U.S. patent application number 12/554846 was filed with the patent office on 2010-04-01 for process for facilitating nucleic acid transfer.
Invention is credited to Yu Aso, Kimi Honma, Shunji Nagahara, Takahiro Ochiya, Akihiko Sano, Masaaki Terada.
Application Number | 20100081202 12/554846 |
Document ID | / |
Family ID | 26617260 |
Filed Date | 2010-04-01 |
United States Patent
Application |
20100081202 |
Kind Code |
A1 |
Terada; Masaaki ; et
al. |
April 1, 2010 |
PROCESS FOR FACILITATING NUCLEIC ACID TRANSFER
Abstract
Means for transferring efficiently a desired nucleic acid into a
cell is provided. The present invention comprises using a complex
comprising a collagen or a collagen derivative, and a desired
nucleic acid.
Inventors: |
Terada; Masaaki; (Tokyo,
JP) ; Ochiya; Takahiro; (Tokyo, JP) ; Aso;
Yu; (Tokyo-to, JP) ; Honma; Kimi; (Tokyo-to,
JP) ; Sano; Akihiko; (Osaka, JP) ; Nagahara;
Shunji; (Osaka, JP) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Family ID: |
26617260 |
Appl. No.: |
12/554846 |
Filed: |
September 4, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10481511 |
Dec 19, 2003 |
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PCT/JP02/06137 |
Jun 20, 2002 |
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12554846 |
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Current U.S.
Class: |
435/455 |
Current CPC
Class: |
A61K 47/6435 20170801;
A61K 48/00 20130101; A61K 48/0008 20130101; C12N 15/87 20130101;
A61P 35/00 20180101 |
Class at
Publication: |
435/455 |
International
Class: |
C12N 15/63 20060101
C12N015/63 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 20, 2001 |
JP |
2001-186320 |
Sep 13, 2001 |
JP |
2001-278293 |
Claims
1. A method of transferring an oligonucleotide to a target organ or
a target tissue comprising administering an electrostatic complex
consisting essentially of an oligonucleotide and a water-soluble
atelocollagen by injection.
2. The method according to claim 1, wherein the oligonucleotide is
5-30 nucleotides in length.
3. The method according to claim 1, wherein the ratio of the number
of atelocollagen molecules to the number of nucleotide monomers of
oligonucleotide is 1:20 to 1:120.
4. The method according to claim 1, wherein the size of the
electrostatic complex is 300 nm to 30 um.
5. The method according to claim 3, wherein the oligonucleotide is
a DNA derivative or an RNA derivative.
6. The method according to claim 3, wherein the oligonucleotide is
a DNA derivative or an RNA comprising a phosphorothioate bond.
7. The method according to claim 3, wherein the oligonucleotide is
a DNA derivative or an RNA comprising an internucleotide comprising
a phosphate, sugar or base moiety chemically modified to avoid
enzymatic degradations.
8. The method according to claim 1, wherein the complex exhibits a
major axis of about 300 nm in length.
9. The method according to claim 1, wherein the complex exhibits a
major axis from 12 to 54 microns in length.
10. The method according to claim 1, wherein the ratio of the
number of oligonucleotide molecules to the number of atelocollagen
molecule is from 0.2 to 6.3.
11. The method according to claim 1, wherein the ratio of the
nucleotide monomers per the number of atelocollagen molecules is
from 4 to 126.
Description
RELATED APPLICATIONS
[0001] This application is a Divisional of co-pending Application
No. 10/481,511 filed on Dec. 19, 2003, and for which priority is
claimed under 35 U.S.C. .sctn.120; and this application claims
priority of Application No. 2001-186320 filed in Japan on Jun. 20,
2001 and for which priority is claimed under 35 U.S.C. .sctn.119;
and this application claims priority of Application No. 2001-278293
filed in Japan on Sep. 13, 2001, and for which priority is claimed
under 35 U.S.C. .sctn.119; and this application claims priority of
Application No. PCT/JP02/06137 filed in Japan on Jun. 20, 2002, and
for which priority is claimed under 35 U.S.C. .sctn.119, for which
the entire contents of all are hereby incorporated by
referenced.
FIELD OF THE INVENTION
[0002] The present invention belongs to medical field, specifically
to gene therapy and genetic fundamental research. More
specifically, the invention relates to preparations for
facilitating the transfer of a desired nucleic acid into a target
cell, and processes therefor.
BACKGROUND ART
[0003] Recently, gene therapy has been actively studied, and
applied practically to clinical therapy of various cancers and
genetic diseases. Gene therapy is an approach to treat a disease by
repairing or correcting a defective gene, and comprises
transferring a gene encoding an intended enzyme, cytokine, or the
like into a cell of a patient, and allowing to produce the intended
substance from the gene in the body, thereby treating the disease.
Gene therapy is a medication that controls a basis of life, and has
a potential to treat various diseases such as AIDS, rheumatoid
arthritis, lifestyle-related diseases, in addition to cancers and
genetic diseases.
[0004] In gene therapy, transfer efficiency of gene into a target
cell is an important factor in the increased efficacy of the
therapy. Gene therapy for cancers includes therapies by virus such
as adenovirus (Cardiovascular Research, 28, 445 (1994); Science,
256, 808 (1992); Gastroenterology, 106, 1076 (1994); TIBTECH, 11,
182 (1993); J. Biol. Chem, 266, 3361 (1991); Nature Medicine, 1,
583 (1995) and the cited references therein) and those by liposome
formulations (Biochem Biophys Acta, 1097, 1 (1991); Human Gene
Therapy, 3, 399 (1992); Proc. Natl. Acad. Sci. USA, 89, 11277
(1992)). Transfer efficiency of genes is generally higher in
therapy using virus vectors than therapy using liposome
formulations. However, therapy using virus vectors suffers from a
problem that multiple administrations are hardly conducted due to
immunological responses to viruses (J. Biol. Chem., 269,
13695(1994), Am. J. Respir. Cell Mol. Biol., 10, 369 (1994)).
[0005] On the other hand, since the analysis on the whole human
genetic information (human genome) was almost completed, the focus
has been shifted to post-genome strategies how to utilize the
accumulated human genetic information in the fields of medication
and industry. Specifically, examinations on human gene functions,
as well as the structures and functions of the proteins encoded by
the gene using the analyzed genetic information have been
emphasized. Such a post-genome examinations require the expression
and the production of proteins, which necessarily involve the
transfer of intended genes into cells. Genes to be transferred into
host cells by adenovirus vectors and liposome vectors or plasmid
DNA vectors are not integrated into the genome of the cells, and
are transiently expressed. Such vectors can not accomplish the
constitutive expression of the genes, which is important in gene
therapy and analysis on gene functions.
DISCLOSURE OF THE INVENTION
[0006] Thus, an approach to efficiently transfer a nucleic acid
representing a gene into a desired cell, and to express the gene
during a long period of time without integration of the gene into
chromosome of host cells is expected to provide a great
utility.
[0007] The inventors of the present application found that
collagens have an unexpected action, and created an approach to
efficiently transfer a nucleic acid into a desired cell.
Specifically, we found that the contact of a collagen and a nucleic
acid such as plasmid DNA surprisingly results in the formation of a
complex, and the formation of a complex facilitates the transfer of
a nucleic acid into a cell and expresses the gene during a long
period of time. Although Japanese Patent Publication (kokai) No.
71542/1997 describes formulations containing a gene wherein the
gene is comprised in a carrier of a biocompatible material such as
a collagen, the formulations are sustained release formulations
that gradually releases the gene in a living body.
[0008] The invention is based on the newly founded use of a
collagen or a collagen derivative.
