIV-3, D00047; MV, ABO16162; RPV, X68311; SeV, AB005796; and
Tupaia, AF079780 for C gene; CDV, M12669; DMV, Z30087; HPIV-1,
S38067; HPIV-2, M62734; HPIV-3, D00130; HPIV-4a, D10241; HPIV-4b,
D10242; Mumps, D86171; MV, AB012948; NDV, AF09819; PDPR, Z47977;
PDV, X75717; RPV, M34018; SeV, U31956; and SV5, M32248 for M gene;
CDV, M21849; DMV, AJ224704; HPN-1, M22347; HPIV-2, M60182; HPIV-3,
X05303; HPIV-4a, D49821; HPIV-4b, D49822; Mumps D86169; MV,
AB003178; NDV, AF048763; PDPR, Z37017; PDV, AJ224706; RPV, M21514;
SeV, D17334; and SV5, AB021962 for F gene; and, CDV, AF112189, DMV,
AJ224705; HPIV-1, U709498; HPIV-2, D000865; HPIV-3, AB012132;
HPIV-4A, M34033; HPIV-4B, AB006954; Mumps, X99040; MV, K01711; NDV,
AF204872; PDPR, Z81358; PDV, Z36979; RPV, AF132934; SeV, U06433;
and SV-5, S76876 for HN (H or G) gene. However, a number of strains
are known for each virus, and genes exist that include sequences
other than those cited above, due to strain variation.
[0049] The ORFs encoding these viral proteins and ORFs of the
foreign genes are arranged in the antisense direction in the
genomic RNAs, via the above-described E-I-S sequence. The ORF
closest to the 3'-end of the genomic RNAs requires only an S
sequence between the 3'-leader region and the ORF, and does not
require an E or I sequence. Further, the ORF closest to the 5'end
of the genomic RNA requires only an E sequence between the
5'-trailer region and the ORF, and does not require an I or S
sequence. Furthermore, two ORFs can be transcribed as a single
cistron, for example, by using an internal ribosome entry site
(IRES) sequence. In such a case, an E-I-S sequence is not required
between these two ORFs. For example, in wild type paramyxoviruses,
a typical RNA genome includes a 3'-leader region, six ORFs encoding
the N, P, M, F, HN, and L protein in the antisense direction and in
this order, and a 5'-trailer region on the other end. The viral
gene orientation in the genomic RNAs of the present invention is
not restricted, but similarly to the wild type viruses, it is
preferable that ORFs encoding the N, P, M, F, HN, and L proteins
are arranged in this order, after the 3'-leader region, and before
the 5'-tailer region. Certain types of viruses have different viral
genes, but even in such cases, it is preferable that each gene be
arranged as in the wild type, as described above. In general,
viruses maintaining the N, P, and L genes can autonomously express
genes from the RNA genome in cells, replicating the genomic RNA.
Furthermore, by the action of genes such as the F and HN genes,
which encode envelope proteins, and the M gene, infectious virions
are formed and released to the outside of cells. Thus, such viruses
become viruses with propagation ability. "With propagation ability"
indicates that when a virus infects a host cell, the virus is
amplified in the cell and infections virions are produced. In the
present invention, a foreign gene may be inserted into a
protein-noncoding region in this genome, as required.
[0050] Further, a minus-strand RNA virus of this invention may be
deficient in any of the wild type virus genes. For example, a virus
that excludes the M, F, or HN gene, or any combination thereof, can
be preferably used in this invention. Such viruses can be
reconstituted, for example, by externally supplying the products of
the deficient genes. Similar to wild type viruses, the viruses thus
prepared adhere to host cells and cause cell fusion, but they
cannot firm daughter virions that retain the same infectivity as
the original virus, because the virus genome introduced into cells
is deficient in viral genes. Therefore, such viruses are useful as
safe viruses that can only introduce genes once (for example,
foreign genes). Examples of genes in which the genome may be
deficient are the F gene and/or HN gene. For example, viruses can
be reconstituted by transfecting host cells with a plasmid
expressing a recombinant minus-strand RNA viral genome deficient in
the F gene, along with an F protein expression vector and
expression vectors for the NP, P, and L proteins (WO00/70055 and
WO00/70070; Li, H. --O. et al., J. Virol. 74(14) 6564-6569 (2000)).
Viruses can also be produced, for example, using host cells that
have incorporated the F gene into their chromosomes. In these
proteins, the amino acid sequences do not need to be the same as
the viral sequences, and a mutant or homologous gene from another
virus may be used as a substitute, so long as the activity in
nucleic acid introduction is the same as, or greater than, that of
the natural type.
[0051] Further, viruses that include an envelope protein other than
that of the virus from which the viral genome was derived, may be
prepared as viruses used in this invention. For example, when
reconstituting a virus, a virus including a desired envelope
protein can be generated by expressing an envelope protein other
than the envelope protein encoded by the basic viral genome. Such
proteins are not particularly limited. A desired protein that
confers an ability to infect cells may be used. Examples of such
proteins include the envelope proteins of other viruses, for
example, the G protein of vesicular stomatitis virus (VSV-G). The
VSV-G protein may be derived from an arbitrary VSV strain. For
example, VSV-G proteins derived from Indiana serotype strains (J.
Virology 39: 519-528 (1981)) may be used, but the present invention
is not limited hereto. Furthermore, the virus of the present
invention may include any arbitrary combination of envelope
proteins derived from other viruses. Preferred examples of such
proteins are envelope protein derived from viruses that infect
human cells. Such proteins are not particularly limited, and
include retroviral amphotropic envelope proteins and the like. For
example, the envelope proteins derived from mouse leukemia virus
(MuLV) 4070A strain can be used as the retroviral amphotropic
envelope proteins. In addition, envelope proteins derived from
MuMLV 10A1 strain may also be used (for example, pCL-10A1 (Imgenex)
(Naviaux, R. K. et al., J. Virol. 70:5701-5705 (1996)). The
proteins of Herpesviridae include, for example, gB, gD, gH, and
gp85 proteins of herpes simplex viruses, and gp350 and gp220
proteins of EB virus. The proteins of Hepadnaviridae include the S
protein of hepatitis B virus. These proteins may be used as fusion
proteins in which the extracellular domain is linked to the
intracellular domain of the F or HN protein. As described above,
the viruses used in this invention include pseudotype viruses that
include envelope proteins, nick as VSV-G, derived from viruses
other than the virus from which the genome was derived. If the
viruses are designed such that these envelope proteins are not
encoded in RNA genomes, the proteins will never be expressed after
virion infection of the cells.
[0052] Furthermore, the viruses used in this invention may be, for
example, viruses that include on the envelope spice thereof,
proteins such as adhesion factors capable of adhering to specific
cells, ligands, receptors, antibodies or fragments, or viruses that
include a chimeric protein with these proteins in the extracellular
domain and polypeptides derived from the virus envelope in the
intracellular domain. These protein may be encoded in the viral
genome, or supplied through the expression of genes not in the
viral genome (for example, genes carried by other expression
vectors, or genes in the host chromosomes) at the time of viral
reconstitution.
[0053] Further, in the viruses, any viral gene contained in the
virus may be modified from the wild type gene in order to reduce
the immunogenicity caused by viral proteins, or to enhance RNA
transcriptional or replicational efficiency, for example.