[0009] More specifically, the invention relates to:
[0010] (1) A preparation for facilitating the transfer of a nucleic
acid into a target cell, which comprises a collagen or a collagen
derivative;
[0011] (2) A preparation for facilitating the transfer of a nucleic
acid into a target cell, which comprises a collagen or a collagen
derivative complexed with a desired nucleic acid, preferably a
preparation for facilitating the transfer of a nucleic acid,
wherein the complex is in a form of particle, more preferably a
preparation for facilitating the transfer of a nucleic acid,
wherein the major axis of the particle is 300 nm to 300 .mu.m,
preferably 300 nm to 100 .mu.m, more preferably 300 nm to 50 .mu.m,
even more preferably 300 nm to 30 .mu.m; Specifically, a
preparation for facilitating the transfer of a nucleic acid wherein
the desired nucleic acid is a plasmid DNA, and wherein the ratio of
the number of a collagen molecule or a collagen derivative molecule
to the number of a nucleotide monomer of the plasmid DNA in the
complex is 1:20 to 1: the number of a nucleotide monomer of the
plasmid DNA, preferably 1:50 to 1: the number of a nucleotide
monomer of the plasmid DNA, more preferably 1:50 to 1:4000, still
more preferably 1:50 to 1:2000, and still more preferably 1:50 to
1:1000, or a preparation for facilitating the transfer of a nucleic
acid wherein the nucleic acid is an oligonucleotide, and which the
ratio of the number of a collagen molecule or a collagen derivative
molecule to the number of a nucleotide monomer of the
oligonucleotide in the complex is 1:1 to 1:200, preferably 1:3 to
1:150, more preferably 1:20 to 1:120, and still more preferably
1:50 to 1:120;
[0012] (3) A particle of the complex comprising a collagen or a
collagen derivative and a desired nucleic acid;
[0013] (4) A process for preparing a particle of the complex
according to the present invention, which comprises mixing a
collagen or a collagen derivative and a desired nucleic acid in a
solution comprising an agent that inhibits the formation of
collagen association body;
[0014] (5) A medical instrument, of which the surface is coated
with a particle of the complex according to above (3) or a cell
culture instrument, of which the surface is coated with the
particle of the complex;
[0015] (6) A process for transferring a desired nucleic acid into a
target cell or a process for improving the expression level of a
desired nucleic acid in a target cell, which comprises using a
particle of the complex according to above (3);
[0016] (7) A process for examining the function of a gene or a
protein in a target cell, which comprises coating a solid surface
with a particle of the complex according to above (3) that
comprises the gene, a gene encoding the protein, or a nucleic acid
inhibiting the expression of the gene or the protein in a cell;
culturing the target cell on the solid surface; and examining the
expression level of the nucleic acid or the expression level of the
gene or the protein in the target cell, or the proliferation ratio
or the phenotype of the cell; and
[0017] (8) A process for screening for a nucleic acid that treats a
disease, which comprises coating a solid surface with a particle of
the complex according to above (3) that comprises a nucleic acid
candidate that inhibits the expression of a gene associated with
the disease in a cell; culturing the cell presenting the condition
of the disease on the solid surface; and examining the expression
level of the gene to be inhibited with each of the nucleic acid
candidate, or the proliferation ratio or the phenotype of the
cell.
[0018] The working examples hereinafter illustrate that the
preparations for facilitating the transfer of a nucleic acid
according to the present invention improved the transfer efficiency
of gene into a target cell as shown to express the nucleic acid in
a cell culture system in vitro where the gene expression is not
observed by mere plasmid DNA. Further, those examples illustrate
that the preparations for facilitating the transfer of a nucleic
acid according to the present invention increased the stability of
a nucleic acid within a cell as shown to sustain the expression of
the nucleic acid during a longer period of time than liposome
formulations.
[0019] According to the invention, it has been found that a
collagen is interacted electrostatically and/or physically with a
nucleic acid to form a complex. Thus, it is believed that the
sustained expression of a nucleic acid as observed in the working
examples would result from the complex formation leading to the
increased stability of nucleic acids within cells. This is quite
different from the mechanism of the sustained release of gene by
collagens that was conventionally understood that a gene
encapsulated in collagen matrix is gradually released according to
the biological degradation of collagen.
BRIEF DESCRIPTION OF DRAWINGS
[0020] FIG. 1 is a photograph substitute for drawing which depicts
an agarose-gel electrophoresis showing the electrostatic
interaction between the defined concentration of plasmid DNA and
the various concentrations of atelocollagen.
[0021] FIG. 2 is a photograph substitute for drawing which depicts
an agarose-gel electrophoresis showing the effect of sodium
chloride on the electrostatic interaction between plasmid DNA and
atelocollagen.
[0022] FIG. 3 is a photograph substitute for drawing which depicts
an agarose-gel electrophoresis showing the effect of heparan
sulfate on the electrostatic interaction between plasmid DNA and
atelocollagen.
[0023] FIG. 4 is a micrograph showing a form of the complexes
between plasmid DNA and atelocollagen in various
concentrations.
[0024] FIG. 5 is a micrograph showing a form of the complexes
between plasmid DNA and atelocollagen in various concentrations
that were stored for a week.
[0025] FIG. 6 is a graph showing a comparison in the duration time
of gene expression among the atelocollagen gel formulation, the
cationic liposome formulation, and the plasmid DNA in a PBS
solution.
[0026] FIG. 7 is a graph showing the relationship between the
complexes comprising a collagen in various concentrations and the
transfer efficiency of plasmid DNA seven days after the
transfection by dropwise addition.
[0027] FIG. 8 is a graph showing a fluorescence intensity
representing the transfer efficiency of plasmid DNA in the
complexes comprising a collagen in various concentrations seven
days after the transfection.
[0028] FIG. 9 is a graph showing the relationship between the
complexes comprising a collagen in various concentrations and the
transfer efficiency of plasmid DNA seven days after the
transfection by solid coating.
[0029] FIG. 10 is a graph showing the relationship between the
complexes comprising a plasmid DNA in various concentrations and
the transfer efficiency of plasmid DNA seven days after the
transfection by dropwise addition.
[0030] FIG. 11 is a graph showing the relationship between the
complexes comprising a plasmid DNA in various concentrations and
the transfer efficiency of plasmid DNA seven days after the
transfection by solid coating.
[0031] FIG. 12 is a graph showing a fluorescence intensity
representing the transfer efficiency of plasmid DNA seven days
after the transfection by dropwise addition, and a micrograph
showing the fluorescence.
[0032] FIG. 13 is a graph showing inhibitory effects of the present
invention on the cell proliferation.
[0033] FIG. 14 is a graph showing that adenovirus was transferred
by solid coating in a dose-dependent manner, and a micrograph
showing the fluorescence.
[0034] FIG. 15 is a graph showing the relationship between the
number of collagen bound to one molecule of plasmid DNA and the
average major axis of the complexes.
[0035] FIG. 16 is a graph showing the relationship between the
number of nucleotide monomer of desired nucleic acids per collagen
molecule and the average major axis of the complexes.
[0036] FIG. 17 is a graph showing the relationship between the
molecular number ratio of oligonucleotide to collagen at the time
of the mixture and the number of oligonucleotide bound to one
molecule of collagen in the complex.
[0037] FIG. 18 is a fluorescence micrograph obtained by observing
the complexes comprising the oligonucleotides released from the
surface of the cell culture instrument according to the present
invention with fluorescence microscopy.
[0038] FIG. 19 is a fluorescence micrograph obtained by observing
the complexes comprising the plasmid DNA released from the surface
of the cell culture instrument according to the present invention
with fluorescence microscopy.
BEST MODE FOR CARRYING OUT THE INVENTION
1) A Preparation for Facilitating the Transfer of a Nucleic
Acid
[0039] As the first embodiment, the invention provides a
preparation for facilitating the transfer of a nucleic acid into a
target cell, which comprises a collagen or a collagen derivative.
The embodiment is based on the effect of a collagen or a collagen
derivative on the facilitation of the transfer of a nucleic acid
into a target cell, which has been found for the first time. In
other words, the present embodiment of the invention provides a new
use of a collagen or a collagen derivative to facilitate the
transfer of a nucleic acid into a target cell.
[0040] As used herein, "a collagen or a collagen derivative"
generally means any kind of collages or collagen derivatives as
used in medical, cosmetic, industrial, and food fields. A soluble
collagen or a solubilized collagen is preferably utilized. Soluble
collagens are soluble in an acidic or neutral water or a water
containing a salt, whereas solubilized collagens include an
enzymatically solubilized collagen which may be solubilized with an
enzyme, an alkali-solubilized collagen which may be solubilized
with an alkali, both collagens being preferably capable of
penetrating through a membrane filter having a pore size of 1
micrometer. Solubility of collagen varies depending on the
crosslinking degree of the collagen, and higher is the crosslinking
degree, more difficult the collagen is solubilized. Accordingly,
the crosslinking degree of a collagen as used in the present
invention is, for example, not more than trimer, more preferably
not more than dimer. Preferable molecular weight of the collagen
is, for example, from about 300,000 to about 900,000, and more
preferably from about 300,000 to about 600,000. Collagens as used
herein include those extracted from any animal species, and it is
desired that preferable collagens are extracted from vertebrates,
more preferable collagens are extracted from a mammal, a bird, or a
fish, and still more preferable collagens are extracted from a
mammal or a bird having a high denaturation temperature. Any type
of collagen may be used, and, because of the type existing in
animal bodies, type I-V collagens are preferable. For example, such
collagens include a type I collagen obtained by acid extraction
from a mammal dermis, and, more preferably, they include, for
example, a type I collagen obtained by acid extraction from calf
dermis, a type I collagen produced by genetic engineering, and the
like. Collagens derived from tendon, which are also type I
collagens, are not suitable because they have a high degree of
crosslinking and are insoluble. Further, an atelocollagen that is
obtained by removing enzymatically a telopeptide having a high
antigenicity or an atelocollagen produced by genetic engineering is
preferable for the sake of safety, and an atelocollagen having
three or less tyrosine residues per 1000 residues is more
preferable. Alternatively, collagens having a modified side chain,
crosslinked collagens or the like may be utilized if desired.