Specifically, for example, modifying at least one of the
replication factors N, P, and L genes, is considered to enhance
transcriptional or replicational function. Furthermore, although
the HN protein, which is an envelope protein, has both
hemagglutinin activity and neuraminidase activity, it is possible,
for example, to improve viral stability in blood if the former
activity is attenuated, and infectivity can be controlled if the
latter activity is modified. Further, it is also possible to
control membrane fusion ability by modifying the F protein. For
example, the epitopes of the F protein and/or HN protein, which can
be cell surface antigenic molecules, can be analyzed, and using
this, viruses with reduced antigenicity to these proteins can be
prepared.
[0054] Furthermore, the minus-strand RNA virus may be deficient in
one or more accessory genes. For example, by knocking out the V
gene, one of the SeV accessory genes, the pathogenicity of SeV
toward hosts such as mice is remarkably reduced, without hindering
gene expression and replication in cultured cells (Kato, A. et al.,
1997, J. Virol. 71: 7266-7272; Kato, A. et al., 1997, EMBO J. 16:
578-587; Curran, J. et al., WO01/04272, EP1067179).
[0055] Minus-strand RNA viruses can be made, for example, to play a
role as a vector to introduce foreign genes that are expected to
have a synergistic effect with their own anticancer effect. These
vectors do not have a DNA phase and carry out transcription and
replication only in the host cytoplasm, and consequently,
chromosomal integration does not occur (Lamb, R. A. and Kolakofsky,
D., Paramyxoviridae: The viruses and their replication. In: Fields
B N, Knipe D M, Howley P M, (eds). Fields of Virology. Vol. 2.
Lippincott-Raven Publishers: Philadelphia, 1996, pp. 1177-1204).
Therefore, safety issues such as formation and immortalization due
to chromosomal abberation do not occur. This characteristic of
minus-strand RNA viruses contributes greatly to safety when it is
used as a vector. For example, results on foreign gene expression
show that even after multiple continuous passages of SeV, almost no
nucletide mutation is observed. This suggests that the viral genome
is highly stable and the inserted foreign genes are stably
expressed over long periods of time (Yu, D. et al., Genes Cells 2,
457-466 (1997)). Further, there are qualitative advantages
associated with SeV not having a capsid structural protein, such as
packaging flexibility and insert gene size. Thus, minus-strand RNA
viruses can be made to play an additional role as a highly
efficient vector for human gene therapy. For example, SeV with
propagation ability are capable of introducing foreign genes of up
to at least 4 kb in size, and can simultaneously express two or
more kinds of genes by adding the transcriptional units.
[0056] Further, SeV is known to be pathogenic in rodents causing
pneumonia, but is not pathogenic for human. This is also supported
by a previous report hat nasal administration of wild type SeV does
not have severely harmful effects on non-human primates (Hurwitz,
J. L. et al., Vaccine 15: 533-540, 1997). These SeV characteristics
suggest that SeV can be applied therapeutically on humans,
supporting the proposition that SeV are a promising choice of gene
therapy for cancer.
[0057] Although not required, minus-strand RNA viruses of this
invention may encode foreign genes in their genomic RNA. A
recombinant virus harboring a foreign gene is obtained by inserting
a foreign gene into an above-described viral genome. The foreign
gene can be any desired gene expected to have a synergistic effect
and such with the anticancer effect of the RNA virus of this
invention, and may be a gene that encodes a naturally-occurring
protein, or protein modified from a naturally-occurring protein by
deletion, substitution, or insertion of amino acid residues. The
foreign gene can be insured at any desired position in a
protein-noncoding region of the virus genome, for example. The
above nucleic acid can be inserted, for example, between the
3'-leader region and the viral protein ORB closest to the 3'-end;
between each of the viral protein ORFs; and/or between the viral
protein ORF closest to the 5'-end and the 5'-trailer region.
Further, in genomes deficient in the F or HN gene or the like,
nucleic acids encoding the foreign genes can be inserted into those
deficient regions. When introducing a foreign gene into a
paramyxovirus, it is desirable to insert the gene such that the
chain length of the polynucleotide to be inserted into the genome
will be a multiple of six (Journal of Virology, Vol. 67, No. 8,
4822-4830, 1993). An E-I-S sequence should be arranged between the
inserted foreign gene and the viral ORF. Two or more genes can be
inserted in tandem via E-I-S sequences.
[0058] Expression levels of a foreign gene carried in a
minus-strand RNA virus can be controlled using the type of
transcriptional initiation sequence added upstream (to the 3'-side
of the negative strand) of the gene (WO01/18223). The expression
levels can also be controlled by the position at which the foreign
gene is inserted in the genome: the nearer to the 3'-end of the
negative strand the insertion position is, the higher the
expression level; while the nearer to the 5'-end the insertion
position is, the lower the expression level. Thus, to obtain a
desired gene expression level, the insertion position of a foreign
gene can be appropriately controlled such that the combination with
genes encoding the viral proteins before and after the foreign gene
is most suitable. In general, since a high foreign gene expression
level is thought to be advantageous, it is preferable to link the
foreign gene to a highly efficient transcriptional initiation
sequence, and to insert it near the 3'-end of the negative strand
genome. Specifically, a foreign gene is inserted between the
3'-leader region and the viral protein ORF closest to the 3'-end.
Alternatively, a foreign gene may be inserted between the ORFs of
the viral gene closest to the 3'-end and the second closest viral
gene. In wild type paramyxoviruses, the viral protein gene closest
to the 3'-end of the genome is the N gene, and the second closest
gene is the P gene. Alternatively, when a high level of expression
of the introduced gene is undesirable, the gene expression level
from the virus can be suppressed to obtain an appropriate effect,
for example, by inserting the foreign gene at a site in the virus
as close as possible to the 5'-side of the negative strand, or by
selecting an inefficient transcriptional initiation sequence.
[0059] To prepare a minus-strand RNA virus, a cDNA encoding a
genomic RNA of a virus is transcribed in mammalian cells, in the
presence of viral proteins (i.e., N, P, and L proteins) essential
for reconstitution of an RNP, which is a component of a virus.
Viral RNP can be reconstituted by producing either the negative
strand genome (that is, the same antisense strand as the viral
genome) or the positive strand (the sense stand encoding the viral
proteins). Production of the positive strand is preferable for
increased efficiency of minus-strand RNA virus reconstitution. The
RNA terminals preferably reflect the terminals of the 3'-leader
sequence and 5'-tailer sequence as accurately as possible, as in
the natural viral genome. To accurately regulate the 5'-end of the
transcript, for example, the RNA polymerase may be expressed within
a cell using the recognition sequence of T7 RNA polymerase as a
transcription initiation site. To regulate the 3'-end of the
transcript, for example, a self-cleaving ribozyme can be encoded at
the 3'-end of the transcript, allowing accurate cleavage of the
3'-end with this ribozyme (Hasan, M. K. et al., J. Gen. Virol. 78:
2813-2820, 1997; Kato, A. et al., 1997, EMBO J. 16: 578-587; and
Yu, D. et al., 1997, Genes Cells 2: 457-466).
[0060] For example, a recombinant Sendai virus carrying a foreign
gene can be constructed as follows, according to descriptions in:
Hasan, M. K. et al., J. Gen. Virol. 78: 2813-2820, 1997; Kato, A.
et al., 1997, EMBO J. 16: 578-587; Yu, D. et al., 1997, Genes Cells
2: 457-466; or the like.
[0061] First, a DNA sample including a cDNA sequence of an
objective foreign gene is prepared. The DNA sample is preferably
one that can be confirmed to be a single plasmid by electrophoresis
at a concentration of 25 ng/.mu.l or more. The following explains
the case of using a NotI site to insert a foreign gene into a DNA
encoding a viral genomic RNA, with reference to examples. When a
NotI recognition site is included in a target cDNA nucleotide
sequence, the nucleotide sequence is altered using site-directed
mutagenesis or the like, such that the encoded amino acid sequence
does not change, and the NotI site is preferably excised in
advance. The objective gene fragment is amplified from this sample
by PCR, and then recovered. By adding the NotI site to the 5'
regions of a pair of primers, both ends of the amplified fragments
become NotI sites E-I-S sequences are designed to be included in
primers such that, after a foreign gene is inserted into the viral
genome, one E-I-S sequence each is placed between the ORF of the
foreign gene, and either side of the ORFs of the viral genes.
[0062] For example, to guarantee cleavage with NotI, the forward
side synthetic DNA sequence has a form in which any desired
sequence of not less than two nucleotides (preferably four
nucleotides not including a sequence derived from the NotI
recognition site, such as GCG and GCC, and more preferably ACTT) is
selected at the 5'-side, and a NotI recognition site `gcggccgc` is
added to its 3'-side. To that 3'-side, nine arbitrary nucleotides,
or nine plus a multiple of six nucleotides are further added as a
spacer sequence. To the further 3' of this, a sequence
corresponding to about 25 nucleotides of the ORF of a desired cDNA,
including and counted from the initiation codon ATG, is added. The
3'-nd of the forward side synthetic oligo DNA is preferably about
25 nucleotides, selected from the desired cDNA such that the final
nucleotide becomes a G or C.
[0063] For the reverse side synthetic DNA sequence, no less am two
arbitrary nucleotides (preferably four nucleotides not including a
sequence derived from a NotI recognition site, such as GCG and GCC,
and more preferably ACTT) are selected from the 5'-side, a NotI
recognition site `gcggccgc` is added to its 3'-side, and to that 3'
is further added an oligo DNA insert fragment for adjusting the
length. The length of this oligo DNA is designed such that the
chain length of the NotI fragment of the final PCR-amplified
product will become a multiple of six nucleotides (the so-called
"rule of six"); Kolakofski, D., et al., J. Virol. 72:891-899, 1998;
Calain, P. and Roux, L., J. Virol. 67:4822-4830, 1993; Calain, P.
and Roux, L., J. Virol. 67:4822-4830, 1993). When adding an E-I-S
sequence to this primer, to the 3'-side of the oligo DNA insertion
fragment is added the complementary strand sequence of the Sendai
virus S, I, and E sequences, preferably 5'-CTTTCACCCT-3' (SEQ ID
NO: 1), 5'-AAG-3', and 5'-TTTTTCTTACTACGG-3' (SEQ ID NO: 2),
respectively; and further to this 3'-side is added a complementary
strand sequence corresponding to about 25 nucleotides, counted
backwards from the termination codon of a desired cDNA sequence,
whose length has been selected such that the final nucleotide of
the chain becomes a G or C, to make the 3'-end of the reverse side
synthetic DNA.
[0064] PCR can be performed according to conventional methods,
using Taq polymerase or other DNA polymerases. Objective amplified
fragments may be digested wit NotI, and then inserted into the NotI
site of plasmid vectors such as pBluescript. The nucleotide
sequences of PCR products thus obtained are confirmed with a
sequencer, and plasmids that include the correct sequence are
selected. The inserted fragment is excised from these plasmids
using NotI, and cloned into the NotI site of a plasmid composed of
genomic cDNA. A recombinant Sendai virus cDNA can also be obtained
by inserting the fragment directly into the NotI site of a genomic
cDNA, without using a plasmid vector.
[0065] For example, a recombinant Sendai virus genomic cDNA can be
constructed according to methods described in the literature (Yu,
D. et al., Genes Cells 2: 457-466, 1997; Hasan, M. K. et al., J.
Gen. Virol. 78: 2813-2820, 1997). For example, an 18 bp spacer
sequence (5'-(G)-CGGCCGCAGATCTTCACG-3') (SEQ ID NO: 3), including a
NotI restriction site, is inserted between the leader sequence and
the ORF of N protein of the cloned Sendai virus genomic cDNA
(pSeV(+)), obtaining plasmid pSeV18.sup.+b(+), which includes an
auto-cleavage ribozyme site derived from the antigenomic strand of
delta hepatits virus (Hasan, M. K. et al., 1997, J. General
Virology 78: 2813-2820). A recombinant Sendai virus cDNA including
a desired foreign gene can be obtained by inserting a foreign gene
fragment into the NotI site of pSeV18.sup.+b(+).
[0066] A virus can be reconstituted by transcribing a DNA encoding
a genomic RNA of a recombinant virus thus prepared, in cells in the
presence of the above-described viral proteins (L, P, and N).
[0067] Moreover, the recombinant viruses can also be reconstituted
by methods known in the art (WO97/16539; WO97/16538; Durbin, A. P.
et al., 1997, Virology 235: 323-332; Whelan, S. P. et al., 1995,
Proc. Natl. Acad. Sci. USA 92: 8388-8392; Schnell. M. J. et al.,
1994, EMBO J. 13: 4195-4203; Radecke, F. et al., 1995, EMBO J. 14:
5773-5784; Lawson, N. D. et al., Proc. Natl. Acad. Sci. USA 92:
4477-4481; Garcin, D. et al., 1995, EMBO J. 14: 6087-6094; Ko, A.
et al., 1996, Genes Cells 1: 569-579; Baron, M. D. and Barrett, T.,
1997, J. Virol. 71: 1265-1271; Bridgen, A. and Elliott, R. M.,
1996, Proc. Natl. Acad. Sci. USA 93: 15400-15404). With these
methods, minus-strand RNA viruses including parainfluenza virus,
vesicular stomatitis virus, rabies virus, measles virus, rinderpest
virus, and Sendai virus can be reconstituted from DNA. The viruses
of this invention can be reconstituted according to these methods.
When a viral DNA is made F gene-, HN gene-, and/or M
gene-deficient, such DNAs do not form infectious virions as is.
However, infectious virions can be formed by separately introducing
host cells with these deficient genes, and/or genes encoding the
envelope proteins of other viruses, and then expressing these genes
therein.
[0068] Specifically, the viruses can be prepared by the steps of:
(a) transcribing cDNAs encoding genomic RNAs of minus-strand RNA
viruses (negative strand RNAs), or complementary strands thereof
(positive strands), in cells expressing N, P, and L proteins; and
(b) harvesting culture supernatants thereof including the produced
minus-strand RNA viruses. For transcription, a DNA encoding a
genomic RNA is linked downstream of an appropriate promoter. The
genomic RNA thus transcribed is replicated in the presence of N, L,
and P proteins to form an RNP complex. Then, in the presence of M,
HN, and F proteins, virions enclosed in an envelope are formed. For
example, a DNA encoding a genomic RNA can be linked downstream of a
T7 promoter, and transcribed to RNA by T7 RNA polymerase. Any
desired promoter can be used as a promoter, in addition to those
including a T7 polymerase recognition sequence. Alternatively, RNA
transcribed in vitro may be transfected into cells.
[0069] Enzymes essential for the initial transcription of genomic
RNA from DNA, such as T7 RNA polymerase, can be supplied by
introducing the plasmid or viral vectors that express them, or, for
example, by incorporating the RNA polymerase gene into a chromosome
of the cell so as to enable induction of its expression, and then
inducing expression at the time of viral reconstitution. Further,
genomic RNA and viral proteins essential for virus reconstitution
are supplied, for example, by introducing the plasmids that express
them. In supplying these viral proteins, helper viruses such as the
wild type or certain types of mutant minus-strand RNA viruses can
be used.