Collagens having a modified side chain includes, for example,
succinylated collagens and methylated collagens, whereas
crosslinked collagens include, for example, collagens treated with
glutaraldehyde, hexamethylene diisocyanat, a polyepoxy compound or
the like (Fragrance Journal 1989-12, 104-109, Japanese Patent
Publication(kokai) No. 59522/1995). Preferred collagen derivatives
are a gelatin or a gelatin-crosslinking complex, or a crosslinking
complex thereof with a collagen.
[0041] Collagens or collagen derivatives may be used in admixture
with another biocompatible material. Biocompatible materials
include, for example, gelatin, fibrin, albumin, hyaluronic acid,
heparin, chondroitin sulfate, chitin, chitosan, alginic acid,
pectin, agarose, hydroxyapatite, polypropylenes, polyethylenes,
polydimethylsiloxane, and a polymer of glycolic acid, lactic acid
or amino acid, and a copolymer thereof, and a mixture containing
two or more of those biocompatible materials.
2) A Preparation for Facilitating the Transfer of a Nucleic Acid,
Which Comprises a Complex
[0042] As the second embodiment, the present invention provides a
preparation for facilitating the transfer of a nucleic acid into a
target cell, which comprises a collagen or a collagen derivative
and a desired nucleic acid, preferably comprises particles of a
complex as an essential component.
[0043] Nucleic acids are hardly transferred into cells when
administered to the cells in vitro solely in the presence of blood
serum. Nucleic acids can be efficiently transferred into cells when
formed with a collagen or a collagen derivative into a complex.
[0044] As used herein, "a nucleic acid" may be any polynucleotide
or any oligonucleotide, and may be any DNA or RNA molecule. DNA
molecules include a plasmid DNA, cDNA, a genomic DNA or a
synthesized DNA. Both DNA and RNA may be double-stranded or
single-stranded. Single-stranded ones include a coding strand and a
non-cording strand. As used herein, "a nucleic acid" includes a DNA
derivative and an RNA derivative, which derivative means a nucleic
acid having a phosphorothioate bond, or a nucleic acid containing
an internucleotide having a phosphate, sugar or base moiety
chemically modified to avoid enzymatic degradations. As used
herein, "a nucleic acid" also includes viruses such as adenovirus
and retrovirus.
[0045] Preferably, "a nucleic acid" is an oligonucleotide or a
ribozyme, and more preferably an oligonucleotide or a ribozyme that
is from 5 to 100 mer, more preferably from 5 to 30 mer. It is
preferred to utilize a plasmid DNA encoding a protein exhibiting a
physiological activity to treat or ameliorate pathological
conditions or a plasmid DNA encoding a protein inducing an
immunological response to treat or ameliorate pathological
conditions.
[0046] When the nucleic acid is a vector as used in gene therapy
such as a plasmid DNA or a virus, it is preferably a system as
constructed to express the encoded genetic information in cells,
such as a vector that comprises an element such as a promoter
necessary to express an intended gene, or an element capable of
integrating into chromosomes. Size of plasmid DNAs as a nucleic
acid used herein is not limited, and may be selected appropriately
from the sizes that allow the encoded genetic information to be
efficiently prepared via genetic engineering, and efficiently
expressed in cells to be transferred.
[0047] The preparation for facilitating the transfer of a nucleic
acid according to the invention may contain a few kinds of separate
vectors incorporated with different desired nucleic acids. Further,
a vector may comprise many genetic information. Amounts of the
vector comprised in the preparation for facilitating the transfer
are not limited.
[0048] Nucleic acids encoding a protein necessary to be expressed
in gene therapy includes any gene capable to be used in the
treatment of a genetic disease, which is exemplified by, but is not
limited to, a gene encoding an enzyme such as adenosine deaminase,
thymidine kinase; a cytokine such as GM-CSF, IL-2; or fibroblast
growth factor HST-1 (FGF4). Nucleic acids encoding other proteins
necessary to be expressed in gene therapy includes, but is not
limited to, a gene aimed at the treatment or the prevention for an
infection or a tumor, which encodes a protein or a peptide serving
as an antigen to induce immune response, i.e., the gene encoding
the protein or the peptide capable of serving as an antigen such as
mentioned above, for example, a gene encoding the surface protein
HA or NA, or the nuclear protein NP of influenza virus, type C
hepatitis virus E2 or NS 1 protein, type B hepatitis virus HBs
antigen protein, type A hepatitis virus capsid protein VP1 or VP3
or capsidoid protein, dengue virus Egp protein, RS virus F or G
protein, G or N protein of the rabies virus structural protein,
herpes virus gD protein, Japanese encephalitis virus E1 or pre-M
protein, rotavirus coat protein VP7 or coat protein VP4, human
immunodeficiency virus gp120 or gp160 protein, Leishmania major
surface antigen protein, malaria circum sporozoite major surface
antigen protein, Toxoplasma 54-kd or CS protein, cell surface
protein PAc of caries-causing Streptococcus mutans; a gene encoding
tumor regression antigens such as MAGE-1, MAGE-3, and BAGE,
tissue-specific antigens such as tyrosinase, Mart-1, gp100 and
gp75, p15, Muc1, CEA, HPV, E6, E7, HPR2/neu, etc.; and the genes
which are described in "Immunization with DNA"; Journal of
Immunological Methods, vol. 176, 1994, pages 145-152.
[0049] In the case that nucleic acids are oligonucleotides, they
includes a base sequence, of which at least the portion binds
complementarily under physiological condition to a sense or
antisense strand of a gene encoding a protein that has a
physiological effect to disrupt the homeostasis of a living body, a
gene specific to pathogenic viruses, bacteria or the like, and,
more specifically, they include a base sequence that binds
complimentarily to the messenger RNA of a gene specific to a
pathogenic virus, a bacterium or the like, or a gene encoding a
protein having a physiological effect to disrupt the homeostasis of
a living body.
[0050] More specifically, oligonucleotides as used in the present
invention includes a sequence complementary to a region containing
an initiation codon of a messenger RNA, or to a splicing site of a
precursor messenger RNA. Examples of the oligonucleotides as used
in the present invention include, for example, those used for
treatment or prevention of a cancer, such as an oligonucleotide
having a sequence of 5'-CTCGTAGGCGTTGTAGTTGT-3' (SEQ ID NO:1) which
specifically inhibits the expression of hst-1; ISIS3521 which
specifically inhibits the expression of protein kinase C.alpha. to
effectively treat progressive cancers such as non-small cell lung
carcinoma and colon cancer, and which has been applied in the
trials to the treatment of prostate cancer, breast cancer, ovary
cancer, pancreas cancer, large intestinal cancer, small cell lung
carcinoma; ISIS5132/CGP69846A which specifically inhibits the
expression of C-raf kinase and has been applied in the trials to
the treatment of prostate cancer, breast cancer, ovary cancer,
cephalophyma, pancreas cancer, large intestinal cancer, small cell
lung carcinoma; ISIS 2503 which specifically inhibits the
expression of Ha-ras and has been applied in the trials to the
treatment of large intestinal cancer, breast cancer, cephalophyma,
pancreas cancer, and small cell cancer; GEM231 which specifically
inhibits the expression of protein kinase A type I; MG98 which
specifically inhibits the expression of DNA methyl transferase;
INXC-6295 which inhibits the expression of c-myc; INX-3001 which
inhibits the expression of c-myb and has been considered to be
applied to the treatment of leukemia; G-3139 (Genasense) which
inhibits expression of bc1-2 and has been considered to be applied
to the treatment of non-Hodgkin's lymphoma, large intestinal
cancer, small cell lung carcinoma, chronic lymphatic leukemia,
acute myeloid leukemia, breast cancer, lymphoma, melanoma, myeloma,
non-small cell lung carcinoma, prostate cancer; an oligonucleotide
which inhibits the expression of MDM2 protein; an oligonucleotide
which inhibits the expression of VEGF and the like. Further,
examples of the oligonucleotides used for treatment or prevention
of infectious diseases include GEM92 and GPI-2A which inhibits the
growth of HIV. ISIS2922 (fomivirsen), Vitravene, ISIS13312 and
GEM132 which inhibits the growth of cytomegalovirus, ISIS14803
which inhibits the growth of hepatitis C virus, and the like.