[0070] Methods for introducing DNAs expressing the genomic RNAs
into cells include, for example, (i) methods for making DNA
precipitates which target cells can internalize; (ii) methods for
making complexes including DNAs that are suitable for
internalization by target cells, and have a low-cytotoxic positive
charge; and (iii) methods for using electric pulses to
instantaneously create holes in the target cell membrane, which are
of sufficient size for DNA molecules to pass through.
[0071] In the context of method (ii), various transfection reagents
can be used. For example, DOTMA (Roche), Superfect (QIACEN
#301305), DOTAP, DOPE, DOSPER (Roche #1811169), and the like can be
cited. Regarding method (i), for example, transfection methods
using calcium phosphate can be cited, and although DNAs transferred
into cells by this method are internalized by phagosomes, a
sufficient amount of DNA is known to enter the nucleus (Graham, F.
L. and Van Der Eb, J., 1973, Virology 52: 456; Wigler, M. and
Silverstein, S., 1977, Cell 11: 223). Chen and Okayama investigated
the optimization of transfer techniques, reporting that (1)
incubation conditions for cells and coprecipitates are 2 to 4%
CO.sub.2, 35.degree. C., and 15 to 24 hours, (2) the activity of
circular DNA is higher than linear DNA, and (3) optimal
precipitation is obtained when the DNA concentration in the
precipitate mixture is 20 to 30 .mu.g/ml (Chen, C. and Okayama, H.,
1987, Mol. Cell. Biol. 7: 2745). The methods of (ii) are suitable
for transient transfections. Methods for performing transfection by
preparing a DEAE-dextran (Sigma #D-9885 M.W. 5.times.10.sup.5)
mixture with a desired DNA concentration ratio have been known for
a while. Since most complexes are decomposed in endosomes,
chloroquine may also be added to enhance the effect (Calos, M. P.,
1983, Proc. Natl. Acad. Sci. USA 80: 3015). The methods of (iii)
are referred to as electroporation methods, and are used more in
general than methods (i) or (ii) because they are not
cell-selective. The efficiency of these methods is presumed to be
good under optimal conditions for: the duration of pulse electric
current, shape of the pulse, potency of electric field (gap between
electrodes, voltage), conductivity of buffer, DNA concentration,
and cell density.
[0072] Of the above three categories, the methods of (ii) are
simple to operate and facilitate examination of many samples using
a large amount of cells, making transfection reagents suitable for
the transduction into cells of DNA for virus reconstitution.
Preferably, the Superfect Transfection Reagent (QIAGEN, Cat No.
301305), or the DOSPER Liposomal Transfection Reagent (Roche, Cat
No. 1811169) is used; however, the transfection reagents are not
limited to these.
[0073] Specifically, virus reconstitution from cDNA can be carried
out, for example, as follows:
[0074] In a plastic plate of about 6 to 24 wells, or a 100-mm Petri
dish or the like, simian kidney-derived LLC-MK2 cells (ATCC CCL-7)
are cultured up to about 100% confluency, using minimum essential
medium (MEM) including 10% fetal calf serum (FCS) and antibiotics
(100 units/ml penicillin G and 100 .mu.g/ml streptomycin). Then
they are infected with, for example, two plaque forming units
(PFU)/cell of the recombinant vaccinia virus vTF7-3, which
expresses T7 RNA polymerase and has been inactivated by 20-minutes
of UW irradiation in the presence of 1 .mu.g/ml psoralen (Fuerst,
T. R. et al., Proc. Natl. Acad. Sci. USA 83: 8122-8126, 1986; Kato,
A. et al., Genes Cells 1: 569-579, 1996). The amount of psoralen
added and the UV irradiation tine can be appropriately adjusted.
One hour after infection, 2 to 60 .mu.g, and more preferably 3 to
20 .mu.g. of DNA encoding the genomic RNA of a recombinant Sendai
virus is transfected along with the plasmids expressing
trans-acting viral proteins essential for viral RNP production (0.5
to 24 .mu.g of pGEM-N, 0.25 to 12 .mu.g of pGEM-P, and 0.5 to 24
.mu.g of pGEM-L) (Kato, A. et al., Genes Cells 1: 569-579, 1996),
using the lipofection method or the like with Superfect (QIAGEN).
For example, the ratio of the amounts of expression vectors
encoding the N, P, and L proteins is preferably 2:1:2, and the
plasmid amounts are appropriately adjusted in the range of 1 to 4
.mu.g of pGEM-N, 0.5 to 2 .mu.g of pGEM-P, and 1 to 4 .mu.g of
pGEM-L.
[0075] The transfected cells are cultured, as desired, in
serum-free MEM composed of 100 .mu.g/ml of rifampicin (Sigma) and
cytosine arabinoside (AraC), more preferably only 40 .mu.g/ml of
cytosine arabinoside (AraC) (Sigma). Optimal drug concentrations
are set so as to minimize cytotoxicity due to the vaccinia virus,
and to maximize virus recovery rate (Kato, A. et al., 1996, Genes
Cells 1: 569-579). After culturing for about 48 to 72 hours after
transfection, cells are harvested, and then disintegrated by
repeating freeze-thawing three times. LLC-MK2 cells are
re-transfected with the disintegrated materials including RNP, and
cultured. Alternatively, the culture supernatant is recovered,
added to a culture solution of LLC-MK2 cells to infect them, and
the cells are then cultured. Transfection can be conducted by, for
example, forming a complex with lipofectamine, polycationic
liposome, or the like, and introducing the complex into cells.
Specifically, various transfection reagents can be used. For
example, DOTMA (Roche), Superfect (QIAGEN #301305), DOTAP, DOPE,
and DOSPER (Roche #1811169) may be cited. In order to prevent
decomposition in the endosome, chloroquine may also be added
(Calos, M. P., 1983, Proc. Natl. Acad. Sci. USA 80: 3015). In cells
transduced with RNP, viral gene expression from RNP and RNP
replication progress, and the virus is amplified. By diluting the
viral solution thus obtained (for example, 10.sup.6-fold), and then
repeating the amplification, the vaccinia virus vTF7-3 can be
completely eliminated. Amplification is repeated, for example, tree
or more times. Viruses thus obtained can be stored at -80.degree.
C. In order to reconstitute a virus having no propagation ability
and lacking a gene an envelope protein, LLC-MK2 cells express the
envelope protein may be used for transfection, or a plasmid
expressing the envelope protein may be cotransfected.
Alternatively, a defective type virus can be amplified by culturing
the transfected cells overlaid with LLK-MK2 cells expressing the
envelope protein (see WO00/70055 and WO00/70070).
[0076] Titers of viruses thus recovered can be determined, for
example, by measuring CIU (Cell-Infected Unit) or hemagglutination
activity (HA) (WO00/70070; Kato, A. et al., 1996, Genes Cells 1:
569-579; Yonemitsu, Y. & Kaneda, Y., Hemaggulutinating virus of
Japan-liposonme-mediated gene delivery to vascular cells. Ed. by
Baker A H. Molecular Biology of Vascular Diseases. Method in
Molecular Medicine: Humana Press: pp. 295-306, 1999). Titers of
viruses carrying GFP (green fluorescent protein) marker genes and
the like can be quantified by directly counting infected cells,
using the marker as an indicator (for example, as GFP-CIU). Titers
thus measured can be treated in the same way as CIU
(WO00/70070).
[0077] So long as a virus can be reconstituted, the host cells used
in the reconstitution are not particularly limited. For example, in
the reconstitution of Sendai viruses and the like, cultured cells
such as LLC-MK2 cells and CV-1 cells derived from monkey kidney,
BHK cells derived from hamster kidney, and cells derived from
humans can be used. By expressing suitable envelope proteins in
these cells, infectious virions including the proteins in the
envelope can also be obtained. Further, to obtain a large quantity
of a Sendai virus, a virus obtained from an above-described host
can be infected to embrionated hen eggs, to propagate the virus.
Methods for manufacturing viruses using hen eggs have already been
developed (Nakanishi, et al., ed. (1993), "State-of-the-Art
Technology Protocol in Neuroscience Research III, Molecular Neuron
Physiology", Koseisha, Osaka, pp. 153-172). Specifically, for
example, a fertilized egg is placed in an incubator, and cultured
for nine to twelve days at 37 to 38.degree. C. to grow an embryo.
After the virus is inoculated into the allantoic cavity, the egg is
cultured for several days (for example, three days) to proliferate
the virus. Conditions such as the period of culture may vary
depending upon the recombinant Sendai virus being used. Then,
allantoic fluids including the virus are recovered. Separation and
purification of a Sendai virus from allantoic fluids can be
performed according to a usual method (Tashiro, M., "Virus
Experiment Protocol," Nagai, Ishihama, ed., Medical View Co., Ltd.
pp. 68-73, (1995)).
[0078] For example, the construction and preparation of Sendai
viruses defective in F gate can be performed as described below
(see WO00/70055 and WO00/70070).
<1> Construction of a Genomic cDNA of an F-Gene Defective
Sendai Virus, and a Plasmid Expressing F gene:
[0079] A full-length genomic cDNA of Sendai virus (SeV), the cDNA
of pSeV18.sup.+b (+) (Hasan, M. K. et al., 1997, J. General
Virology 78: 2813-2820) ("pSeV18.sup.+b (+)" s also referred to as
"pSeV18.sup.+"), is digested with SphI/KpnI to recover a fragment
(14673 bp), which is cloned into pUC18 to prepare plasmid pUC18/KS.
Construction of an F gene-defective site is performed on this
pUC18/KS. An F gene deficiency is created by a combination of
PCR-ligation methods, and, as a result, the F gene ORF
(ATG-TGA=1698 bp) is removed. Then, for example,
`atgcatgccggcagatga (SEQ ID NO: 4)` is ligated to construct an F
gene-defective type SeV genomic cDNA (pSeV18.sup.+/.DELTA.F). A PCR
product formed in PCR by using the pair of primers [forward:
5'-gttgagtactgcaagagc/SEQ ID NO: 5, reverse:
5'-tttgccggcatgcatgtttcccaaggggagagtttgcaacc/SEQ ID NO: 6] is
connected upstream of F, and a PCR product formed using the pair of
primers [forward: 5'-atgcatgccggcagatga/SEQ ID NO: 7, reverse:
5'-tgggtgaatgagagaatcagc/SEQ ID NO: 8] is connected downstream of
the F gene with EcoT22I. The plasmid thus obtained is digested with
SacI and SalI to recover a 4931 bp fragment of the region including
the F gene-defective site, which is cloned into pUC1 to form
pUC18/dFSS. This pUC18/dFSS is digested with DraIII, the fragment
is recovered, replaced with the DraIII fragment of the region
including the F gene of pSeV18.sup.+, and ligated to obtain the
plasmid pSeV18.sup.+/.DELTA.F.
[0080] A foreign gene is inserted, for example, into the NsiI and
NgoMIV restriction enzyme sites in the F gene-defective site of
pUC18/dFSS. For this, a foreign gene fragment may be, for example,
amplified using an NsiI-tailed primer and an NgoMIV-tailed
primer.
<2> Preparation of Helper Cells That Induce SeV-F Protein
Expression;
[0081] To construct an expression plasmid of the Cre/IoxP induction
type that expresses the Sendai virus F gene (SeV-F), the SeV-F gene
is amplified by PCR, and inserted to the unique SwaI site of the
plasmid pCALNdlw (Arai, T. et al., J. Virology 72, 1998, p
1115-1121), which is designed to enable the inducible expression of
a gene product by Cre DNA recombinase, thus constructing the
plasmid pCALNdLw/F.
[0082] To recover infectious virions from the F gene-defective
genome, a helper cell line expressing SeV-F protein is established.
The monkey kidney-derived LLC-MK2 cell line, which is commonly used
for SeV propagation, can be used as the cells, for example. LLC-MK2
cells are cultured in MEM supplemented with 10% heat-inactivated
fetal bovine set (FBS), penicillin G sodium (50 units/ml), and
streptomycin (50 .mu.g/ml) at 37.degree. C. in 5% CO.sub.2. Since
the SeV-F gene product is cytotoxic, the above-described plasmid
pCALNdLw/F, which was designed to enable inducible expression of
the F gene product with Cre DNA recombinase, is transfected to
LLC-MK2 cells by the calcium phosphate method (using a mammalian
transfection kit (Stratagene)), according to protocols well known
in the art.
[0083] The plasmid pCALNdLw/F (10 .mu.g) is introduced into LLC-MK2
cells grown to 40% confluency using a 10cm plate, and the cells are
then cultured in MEM (10 ml) including 10% FBS, in a 5% CO.sub.2
incubator at 37.degree. C. for 24 hours. After 24 hours, the cells
are detached and suspended in the medium (10 ml). The suspension is
then seeded into five 10-cm dishes, 5 ml into one dish, 2 ml each
into two dishes, and 0.2 ml each into two dies, and cultured n MEM
(10 ml) including G418 (GIBCO-BRL) (1200 .mu.g/ml) and 10% FBS. The
cells were cultured for 14 days, exchanging the medium every two
days, to select cell lines into which the gene is stably
introduced. The cells grown from the above medium that show G418
resistance are recovered using a cloning ring. Culture of each
clone thus recovered is continued in 10-cm plates until
confluent.
[0084] After the cells have grown to confluency in a 6-cm dish, F
protein expression can be induced by infecting the cells with
adenovirus AxCANCre, for example, at MOI=3, according to the method
of Saito, et al. (Saito et al., Nucl. Acids Res. 23: 3816-3821
(1995), Arai, T. et al., J. Virol 72, 1115-1121 (1998)).
<3> Reconstruction and Amplification of F Gene-Deficient SeV
Virus:
[0085] The above-described plasmid pSeV18.sup.+/.DELTA.F inserted
with the foreign gene is transfected into LLC-MK2 cells by the
procedure described below. LLC-MK2 cells are seeded on 100-mm
dishes at 5.times.10.sup.6 cells/dish. To transcribe the genomic
RNA using T7 RNA polymerase, the cells are cultured for 24 hours,
and then recombinant vaccinia virus, which expresses T7 RNA
polymerase (PLWUV-VacT7: Fuerst, T. R. et al., Proc. Natl. Acad.