Examples of the oligonucleotides used for treatment of inflammation
include ISIS2302 which specifically inhibits the expression of
ICAM-1, and which has been applied in the trials to the treatment
of Crohn's disease, ulcerative colitis, kidney transplantation
rejection inhibition, psoriasis, asthma; EPI-2000 which inhibits
the expression of adenosine A1 receptor and has been applied in the
trials to the treatment of asthma; and oligonucleotides which
inhibit the expression of TNF-.alpha., CD49d (VLA-4), VCAM-1,
PECAM-1 and the like. Further, examples of the oligonucleotides
that prevent the restenosis after percutaneous transluminal
coronary angiogenesis include Resten-NG that inhibits the
expression of c-myc.
[0051] Genes encoding a protein that exhibits a physiological
effect to disrupt the homeostasis include, for example, a series of
genes, so-called cancer genes. Specifically, they include genes for
growth factors, receptor type tyrosine kinases, non-receptor type
tyrosine kinases, GTP-binding proteins, serine-threonine kinases,
transcription factors and the like. More specifically, they include
genes coding for hst-1 or ornithine decarboxylase and the like.
[0052] The preparation for facilitating the transfer of a nucleic
acid according to the present invention may further comprise a
pharmaceutically acceptable additive as appropriate in addition to
the complex of the present invention. Pharmaceutically acceptable
additives include an agent making isotonic, a pH modifier, and a
soothing agent in case of the use of the complex as injection, and
an excipient, a disintegrator, and a coating agent in case of the
use of the complex as solid, as well as those described in Japanese
Handbook of Pharmaceutical Excipients (Japan Pharmaceutical
Excipients Council). Specific examples include salts and
saccharides, which are used to keep the pH 6-8, or to make isotonic
with cells.
[0053] The preparation for facilitating the transfer of a nucleic
acid according to the present invention may be in a form of solid
or solution. The preparation in a form of solid is loaded to a
desired cell as it is, or after making a solution with a purified
water, a physiological solution, a buffer isotonic with living
bodies, or the like. Such preparation in a solution form also
constitutes a part of the present invention.
[0054] The administration of the preparation for facilitating the
transfer of a nucleic acid according to the present invention may
be selected from oral route, injection, eye drop, nasal drop,
transpulmonary route, transdermal absorption, and oral route and
injection are preferred. The preparation may be administered to
various sites depending on diseases, and may be placed on the
necessary site at the time of operation.
[0055] In the present embodiment, the invention provides: [0056]
(1) A preparation for facilitating the transfer of a nucleic acid
into a target cell, which comprises as an essential component a
complex, preferably particles of a complex comprising a desired
nucleic acid and a collagen or a collagen derivative, wherein the
major axis of the particle is preferably 300 nm to 300 .mu.m, more
preferably 300 nm to 100 .mu.m, even more preferably 300 nm to 50
.mu.m, still more preferably 300 nm to 30 .mu.m; [0057] (2) The
preparation for facilitating the transfer of a nucleic acid
according to (1) wherein the target cell is an animal cell; [0058]
(3) The preparation for facilitating the transfer of a nucleic acid
according to (2) wherein the target cell is an organ or a tissue
which requires to be treated, or its cell around;
[0059] (4) The preparation for facilitating the transfer of a
nucleic acid according to any one of (1) to (3) wherein the
collagen is atelocollagen:
[0060] (5) The preparation for facilitating the transfer of a
nucleic acid according to any one of (1) to (4) wherein the
molecular weight of the collagen is from about 300,000 to about
900,000; [0061] (6) The preparation for facilitating the transfer
of a nucleic acid according to any one of (1) to (5) wherein the
collagen derivative is a gelatin or a gelatin-crosslinking complex;
[0062] (7) The preparation for facilitating the transfer of a
nucleic acid according to any one of (1) to (5) wherein the
collagen derivative is a collagen-crosslinking complex; [0063] (8)
The preparation for facilitating the transfer of a nucleic acid
according to any one of (1) to (7) wherein the nucleic acid is an
oligonucleotide; [0064] (9) The preparation for facilitating the
transfer of a nucleic acid according to (8) wherein the
oligonucleotide is from 5 to 30 mer in length; [0065] (10) The
preparation for facilitating the transfer of a nucleic acid
according to (8) or (9) wherein the oligonucleotide is a DNA or a
DNA derivative; [0066] (11) The preparation for facilitating the
transfer of a nucleic acid according to (8) or (9) wherein the
oligonucleotide is an RNA or an RNA derivative; [0067] (12) The
preparation for facilitating the transfer of a nucleic acid
according to (10) or (11) wherein the DNA derivative or the RNA
derivative has at least one phosphorothioate bond; [0068] (13) The
preparation for facilitating the transfer of a nucleic acid
according to any one of (1) to (7) wherein the nucleic acid is a
ribozyme or an oligonucleotide; [0069] (14) The preparation for
facilitating the transfer of a nucleic acid according to any one of
(1) to (7) wherein the nucleic acid is a plasmid DNA; [0070] (15)
The preparation for facilitating the transfer of a nucleic acid
according to (13) wherein the plasmid DNA encodes a protein that
exhibits a physiological activity to treat or ameliorate
pathological conditions; [0071] (16) The preparation for
facilitating the transfer of a nucleic acid according to (13)
wherein the plasmid DNA encodes a protein that induces an
immunological response to treat or ameliorate pathological
conditions; [0072] (17) The preparation for facilitating the
transfer of a nucleic acid according to (1) to (16) wherein the
preparation is in a form of solution, and the complex comprises 10
mg/ml or less of a nucleic acid; [0073] (18) The preparation for
facilitating the transfer of a nucleic acid according to (17)
wherein the complex comprises 1 mg/ml or less of a nucleic acid;
[0074] (19) The preparation for facilitating the transfer of a
nucleic acid according to (18) wherein the complex comprises 500
.mu.g/ml or less of a nucleic acid; [0075] (20) The preparation for
facilitating the transfer of a nucleic acid according to (1) to
(19) wherein the preparation has pH5 to pH9, preferably, pH6 to
pH8; [0076] (21) The preparation for facilitating the transfer of a
nucleic acid according to (1) to (20) further comprising phosphoric
acid in a concentration from 0.001M to 0.1M; [0077] (22) The
preparation for facilitating the transfer of a nucleic acid
according to (21) further comprising phosphoric acid in a
concentration from 0.01M to 0.1M; [0078] (23) The preparation for
facilitating the transfer of a nucleic acid according to (1) to
(22) further comprising an agent that inhibits the formation of
collagen association body, for example sucrose or arginine; and
[0079] (24) The preparation for facilitating the transfer of a
nucleic acid according to (1) to (24) further comprising an
appropriate amount of a pharmaceutically acceptable additive.
3) Complexes
[0080] As the third embodiment, the present invention provides a
complex comprising a collagen or a collagen derivative and a
desired nucleic acid. The complexes include electrostatic complexes
that are bound and formed by the electrostatic force, and physical
complexes that are bound and formed by the physical force such as
hydrophobic binding. Both binding systems may coexist, and this
embodiment is also fallen in the scope of the present
invention.
[0081] It has been found for the first time that a collagen is
electrostatically interacted with a nucleic acid to form a
complex.
[0082] As used herein, "electrostatic complexes" means polyionic
complexes between a collagen or a collagen derivative having many
electric charges in the molecule and a nucleic acid, and
specifically means binding bodies wherein a collagen or a collagen
positively charged electrically attract to a nucleic acid
negatively charged. In the polyionic complexes, many counter ions
are released from the molecule on the complex formation, and
therefore very large increase in entropy is produced. In view of
the formation of such electrostatic complexes, it is understood
that the sustained expression of a nucleic acid as observed in the
working examples would result from the complex formation leading to
the increased stability of nucleic acids within cells.
[0083] Minimum unit of the complex of the present invention is a
complex formed by one molecule of collagen and one molecule of
nucleic acid. We found that a collagen forms a complex with a
nucleic acid, and stimulatingly associates with another collagen to
form an association body. The association body is formed in a
manner that a collagen molecule having a cylindrical shape wherein
the major axis is about 300 nm and the diameter is 1.5 nm is
predominantly associated parallel to the longitudinal axis of the
molecule. Accordingly, the complexes are formed by the noncovalent
binding between many association bodies and many nucleic acid
molecules, and include filamentous complexes having a longitudinal
axis of 1 nm or more as a result of the largely developed extension
of the bodies, filamentous complexes wherein fine bodies are each
bound via nucleic acids, and particulate complexes formed by more
fine complexes and nucleic acids.