Sci. USA 83, 8122-8126 (1986)) and is treated with psoralen and
long-wavelength ultraviolet light (365 nm) for 20 minutes, is
inoculated to the cells at a MOI of about 2 at room temperature for
one hour. The ultraviolet light irradiation to the vaccinia virus
can be achieved, for example, by using UV Stratalinker 2400 with
five 15-watt bulbs (Catalog No. 400676 (100V); Stratagene, La
Jolla, Calif., USA). After the cells are washed with serum-free
MEM, plasmid expressing the genomic RNA and expression plasmids
each expressing N, P, L, F, or HN protein of the minus-strand RNA
virus are transected into the cells using an appropriate
lipofection reagent. The plasmid ratio is preferably, but is not
limited to, 6:2:1:2:2:2 in this order. For example, the expression
plasmid for the genomic RNA, and the expression plasmids each of
which expresses N, P, or L protein, or F and HN proteins (pGEM/NP,
pGEM/P, pGEM/L, and pGEM/F-HN; WO00/70070, Kato, A. et al., Genes
Cells 1, 569-579 (1996)) are transfected at amounts of 12, 4, 2, 4,
and 4 .mu.g/dish, respectively. After a few hours of culture, the
cells are washed twice with serum-free MEM, and then cultured in
MEM supplemented with 40 .mu.g/ml cytosine
.beta.-D-arabinofuranoside (AraC: Sigma, St. Louis, Mo.) and 7.5
.mu.g/ml trypsin (Gibco-BRL, Rockville, Md.). The cells are
recovered, and the resulting pellet is suspended in OptiMEM
(10.sup.7 cells/ml). The suspension is subjected to three
freeze-thaw cycles, and mixed with lipofection reagent DOSPER
(Boehringer Mannheim) (10.sup.6 cells/25 .mu.l DOSPER). After the
mixture is allowed to stand at room temperature for 15 minutes, it
is transfected to F-expressing helper cells (10.sup.6 cells/well in
12-well-plate) cloned as described above. The cells are cultured in
serum-free MEM (containing 40 .mu.g/ml AraC and 7.5 .mu.g/ml
trypsin), and the supernatant is collected. Viruses deficient in
genes other than F, for example, HN and M genes, can be prepared by
a similar method as described above.
[0086] Instead of a recombinant virus, a naturally derived vim may
also be used as a minus-strand RNA virus of the present invention,
and one can refer to "Uirusu Jikken-gaku kakuron (Virology
Experiments in detail)", second revised edition (edited by
Researcher's Associates at the National Institute of Health,
Maruzen, 1982) for methods of purifying, multiplying, and obtaining
isolated stains of each of the RNA viruses. For example, each type
of parainfluenza virus, such as Sendai virus belonging to the
Paramyxoviridae family, can propagate well in primary culture cells
of simian kidney (MK2), human embryonic lung, kidney, and amnion,
and in trypsin-supplemented Vero cells (same as above, p 334; Itoh
H. et al., Jap. J. Med. Sci. Biol. 23, 227 (1970)), and can then be
collected. Purification can be carried out by the sucrose density
gradient centrifugation method, and equilibrium centrifugation
method (p 336). Measles virus can propagate well in various cells
derived from monkeys (Matsumoto M., Bact. Rev. 30, 152 (1966)), and
while Vero cells are most widely used, it can be propagated using
CV1, FL, KB, HeLa, HEp2, and such (p 351). Viruses belonging to the
Rhabdoviridae family, such as the rabies virus, are propagated by
tissue culture methods using BHK, CE, Vero cells, and the like.
Purification methods involve the steps of adjusting the pH of a
culture solution on the third to fourth day of infection to 7.4 or
more, and concentrating the solution after removing cell debris by
low-speed centrifugation (p 376). Viruses belonging to the
Arenaviridae family, such as the lassa virus, propagate well in
most cultured cells that are subcultured in vitro, and it can be
propagated by infection into HK-21/13S cells, followed by culturing
as a suspension in agar (Sedwik W. D., J. Virol. 1, 1224 (1967)) (p
240). Viruses belonging to the Orthomyxoviridae family, such as the
influenza virus, can be propagated in developing hen eggs and MDCK
cells (p 295). Purification can be carried out by methods such as
centrifugation, and adsorption to and release from red blood cells
(Layer W. C., Fundamental Techniques in Virology, 82 (1969)) (p
317).
[0087] There is no limitation on the foreign gene to be introduced
using the minus-strand RNA virus, and examples of naturally
occurring proteins include, for example, hormones, cytokines,
growth factors, receptors, intracellular signaling molecules,
enzymes, and peptides. The proteins may be secretory proteins,
membrane proteins, cytoplasmic proteins, nuclear proteins, and the
like. Artificial proteins include, for example, fusion proteins of
chimeric toxins and such, dominant negative proteins (including
soluble receptor molecules or membrane-bound dominant negative
receptors), cell surface molecules and truncated cell adhesion
molecules. The proteins may also be proteins to which a secretory
signal, membrane-localization signal, nuclear translocation signal,
or the like has been attached. Functions of a particular gene can
be suppressed by expressing antisense RNA molecules, RNA-cleaving
ribozymes, or the like as the introduced gene. When a virus of this
invention is prepared using a gene for treating diseases as the
foreign gene, gene therapy can be performed by administering this
virus.
[0088] According to the method for producing viruses as described
herein, the virus of the present invention can be released into
extracellular fluid of virus producing cells at a titer of, for
example, 1.times.10.sup.5 CIU/ml or higher, preferably
1.times.10.sup.6 CIU/ml or higher, more preferably 5.times.10.sup.6
CIU/ml or higher, more preferably 1.times.10.sup.7 CIU/ml or
higher, more preferably 5.times.10.sup.7 CIU/ml or higher, more
preferably 1.times.10.sup.8 CIU/ml or higher, and more preferably
5.times.10.sup.8 CIU/ml or higher. The titer of virus can be
determined according to methods described herein or elsewhere
(Kiyotani, K. et al., Virology 177(1), 65-74 (1990); and
WO00/70070).
[0089] The recovered viruses can be purified to be substantial
pure. Tee purification can be achieved using known
purification/separation methods, including filtration,
centrifugation, adsorption, and column purification, or any
combinations thereof. The phrase "substantially pure" means that
the virus component constitutes a major proportion of a solution
comprising the virus. For example, a viral composition can be
confirmed to be substantially pure by the fact that the proportion
of protein contained as the viral component to the total protein
(excluding proteins added as carriers and stabilizers) in the
solution is 10% (w/w) or greater, preferably 20% or greater, more
preferably 50% or greater, preferably 70% or greater, more
preferably 80% or greater, and even more preferably 90% or greater.
Specific purification methods for, for example, the paramyxovirus
include methods using cellulose sulfate ester or cross-linked
polysaccharide sulfate ester (Japanese Patent Application Kokoku
Publication No. (JP-B) S62-30752 (examined, approved Japanese
patent application published for opposition), JP-B S62-33879, and
JP-B S62-30753) and methods including adsorbing to fucose
sulfate-containing polysaccharide and/or degradation products
thereof (WO97/32010), but are not limited thereto.
[0090] The minus-strand RNA viruses of the present invention may
be, for example, infectious virions or non-infectious virions. The
RNA viruses of the present invention may also be genomic
RNA-protein complexes (ribonucleoprotein complexes; RNPs). When
using such virions or complexes, these virions or complexes are
preferably mixed with a lipofection reagent and administered in
vivo. For example, virions and complexes can be mixed with
lipofectamine or a polycationic liposome and this mixture can be
administered in vivo (WO00/70055). In this method, various
transfection reagents can be used. Examples include DOTMA
(Boehringer), Superfect (QIAGEN #301305), DOTAP, DOPE, and DOSPER
(Boehringer #1811169). Chloroquine can be added to prevent
degradation in endosomes (Calos, M. P., 1983, Proc. Natl. Acad.
Sci. USA 80: 3015).
[0091] In the production of an anticancer agent comprising a
minus-strand RNA virus in the present invention, the minus-stan RNA
virus can be mixed (combined as necessary with a desired
pharmaceutically acceptable carrier or medium.
[0092] Specifically, the present invention provides methods for
producing an anticancer agent, which comprises the step of mixing a
minus-strand RNA virus with a pharmaceutically acceptable carrier
or medium.