[0084] The complexes of the present invention can be various in
shape.
[0085] The complexes of the present invention are preferably in a
form of particle. As used herein "particle" means a shape that a
collagen or a collagen derivative could assume, and does not
necessarily mean a spherical shape. Minimum size of the complexes
of the present invention is 300 nm that corresponds to the major
axis of a complex formed by one molecule of collagen. The particles
have a major axis of 300 nm to 1 mm, and in view of transfer
efficiency of nucleic acids, they preferably have a major axis of
300 nm to 300 .mu.m, more preferably 300 nm to 100 .mu.m, even more
preferably 300 nm to 50 .mu.m, still even more preferably 300 nm to
30 .mu.m.
[0086] We found that the ratio of a collagen or a collagen
derivative to a nucleic acid composed in a complex is responsible
for the fact that the complex can assume various forms or shapes,
and that the form of the complex is dependent exclusively on the
development of the association (fibrosis) of the collagen or
collagen derivative promoted by the formation of the complex with
the nucleic acid. We also found that the excess formation of the
association bodies is not suitable for the transfer of the nucleic
acid, and found that the form or the shape of the complex could be
controlled by adjusting the concentration of the collagen or
collagen derivative and the desired nucleic acid to be mixed, as
well as environmental factors such as salt concentration,
temperature, pH, and glucose concentration. Further, we observed
that a plasmid DNA having 1000 by or more is extremely promoted in
the association, whereas a plasmid DNA having a 100 by or less is
weakly promoted in the association, and found that the association
of collagen or collagen derivative involved in the complex
formation is affected by the length of the nucleic acid, concluding
that there is a composition of a collagen or collagen derivative
and a nucleic acid optimal for the association depending on the
length of the nucleic acid.
[0087] The form of the shape of the complex may affect the transfer
efficiency of nucleic acids into cells. The finding that collagens
are complexed with nucleic acids, and the shape of the complex
affects the transfer efficiency and the expression efficiency of
nucleic acids in cells provides a strategy for optimizing the
transfer efficiency of nucleic acids. Liposomes as used in
laboratory to transfer nucleic acids into target cells are
difficult to be widely utilized from practical view points, since
they tend to aggregate together immediately when combined with
nucleic acids, are varied in transfer efficiency on use, and are
necessarily prepared just before use. However, the complexes of the
present invention are stable in the form or the shape when stored
in cold space. The most important issue in gene transfer technique
is that widely used methods are quite few. The complexes of the
present invention are stable in the form or the shape and could be
used practically.
[0088] In the present embodiment, the invention provides: [0089]
(1) A particle of the complex comprising a desired nucleic acid and
a collagen or a collagen derivative; [0090] (2) The particle of the
complex according to (1), wherein the major axis is 300 nm to 300
.mu.m; [0091] (3) The particle of the complex according to (2),
wherein the major axis is 300 nm to 100 .mu.m; [0092] (4) The
particle of the complex according to (3), wherein the major axis is
300 nm to 50 .mu.m, preferably 300 nm to 30 .mu.m; [0093] (5) The
particle of the complex according to (1) to (4), wherein the
nucleic acid is an plasmid DNA; [0094] (6) The particle of the
complex according to (5), wherein the ratio of the number of a
collagen molecule or a collagen derivative molecule to the number
of a nucleotide monomer of the plasmid DNA is 1:20 to 1: the number
of a nucleotide monomer of the plasmid DNA, preferably 1:50 to 1:
the number of a nucleotide monomer of the plasmid DNA, more
preferably 1:50 to 1:4000, even more preferably 1:50 to 1:2000,
still even more preferably 1:50 to 1:1000; [0095] (7) The particle
of the complex according to (6), wherein the ratio is 1:96 to 1:
the number of a nucleotide monomer of the plasmid DNA; [0096] (8)
The particle of the complex according to (7), wherein the ratio is
1:96 to 1:1122; [0097] (9) The particle of the complex according to
(8), wherein the ratio is 1:96 to 1:701; [0098] (10) The particle
of the complex according to (1) to (4), wherein the nucleic acid is
an oligonucleotide; [0099] (11) The particle of the complex
according to (10), wherein the ratio of the number of a collagen or
a collagen derivative to the number of a nucleotide monomer of the
oligonucleotide in the complex is 1:1 to 1:200, preferably 1:3 to
1:150, more preferably 1:3 to 1:120; [0100] (12) The particle of
the complex according to (11), wherein the ratio is 1:20 to 1:120;
[0101] (13) The particle of the complex according to (12), wherein
the ratio is 1:50 to 1:120; [0102] (14) The particle of the complex
according to (1) to (13), which is comprised in a solution of pH 5
to pH 9, preferably pH 6 to pH 8; [0103] (15) A process for
preparing a particle of the complex according to (1) to (13), which
comprises mixing a collagen or a collagen derivative and a desired
nucleic acid in a solution cooled to 10 .degree. C. or less; [0104]
(16) A process for preparing a particle of the complex according to
(1) to (13), which comprises mixing a collagen or a collagen
derivative and a desired nucleic acid in a solution comprising
phosphoric acid in a concentration from 0.001M to 0.1M; [0105] (17)
A process for preparing a particle of the complex according to (1)
to (13), which comprises mixing a collagen or a collagen derivative
and a desired nucleic acid in a solution comprising phosphoric acid
in a concentration from 0.01M to 0.1M; [0106] (18) A process for
preparing a particle of the complex according to (1) to (13), which
comprises mixing a collagen or a collagen derivative and a desired
nucleic acid in a solution comprising an agent that inhibits the
formation of collagen association body, for example sucrose or
arginine; [0107] (19) A process for preparing a particle of the
complex according to (1) to (13), which comprises mixing a collagen
or a collagen derivative and a desired nucleic acid in a solution
comprising an appropriate amount of a pharmaceutically acceptable
additive wherein the pharmaceutically acceptable additive is as
defined above; [0108] (20) A process for preparing a particle of
the complex according to (1) to (13), which comprises combining two
or more of the processes according to (14) to (19); [0109] (21) A
process for preparing a particle of the complex according to (1) to
(13), which further comprises penetrating the complex through a
filter having a pore size of 100 .mu.m or less for size selection.
[0110] (22) The process according to (21), which comprises
penetrating the complex through a filter having a pore size of 10
.mu.m or less for size selection; [0111] (23) A process for
preparing a particle of the complex according to (1) to (13), which
further comprises centrifuging the complex at 10,000 rpm or more
for concentration and isolation; [0112] (24) The process according
to (23), which comprises centrifuging the complex at 50,000 rpm or
more.
[0113] As used herein, "the number of a nucleotide monomer" means
the number of nucleotide monomer units composing of a desired
nucleic acid. As used herein, "the ratio of the number of a
collagen molecule or a collagen derivative molecule to the number
of a nucleotide monomer" means the number of nucleotide monomers in
a desired nucleic acid relative to one molecule of a collagen or
collagen derivative comprising an electrostatically or physically
bound complex. The number of a nucleotide monomers is almost the
same as "negative charge of a desired nucleic acid". As used
herein, "negative charge of a desired nucleic acid" means the
number of phosphate groups intervening between nucleotides
composing of a nucleic acid. The numeral values of the negative
charge of a nucleic acid is equal to the number of phosphate groups
of the nucleic acid, phosphate groups intervening between
nucleotides, and phosphorus-containing groups intervening between
nucleotides (phosphate groups and phosphorothioate groups).
[0114] As used herein, "an agent that inhibits the formation of
collagen association body" includes an agent that inhibits
electrostatic interactions that would cause the formation of
collagen association body via charges of basic and acidic amino
acids comprised mainly in a collagen molecule, and an agent that
inhibits the ordered arrangement of collagen molecules. Examples of
the former include salts amino acids, and urea, and examples of the
latter include saccharides such as sucrose, and arginine.
[0115] The particle of the complex according to the invention may
be comprised in the preparation for facilitating the transfer of a
nucleic acid according to the invention.
4) Medical Instruments and Cell Culture Instruments
[0116] As the fourth embodiment, the invention provides a medical
instrument or a cell culture instrument, of which the surface is
coated with a particle of the complex according to the present
invention.
[0117] It has been found that the transfer efficiency of a nucleic
acid is improved when a particle of the complex according to the
present invention is coated onto a solid surface, and target cells
are contacted thereto, compared to dropwise addition of a particle
of the complex according to the present invention to the target
cells, in other words, solid coating of the complex particles is
superior to dropwise addition in terms of transfer efficiency (as
shown in Example 6).
[0118] Specifically, medical instruments and cell culture
instruments according to the present embodiment include artificial
vascular grafts, medical stents, and artificial hearts. Artificial
vascular grafts are required to allow vascular endothelial cells to
proliferate and spread in their inner side in order to inhibit
fibrin development and suppress the complement activation within
the vessels. Thus, when coated onto artificial vascular grafts, a
particle of the complex according to the present invention wherein
nucleic acids encoding endothelial growth factors such as a
vascular endothelial growth factor (VEGF) are bound to a collagen
is expected to readily and rapidly proliferate vascular endothelial
cells.
[0119] Cell culture instruments, of which the surface is coated
with a particle of the complex comprising a desired nucleic acid
and a collagen or a collagen derivative include plates, flasks,
96-well microplates as usually used in cell culture experiments.
Example 6 hereinafter demonstrated that amounts of the complex
particles coated onto the solid surface per unit area drastically
affect transfer efficiency of nucleic acids into cells.
Accordingly, the amounts of the complex particles coated onto the
solid surface per unit area constitute a part of the present
invention.
[0120] In the present embodiment, the invention specifically
provides: [0121] (1) A medical instrument or a cell culture
instrument that is coated with a particle of the complex comprising
a desired nucleic acid and a collagen or a collagen derivative;
[0122] (2) The medical instrument or the cell culture instrument
according to (1), wherein the particle of the complex is coated so
that an amount from 0.1 .mu.g to 50 .mu.g of a nucleic acid is
comprised in 1 square centimeter; [0123] (3) The medical instrument
or the cell culture instrument according to (1), wherein the
particle of the complex is coated so that an amount from 0.1 .mu.g
to 50 .mu.g of a nucleic acid is comprised in 1 square centimeter;
[0124] (4) The medical instrument or the cell culture instrument
according to (3), wherein the particle of the complex is coated so
that an amount from 1 .mu.g to 10 .mu.g of a nucleic acid is
comprised in 1 square centimeter; [0125] (5) The medical instrument
or the cell culture instrument according to (1), wherein the
particle of the complex having a major axis of 300 nm to 300 .mu.m
is released from the solid surface when exposed in a solution
isotonic with a living body; and [0126] (6) The medical instrument
or the cell culture instrument according to (5), wherein the
solution isotonic with a living body is a phosphate buffer
comprising sodium chloride.
[0127] Feasible distribution of the invention on the market is
important to carry out the present embodiment. As described above,
liposomes are required to be prepared just before use, meaning
extremely poor distribution. In general, it is believed difficult
to distribute adenovirus as coated onto the solid surface according
to the present embodiment. However, it has been found unexpectedly
that adenovirus as coated and dried on solid surface together with
a collagen according to the present invention retain the
infectivity at room temperature for 7 days. This shows that the
medical instrument and the cell culture instrument according to the
invention could be feasibly distributed on the market.
5) A Process for Transferring a Desired Nucleic Acid into a Target
Cell or a Process for Improving the Expression Level of a Desired
Nucleic Acid in a Target Cell
[0128] As described above, a particle of the complex according to
the present invention can be used to facilitate the transfer of a
desired nucleic acid into a target cell. Thus, as the fifth
embodiment, the present invention provides a process for
transferring a desired nucleic acid into a target cell or a process
for improving the expression level of a desired nucleic acid in a
target cell, which comprises using a particle of the complex
according to the present invention. As used herein, "improving the
expression level" means the increasing of the expression level of a
desired nucleic acid or the extension of the duration time of the
expression.
[0129] Thus, in the present embodiment, the invention provides:
[0130] (1) A process for transferring a desired nucleic acid into a
target cell, which comprises using a particle of the complex as
defined above comprising a desired nucleic acid and a collagen or a
collagen derivative; [0131] (2) A process for improving the
expression level of a desired nucleic acid in a target cell, which
comprises using a particle of the complex as defined above
comprising a desired nucleic acid and a collagen or a collagen
derivative; [0132] (3) The process according to (2), wherein the
process is to improve the expression level, or to extend the
duration time of the expression; [0133] (4) The process according
to any one of (1) to (3), wherein the particle of the complex as
defined above is coated onto the solid surface, and the target cell
is cultured on the solid surface. [0134] 6) A process for examining
the function of a gene or a protein in a target cell
[0135] According to the present invention wherein the transfer of a
nucleic acid into a cell is facilitated, it is possible to readily
examine the function of a gene or a protein in a target cell. For
example, human genome project identify a large number of genes, and
showed the base sequences thereof. It is necessary to clarify the
functions of identified genes in order to utilize those information
practically in the field of medication or food industry. However,
it has been clear that the conventional approach to examine the
function by producing and purifying proteins from genes one by one
is time-consuming and not practical. Accordingly, a process for
examining the function of a gene which comprises transferring and
expressing a plasmid DNA incorporated with a gene to be examined
into a cell, or a process for examining the function of a gene
which comprises transferring an antisense oligonucleotide that
inhibits the expression of a gene to be examined into a cell and
inhibiting the gene expression should be useful. In case of the
examination of the gene function by phenotype of the cell as shown
above, it is necessary to conduct the process for transferring a
plasmid DNA or an antisense DNA into a cell without adverse
affection to the cell as much as possible. Thus, a liposome, which
is high in cytotoxicity, would affect the information as obtained
due to its cytotoxicity. On the other hand, a collagen as used
herein hardly affect the cells since a collagen originally exist in
a living body and contacts with the cells, and therefore the
invention enables the measurement of gene functions without noise.
Particular measurements comprise mixing a plasmid DNA or an
adenovirus expressing a gene to be examined, or an antisense
oligonucleotide inhibiting the expression of a gene to be examined,
allowing the formation of particles of the complex, then coating
and arranging the same on the solid surface of the culture plate.
Solid plates as used herein include 96-well multiwellplates and
microplates. After the coated complex particles are dried and
immobilized on the solid, cells are seeded and cultured on the
plate for several days. The coated complex particles are
transferred efficiently into cells attached to the coated part, and
allow the expression of a gene to be examined or the inhibition of
the same for a long period of time. After a few days, the functions
of target genes can be clarified by examining the morphology of the
cells, the level of the gene expression in the cells, or the kinds
or the amounts of the proteins produced by the cells. The features
of the present invention also include selective and efficient
transfer of the complex particles coated onto the solid into the
cells attached to the coated part. In other words, it is possible
to examine the functions of a large number of genes on microplates
at a time without computing of the cells by wells.
[0136] In the present embodiment, the invention provides: [0137]
(1) A process for examining the function of a gene or a protein in
a target cell, which comprises coating a solid surface with a
particle of the complex according to above (3) of the present
invention that comprises the gene, a gene encoding the protein, or
a nucleic acid inhibiting the expression of the gene or the protein
in a cell; culturing the target cell on the solid surface; and
examining the expression level of the nucleic acid or the
expression level of the gene or the protein in the target cell, or
the proliferation ratio or the phenotype of the cell; [0138] (2)
The process according to (1), wherein the gene or a nucleic acid
encoding the protein is a plasmid DNA, and the expression level of
the nucleic acid is examined; and [0139] (3) The process according
to (1), wherein the gene or a nucleic acid that inhibits the
expression of the protein in a cell is an antisense oligonucleotide
or a ribozyme, and the expression level of the gene or the protein
is examined. [0140] (7) A process for screening for a nucleic acid
that treats a disease
[0141] According to the present invention wherein the transfer of a
nucleic acid into a cell is facilitated, it is possible to
screening for a nucleic acid that is capable to compensate normal
genes, repair or correct defective genes so as to treat various
diseases such as genetic diseases, cancers, AIDS, rheumatoid
arthritis, lifestyle-related diseases. For example, after the
complex particles are formed with a nucleic acid that is examined
for a therapeutic effect on diseases and a collagen, and are
coated, dried and immobilized on the solid as described in 6)
above, cells presenting pathological condition are cultured on the
plate, transferring efficiently the nucleic acid into the cell
presenting pathological condition. Effects of the nucleic acid may
be examined on the basis of change in the morphology of the cells,
cell death, cell proliferation, pattern of the gene expression in
the cells, or the kinds or the amounts of the proteins produced by
the cells. It is evident that, in the nucleic acid transfer in the
embodiment, affection by vectors to be transferred should be
minimized, and a collagen as used herein makes it possible to
examine the effect on pathological condition without noise. The
features of the present invention also include coincidental
examinations of a large number of genes, as shown by the fact that
nucleic acids that is to be examined for function can be
immobilized and arranged on a cell culture solid carrier having a
tiny area.