[0093] The "pharmaceutically acceptable carrier or medium" refers
to materials that can be administered together with a minus-strand
RNA virus and which do not significantly inhibit infection by the
minus-strand RNA virus. Such carrier or medium includes, for
example, deionized water, sterilized water, sodium chloride
solution, dextrose solution, culture medium, serum, phosphate
buffered saline (PBS), and Ringer's solution containing dextrose,
sodium chloride, and lactic acid, and they may be appropriately
combined with a minus-strand RNA virus in formulations. Further,
they may be concentrated by centrifugation when necessary, and
resuspended in a physiological solution such as a culture solution
or physiological saline solution. They may also include membrane
stabilizers for liposomes (for example, sterols such as
cholesterol) or antioxidants (for example, tocopherol or vitamin
E). In addition, vegetable oils, suspending agents, detergents,
stabilizes, biocidal agents, and such may also be included.
Preservatives and other additives can also be added. A composition
of the present invention may take the form of an aqueous solution,
capsule, suspension, syrup, or such. A virus composition of the
present invention may be a composition in the form of a solution,
freeze-dried product, or aerosol. In the case of a freeze-dried
product, it may include sorbitol, sucrose, amino acids, various
proteins, and such as stabilizers.
[0094] The minus-strand RNA viruses of the present invention have
anticancer activity, and they can exhibit anticancer
(carcinostatic) effects when administered to tumors (cancer
tissue). Therefore, the present invention relates to a method for
suppressing cancer (method for suppressing cancer cell growth, or
method for treating cancer), which comprises the step of
administering a minus-strand RNA virus to cancer tissues (in vivo
administration).
[0095] For example, cancer therapy can be performed on cancer
patients. This method comprises the step of administering a
minus-strand RNA virus (an anticancer agent of the present
invention). Specifically, the method comprises the step of
administering a therapeutically effective amount of a minus-strand
RNA virus to a patient. Use of the present method is expected to
suppress the growth of cancer cells compared to when a minus-strand
RNA virus of the present invention is not administered. The
minus-strand RNA virus may not carry a foreign gene, or it may
carry a gene (foreign gene) encoding one or more cancer antigens,
immunostimulatory cytokines, proteins that inhibit angiogenesis, or
such.
[0096] The present invention can be applied to desired solid
cancers, and such examples include tongue cancer, gum cancer,
malignant lymphoma, malignant melanoma, upper jaw cancer, nose
cancer, nasal cavity cancer, larynx cancer, pharyngeal cancer,
glioma, meningioma, glioma, lung cancer, breast cancer, pancreatic
cancer, gastrointestinal cancer (esophageal cancer, stomach cancer,
duodenal cancer, colon cancer), squamous cancer, adenocarcinoma,
alveolar cell cancer, testis tumor, prostate cancer, thyroid
cancer, liver cancer, ovarian cancer, rhabdomyosarcoma,
fibrosarcoma, osteosarcoma, and chondrosarcoma. The target cancer
is preferably epithelial cancer, and is more preferably skin cancer
including skin squamous cancer, skin basal cell cancer, Bowen's
disease, Paget's disease, and skin malignant melanoma.
[0097] The in vivo dose of the minus-strand RNA virus of this
invention (anticancer agent of this invention) varies depending on
the disease, patient's weight, age, sex, and symptom, purpose of
administration, form of administered composition, administration
method, introduced gene, and the like, but can be appropriately
determined by those skilled in the art. The route of administration
can be appropriately selected, and includes, for example,
percutaneous, intranasal, transbronchial, intramuscular,
intraperitoneal, intravenous, intraarticular, and subcutaneous
administration. The administration may be local or systemic. It is
preferred to administer the virus at a dose within the range of
preferably about 10.sup.5 to about 10.sup.11 CIU/ml, more
preferably about 10.sup.7 to about 10.sup.9 CIU/ml, and most
preferably about 1.times.10.sup.5 to about 5.times.10.sup.8 CIU/ml,
in a pharmaceutically acceptable carrier. The amount per dose for
human is preferably 2.times.10.sup.5 to 2.times.10.sup.11 CIU,
which is administered once or more within a range where the side
effects are clinically acceptable. The same applies to the number
of doses per day. Regarding nonhuman animals, for example, a dose
converted from the above-described dose based on the body weight
ratio between the subject animal and human or the volume ratio
(e.g., mean value) of the target site for administration. In
addition, when it becomes necessary to suppress the propagation of
the transmissible minus-strand RNA virus after administration to
subjects or cells due to the completion of treatment or the like,
through the administration of an RNA-dependent RNA polymerase
inhibitor, the propagation of the virus can be specifically
suppressed without damaging the host.
[0098] The minus-strand RNA viruses of the present invention are
preferably administered to the cancerous lesion of a patient.
"Cancerous lesion" refers to a region of cancer tissue or its
surrounding area (for example, within 5 mm of the cancer, or
preferably within 3 mm of the cancer). The dose may be
appropriately adjusted depending on the type and stage of the
cancer, the presence or absence of an introduced gene, and such.
Although antitumor effects are expected of minus-strand RNA viruses
even if they do not carry foreign genes, higher synergistic effects
can be expected by loading RNA viruses with, for example, an
IFN-beta gene or soluble FGF receptor gene.
[0099] The minus-strand RNA virus can be administered one or more
times as long as the side effects are clinically acceptable, and
the same applies to the number of doses administered per day.
Subjects receiving the administration are not particularly limited,
and examples include birds and mammals (human and non-human
mammals) such as chicken, quail, mouse, rat, dog, pig, cat, bovine,
rabbit, sheep, goat monkey, and human. When administering to
non-human animals, for example, an amount calculated from the
above-mentioned dose according to the body weight ratio between
human and the animal of interest can be administered.
[0100] The present invention also relates to the use of a
minus-strand RNA virus in cancer therapy. Furthermore, the present
invention relates to the use of a minus-strand RNA virus in the
production of anticancer agents (or carcinostatic agents, agents
for suppressing cancer growth, or such).
[0101] In addition, the present invention relates to a package
comprising a minus-strand RNA virus, which includes descriptions on
the use of the minus-strand RNA virus for cancer suppression (as an
anticancer agent). The minus-strand RNA virus may be suspended in a
solution such as culture solution or physiological saline solution.
"Use in cancer suppression" means that for example, a minus-strand
RNA virus, or a composition comprising such RNA virus, is used in
tumor growth suppression, cancer degeneration, cancel therapy,
treatment and therapy of cancer patients, prolongation of the life
of cancer patients, or as an anticancer agent. The descriptions can
be printed directly on the package; alternatively, a piece of paper
or a sticker containing the descriptions may be comprised in the
package. The package may be a container comprising a minus-strand
RNA virus, and in such case, the container may be, for example, a
bottle, tube, plastic bag, vial syringe, or such. Furthermore, the
package of the present invention may comprise a bag, outer box, or
such that stores the container. The package may comprise
instructions describing the method for administering a minus-strand
RNA virus, and may further comprise a syringe, catheter, and/or
injection needle for administering the minus-strand RNA virus.
[0102] Furthermore, the present invention relates to a kit for
treating cancer or for producing an anticancer agent, which
comprises at least a minus-strand RNA virus as a component. In
addition to a minus-strand RNA virus, a kit of the present
invention may appropriately comprise, for example, carries,
physiological saline solution, buffer, bottle, tube, plastic bag,
vial, and syringe. Instructions for use can also be packaged into
the kit.
[0103] All references cited herein are incorporated as a part of
this description.
EXAMPLES
[0104] Hereinbelow, the present invention will be specifically
described with reference to the Examples, but it is not to be
construed as being limited thereto.