[0142] In the present embodiment, the invention provides: [0143]
(1) A process for screening for a nucleic acid that treats a
disease, which comprises coating a solid surface with a particle of
the complex according to the present invention that comprises a
nucleic acid candidate that inhibits the expression of a gene
associated with the disease in a cell; culturing the cell
presenting the condition of the disease on the solid surface; and
examining the expression level of the gene to be inhibited with
each of the nucleic acid candidate, or the proliferation ratio or
the phenotype of the cell; [0144] (2) A process for examining a
therapeutic effect of a nucleic acid to be expected to inhibit the
expression of a gene associated with a disease, which comprises
coating a solid surface with a particle of the complex according to
the present invention that comprises the nucleic acid; culturing
the cell presenting the condition of the disease on the solid
surface; and examining the expression level of the gene, or the
proliferation ratio or the phenotype of the cell; [0145] (3) The
process according to (1) or (2), wherein the nucleic acid is to
inhibit the expression of the gene associated with the disease in
the cell, or to have a function that inhibits the expression of the
gene associated with the disease in the cell; and [0146] (4) The
process according to (3), wherein the nucleic acid that inhibits
the expression of the gene associated with the disease in the cell
is a plasmid DNA encoding the gene, or the nucleic acid that have a
function that inhibits the expression of the gene associated with
the disease in the cell is an antisense oligonucleotide or a
ribozyme.
8) Other Embodiments
[0147] In the present embodiments, the invention provides: [0148]
(1) A cell culture instrument, of which the surface is coated with
a desired nucleic acid together with a collagen or a collagen
derivative; preferably the cell culture instrument wherein the
desired nucleic acid is complexed with the collagen or the collagen
derivative to form a particle of the complex; [0149] (2) A cell
culture instrument, of which the surface is coated with a film
comprising a collagen or a collagen derivative containing a desired
nucleic acid; preferably the cell culture instrument wherein the
desired nucleic acid is complexed with the collagen or the collagen
derivative to form a particle of the complex; [0150] (3) A cell
culture instrument, on which the surface is coated with a film
comprising a collagen or a collagen derivative, and a desired
nucleic acid; [0151] (4) The cell culture instrument according to
(1) to (3), wherein the nucleic acid constitutes a library; [0152]
(5) The cell culture instrument according to (4), wherein the
nucleic acid is a cDNA or oligonucleotide that constitutes a
library; [0153] (6) The cell culture instrument according to (4) or
(5), wherein the nucleic acid that constitutes a library is
comparted each other at a distance; [0154] (7) A film comprising a
collagen or a collagen derivative and a desired nucleic acid
wherein the nucleic acid that constitutes a library is comparted
each other at a distance; preferably the film wherein the nucleic
acid is complexed with the collagen or the collagen derivative to
form a particle of the complex; [0155] (8) The film according to
(7), wherein the nucleic acid is a cDNA or oligonucleotide that
constitutes a library; [0156] (9) The cell culture instrument
according to (2) or (3), on which the surface is coated with the
film according to (7) or (8); [0157] (10) The cell culture
instrument according to (9), that is used for the expression of
proteins; [0158] (11) The cell culture instrument according to (9),
that is used for the inhibition of gene expression; [0159] (12) A
process for examining the function of a gene in a cell, which
comprises culturing the cell on the cell culture instrument
according to (1) to (6) on which a nucleic acid containing a cDNA
of the gene is immobilized; and examining the proliferation ratio
or the phenotype of the cell, or the production level of certain
proteins; [0160] (13) A process for examining the function of a
gene in a cell, which comprises culturing the cell on the cell
culture instrument according to (1) to (6) on which an
oligonucleotide containing a base sequence complementary to a
messenger RNA of the gene is immobilized; and examining the
proliferation ratio or the phenotype of the cell, or the production
level of certain proteins; and [0161] (14) The cell culture
instrument according to (1) to (6), wherein the surface of the cell
culture instrument outside the part immobilized by the nucleic acid
is hydrophilic or hydrophobic as high as the cell is not
attached;
[0162] In order to transfer a nucleic acid into a target cell on
solid phase, the cell may be cultured on a cell culture instrument,
on which a desired nucleic acid together with a collagen or a
collagen derivative, or a desired nucleic acid complexed with the
collagen or the collagen derivative to form a particle of the
complex is directly immobilized. Alternatively, target cells may be
cultured on a cell culture instrument, of which the surface is
covered with a film comprising a collagen or a collagen derivative
containing a desired nucleic acid, or a film comprising a particle
of the complex, and a film made of agarose and albumin comprising a
collagen or a collagen derivative containing a desired nucleic
acid, or a particle of the complex may be used.
[0163] The present invention comprises making a library that is
constituted with various nucleic acids complexed with a collagen or
a collagen derivative independently on the solid phase, and makes
it possible to examine the functions of the gene in cells at the
same time and in the same condition. Further, the invention provide
a library for gene expression or a library for inhibition of gene
expression wherein a nucleic acid or an oligonucleotide comprising
the libraryed cDNA is arranged on the solid phase, allowing the
examination of the gene functions at a stage of cell. Nucleic acids
comprising the cDNA that constitutes a library are not limited to a
specific species, and include Gene Storm pcDNA3.1 vector (In
Vitrogen, Inc.).
[0164] When a complex comprising a nucleic acid is arranged on the
solid phase, and cells are cultured thereon to examine the
functions of the transferred gene, it is necessary to avoid the
contamination of the cells, each of which is transferred with
respective nucleic acid independently arranged.
[0165] The features of the present invention also include selective
and efficient transfer of the complex particles coated onto the
solid into the cells attached to the coated part. In other words,
it is possible to examine the functions of a large number of genes
on microplates at a time without computing of the cells by wells.
To do so, wells may be used to compart each part arranged by the
nucleic acids, and 6 to 384 wells may compart one plate. In
addition to the wells, alternatively, surface on the solid phase
that is hydrophilic or hydrophobic as high as the cell is not
attached may be used to prevent the cells from moving across the
parts arranged by the nucleic acids. As used herein, the surface
that is highly hydrophilic means surface having a water-contact
angle of 40 degree or less, and the surface that is highly
hydrophobic means surface having a water-contact angle of 110
degree or more. Distance between the arranged nucleic acids should
be kept over the length of the most extension of the seeded cells.
Thus, when the surface of the cell culture instrument outside the
part immobilized by the nucleic acid or the part coated with a film
comprising a nucleic acid, it is not necessary to carry out culture
by computing the cells with wells, and it is possible to examine
the functions of a large number of the genes on a microplate.
Examples
[0166] The present invention is further illustrated by the
following Examples and Experiments, but is not restricted by these
Examples and Experiments in any way.
Example 1
Formation of Complexes Between a Plasmid DNA and a Collagen
[0167] Both equal amounts of an aqueous solution containing a
plasmid DNA (pCAHST-1: 7.9 Kbp) incorporated with a gene of
fibroblast growth factor HST-1 (FGF4) gene (Proc. Natl. Acad. Sci.
USA, 84, 2890-2984 (1987)) at 10 .mu.g/ml, and of a neutral aqueous
solution containing 200, 60, 20, 6, 2, 0.6, and 0 .mu.g of an
atelocollagen (KOKEN CO., LTD.) were mixed together, and the
mixtures were analyzed on agarose gel electrophoresis. Agarose gel
electrophoresis was carried out with 0.8% agarose gel in TAE
(Tris-acetate) buffer at Horizontal Electrophoresis Unit (Mupid,
Advance CO.). After electrophoresis, the gel was stained with
ethidium bromide, and photographed with a transilluminator. The
results are shown in FIG. 1. pCAHST-1 in the presence of the
collagen at 10 .mu.g/ml or more did not electrophorese, and
retained on the well. This means that pCAHST-1 at 5 .mu.g/ml was
complexed with the collagen at 10 .mu.g/ml.
[0168] Similarly, both equal amounts of 10 .mu.g/ml pCAHST-1 and
100 .mu.g/ml collagen were mixed together in 10 mM Tris
hydrochloride buffer (pH7.5) containing 1.0 M sodium chloride, and
the mixture was electrophoresed, which results are shown in FIG. 2.
When 1.0 M sodium chloride coexisted, pCAHST-1 irrespective of the
presence of the collagen at 100 .mu.g/ml did electrophorese. This
means that a complex formed by pCAHST-1 and the collagen should be
an electrostatic one.