Example 1
Antitumor Effects by a Sendai Virus that Does not Carry a
Therapeutic Gene
[0105] 1.times.10.sup.5 cells of the melanoma cell line B16F1 (ATCC
CRL6323) were inoculated subcutaneously into the ventral part of
C57BL/6 mice (6 to 8-week old, female) (n=4). Five days (day 5) and
12 days (day 12) after inoculation, 1.times.10.sup.8 PFU of Sendai
virus that does not comprise any specific therapeutic gene
(GFP-expressing SeV; SeV-GFP). Sendai virus expressing a human
soluble FGF receptor (SeV-sFGFR), or Sendai virus expressing human
soluble PDGFR.alpha. (SeV-hsPDGFR.alpha.) was injected into the
tumor. Then, the tumor size was measured over time.
[0106] As a result, in all SeV-administered groups, the tumor size
significantly decreased compared to the tumor size of the
nonadministered group (FIG. 1). Therefore, tn vivo administration
of SeV was able to exhibit antitumor effects, even when the SeV did
not carry any therapeutic gene. When SeV expressing a soluble FGF
receptor was administered, a stronger effect of tumor growth
suppression was confirmed as compared to that in the
SeV-GFP-administered group. The antitumor effect was most
significant when the soluble PDGFR.alpha.-expressing SeV was
administered, since the tumor size hardly increased.
[0107] The above revealed that SeV exhibits a sufficient antitumor
effect even if it does not carry any foreign gene such as a
therapeutic gene.
Example 2
Antitumor Effects by a Therapeutic Gene (IFN.beta.)-comprising
Sendai virus
[0108] This Example shows an example of a method for beating a
tumor by in vivo and ex vivo administration of an RNA virus
[0109] A B16 melanoma-transplanted model that expresses MHC class I
at only a very low level and exhibits poor immunogenicity was used
as a tumor model. C57BL/6 mice (6- to 8-week-old; female) (CHARLES
RIVER JAPAN, INC.) were used as the tumor model mice, and dendritic
cells were collected from C57BL/6 mice (8week-old; female) (CHARLES
RIVER JAPAN, INC.). The dendritic cells were obtained by collecting
bone marrow from thigh bones of C57BL/6 mice; removing T cells
using SpinSep.TM., murine hematopoietic progenitor enrichment
cocktail (anti-CD5 antibody, anti-CD45R antibody, anti-CD11b
antibody, anti-Gr-1 antibody, anti-TER119 antibody, anti-7/4
antibody; Stem Cell technology); then culturing the cells for one
week with the addition of IL-4 and GM-CSF. On day 0,
1.times.10.sup.5/100 .mu.L of B16 melanoma cells were
subcutaneously (s.c.) inoculated into the abdominal area of the
mice. On days 10, 17, and 24, dendritic cells without stimulation
for activation, dendritic cells activated with LPS (LPS DC), or
dendritic cells activated by introducing SeV-GFP or SeV-IFN.beta.
expressing mouse interferon .beta. (SeV GFP DC and SeV IFN.beta.
DC, respectively) were administered in the area surrounding the
tumor. Simultaneously, another experiment was carried out, wherein
the dendritic cells were administered after the pulsing with tumor
antigens (tumor lysate obtained by freeze and thaw of B16). In
addition, the antitumor effect of direct intratumoral injection of
SeV-IFN.beta. into the tumor 10 days after tumor injection (day 10)
was examined.
[0110] SeV was introduced into dendritic cells by infecting
dendritic cells cultured for one week as described above with
SeV-IFN.beta. at a MOI of 40, and culturing the cells for 8 hours.
When pulsing dendritic cells with tumor antigens, dendritic cells
cultured for one week as described above were recovered and pulsed
with tumor lysate as the tumor antigens (DC:tumor lysate=1:3),
cultured for 18 hours, infected with SeV-IFN.beta. at a MOI of 40,
and cultured for 8 hours. Then, these dendritic cells were
recovered and administered at a cell number of 5.times.10.sup.5 to
10.times.10.sup.5 cells in an area surrounding the tumor of the
mice.
[0111] As shown in FIG. 2, SeV-IFN.beta. suppressed tumor growth,
both in the case of direct intratumoral injection and in the case
of ex vivo administration through dendritic cells In particular, a
significantly strong tumor suppressing effect was observed in mice
treated with DC/SeV-IFN.beta..
[0112] The antitumor effect in each of the therapeutic groups
described above was closely examined. To assay natural killer (NK)
cell activity, spleens were excised from mice of each of the
therapeutic groups described above after 7 days from the end of
three rounds of DC therapy to prepare effector cells. .sup.51Cr
release assay was performed using Yac-1 as the target. Further, to
assay the cytotoxicity of T lymphocytes, the spleen cells remaining
from the NK cell activity assay described above were cultured for 5
days with TRP-2 peptide, a B16 tumor antigen, to use them as
effector cells. The effector cells were co-cultured with EL-4
target cells pulsed with mTRP-2 peptide, and then .sup.51Cr release
assay was performed. The rate of specific .sup.51Cr release was
calculated as follows: [(sample (cpm)-spontaneous emission
(cpm))/(maximum emission (cpm)-spontaneous emission
(cpm))].times.100 where the maximum emission was determined using
target cells incubated with 1% triton X, while spontaneous emission
was determined using target cells incubated with culture medium
alone.
[0113] The activation of natal killer (NK) cells was detected only
in mice that were directly injected with SeV, and not in he
dendritic cell injection group (FIG. 3). In contrast, the
activation of cytotoxic T lymphocytes (CTLs) was maximal in the
DC/LPS treated group and mice treated with DC/SeV-IFN.beta.,
slightly lower in the DC/SeV-GFP treated group, and was not
detected in the group of SeV-IFN.beta. direct injection (FIG. 4).
The tumor lysate pulsing had no significant influence on tumor
growth nor on CTL response. Thus, it was revealed that direct
administration of SeV-IFN.beta. strongly activates NK cells.
[0114] Therefore, the effect of tumor suppression by SeV
administration was suggested to take place through a mechanism of
NK cell activation. Furthermore, it was shown that an effective
anticancer therapeutic effect is exhibited by both the
administration of an SeV that does not carry a therapeutic gene and
the administration of an SeV carrying a gene that is expected to be
therapeutically effective.
INDUSTRIAL APPLICABILITY
[0115] The present invention provides anticancer agents that
contain a minus-strand RNA virus as an active ingredient. The RNA
viruses of the present invention exhibit effective anticancer
therapeutic effects without carrying any foreign gene that has
therapeutic effects. Therefore, they may contribute greatly to the
reduction of the cost for introducing foreign genes and to the
reduction of the operational time for virus preparation. The
present invention enables new virotherapies that use minus-strand
RNA viruses.
Sequence CWU 1
1
8 1 10 DNA Artificial artificially synthesized sequence 1
ctttcaccct 10 2 15 DNA Artificial artificially synthesized sequence
2 tttttcttac tacgg 15 3 18 DNA Artificial artificially synthesized
sequence 3 cggccgcaga tcttcacg 18 4 18 DNA Artificial artificially
synthesized sequence 4 atgcatgccg gcagatga 18 5 18 DNA Artificial
artificially synthesized sequence 5 gttgagtact gcaagagc 18 6 42 DNA
Artificial artificially synthesized sequence 6 tttgccggca
tgcatgtttc ccaaggggag agttttgcaa cc 42 7 18 DNA Artificial
artificially synthesized sequence 7 atgcatgccg gcagatga 18 8 21 DNA
Artificial artificially synthesized sequence 8 tgggtgaatg
agagaatcag c 21
* * * * *