[0169] Further, the formation of a complex between 10 .mu.g/ml
pCAHST-1 and the collagen in 10 mM Tris hydrochloride buffer
(pH7.5) containing 1.0 M sodium chloride was compared between the
presence and the absence of heparan sulfate. The results are shown
in FIG. 3. In the absence of heparan sulfate, all of 5 .mu.g/ml
pCAHST-1 were complexed with 100 .mu.g/ml collagen, whereas in the
presence of heparan sulfate, it found that there were some pCAHST-1
that were not complexed with collagen even at 300 .mu.g/ml. This
means that heparan sulfate having a negative charge similar to
pCAHST-1 cause the competition in complex formation with the
collagen between pCAHST-1 and heparan sulfate, suggesting that
pCAHST-1 and a collagen form a electrostatic complex.
Example 2
Microscopy of Complexes
[0170] Both equal amounts of an aqueous solution containing
pCAHST-1 at 200 .mu.g/ml, and an aqueous solution containing an
atelocollagen at 0, 20, 200, and 1000 .mu.g/ml were mixed, and the
mixture was added with PicoGreen dsDNA Quantitation Reagent
(Molecular Probes) to stain pCAHST-1 and observed by microscopy.
The results are shown in FIG. 4. In case that the collagen
concentration after the mixing is 500 .mu.g/ml (0.05% collagen in
the figure), the filamentous complexes having a major axis of more
than 1 mm were predominantly observed, whereas the particulate
complexes having a major axis of 10 to 100 .mu.m were predominantly
observed in case that the collagen concentration is 100 .mu.g/ml
(0.01% collagen in the figure), and the particulate complexes
having a major axis of 10 .mu.m or less were predominantly observed
in case that the collagen concentration is 10 .mu.g/ml (0.001%
collagen in the figure). This means that the formation of the
complexes can be controlled by the concentrations of plasmid DNAs
and collagens at the time of mixing. These complexes maintained
stably their shape over a week or more when stored at 5.degree. C.
(FIG. 5). This means that the complexes can be stored for a long
period of time, and be distributed on the market at they are, not
requiring the preparation just before use.
Example 3
Extension Effect of Complexes on the Expression Duration Time
[0171] Both equal amounts of an aqueous solution containing a
plasmid DNA encoding a fluorescent protein (EGFP)
(pCMV-EGFP/pEGFP-N1, Clontech Co.) at 200 .mu.g/ml, and an aqueous
solution containing an atelocollagen at 0.02%(w/w) were mixed
together to prepare a formulation in a gel form.
[0172] To human embryonic kidney cells, 293 cells cultured on a
dish (diameter: 6 cm), 100 .mu.l of the gel formulation (containing
10 .mu.g of pCMV-EGFP) was added in the presence of 2 ml of
serum-free medium. Then, after cultured at 37.degree. C. overnight,
the cells were washed with PBS to remove the serum-free medium and
the formulation, and cultured in a medium containing 10% calf
serum. The cells were observed by fluorescence microscopy with time
course to check the EGFP expression. As a control, a PBS solution
containing an equal amount of pCMV-EGFP, and a complex of a
cationic liposome formulation and pCMV-EGFP were each added to the
293 cells.
[0173] The results are shown in FIG. 6. In case of the gel
formation of pCMV-EGFP, the expression of EGFP was observed, and
the expression was found to maintain during a long period of time
up to 52 days after the addition. On the other hand, in case of the
PBS solution containing pCMV-EGFP, no expression of EGFP was
observed, and in case of the cationic liposome formulation, the
expression of EGFP was found to merely maintain during a short
period of time for 2 days. These results shows that the formulation
of a plasmid DNA with a collagen promotes the expression efficiency
of a plasmid DNA, and maintains the expression during a longer
period of time.
[0174] This means that a complex as formed with a plasmid DNA and a
collagen promotes the transfer of a plasmid DNA into a cell, and
enhances the stability of the plasmid DNA inside and outside the
cell, resulting in extension of the gene expression in the cell.
Without being limited to a particular theory of operation, it is
believed that the fact that the expression was extended for a
longer period of time in the absence of any complex in the system
shows that the complexes interact with the cells as strongly as
that the complexes are not removed by the washing, or that the
complexes are incorporated into the cells.
Example 4
Effect of the Composition of Complexes on Transfer Efficiency of
Plasmid DNAs
[0175] Using pCMV-EGFP as a plasmid DNA, complexes having the
composition as shown in Table 1 were prepared.
TABLE-US-00001 TABLE 1 complex atelocollagen (%) pDNA (.mu.g/ml)
EGFG-1 0 100 EGFG-2 0.001 100 EGFG-3 0.01 100 EGFG-4 0.05 100
EGFG-5 0.1 100
[0176] The results show that their transfer efficiencies are
promoted in order of EGFG-3 complex>EGFG-2 complex>EGFG-4
complex=EGFG-5 complex. No transfer was found in EGFG-1. This shows
that the formation of a plasmid DNA with a atelocollagen promotes
the expression efficiency of a plasmid DNA, and that the expression
efficiency in the complexes containing the equal amount of plasmid
DNA varies depending on the content of atelocollagen.
Example 5
[0177] Effects of the Amounts of Plasmid DNA on the transfer
Efficiency of DNA Plasmid.
[0178] Zero, 10, 50, 100, 250, and 500 .mu.l of EGFG-3 complex
(0.01% atelocollagen, 100 .mu.m/ml pCMV-EGFP), which was found in
Example 4 that the transfer efficiency is the highest, was added to
the 293 cells cultured on a 6-well dish in the presence of 1 ml of
serum-free medium. Then, after cultured at 37.degree. C. overnight,
the cells were washed with PBS to remove the serum-free medium and
the complex, and the medium was replaced for a 10% FBS (a medium
containing 10% calf serum). The cells were observed by fluorescence
microscopy with time course over 6 days to check the EGFP
expression.
[0179] When the amount of plasmid DNA is increased, then the
transfer efficiency was also improved. Particular data are shown in
the followings. In the table, expression efficiency was estimated
by counting the number of the cells emitting fluorescence caused by
EGFP expression among the counted number of all cells existing in
the compartment defined by the microscopic range.
TABLE-US-00002 TABLE 2 Transfection efficiency EGFG-3 (.mu.l) 100
250 500 EGFP expressing cells 262 1057 1081 (cells/wel) efficiency
0.006 0.03 0.03
Example 6
Comparison in Extension Effects of Complexes on Duration Time of
Expression Between the Dropwise Addition and Solid Coating of the
Complexes
[0180] First, the composition of a collagen and a plasmid DNA was
optimized on the basis of the composition of EGFG-3 complex (0.01%
atelocollagen, 100 .mu.g/ml pCMV-EGFP), which was found that the
transfer efficiency is the highest, and then, the duration times of
expression were compared between the dropwise addition and the
solid coating. [0181] (1) Using pCMV-EGFP as a plasmid DNA,
complexes having the various compositions of atelocollagen
concentrations as shown in Table 3 were prepared.
TABLE-US-00003 [0181] TABLE 3 Experiment 6: Gene-transfection
efficiency relative to atelocollagen complex atelocollagen (%) pDNA
(.mu.g/ml) EGFG-3A 0.005 100 EGFG-3B 0.008 100 EGFG-3 0.01 100
EGFG-3C 0.015 100 EGFG-3D 0.02 100 EGFG-3E 0.03 100 pDNA solely 0
100
[0182] The complexes prepared were added dropwise to the 293 cells
cultured on a 6-well dish in the presence of serum-free medium.
Then, after cultured at 37.degree. C. overnight, the cells were
washed with PBS to remove the serum-free medium and the complex,
and the medium was replaced for a medium containing 10% calf serum.
Seven days later, the cells were observed by fluorescence
microscopy, and the cells expressing EGFP were counted. The results
are shown in FIG. 7. For EGFG-3B, 3D, and 3E, fluorescence
intensities of the expressed EGFP were measured (Array Scan II
System, Cellomics, Co.). Relative fluorescence intensities are
shown in FIG. 8.
[0183] The complexes prepared were coated onto a 6-well dish at 250
.mu.l/well, and the dish was air-dried for 30 minutes. Then, the
293 cells (4-5x10.sup.5 cells/well) in a medium containing 10% FBS
were added thereto, and cultured at 37.degree. C. overnight. Seven
days later, the cells were observed by fluorescence microscopy, and
the cells expressing EGFP were counted. The results are shown in
FIG. 9.
[0184] FIG. 7 showing the results of dropwise addition and FIG. 9
showing the results of solid coating indicate that the
concentration of collagen affects the transfer efficiency, and the
solid coating is superior to the dropwise addition in transfer
efficiency. [0185] (2) Complexes having the various compositions of
plasmid DNA concentrations as shown in Table 4 were prepared, and
transfer efficiencies were compared between dropwise addition and
solid coating.
TABLE-US-00004 [0185] TABLE 4 complex atelocollagen (%) pDNA
(.mu.g/ml) EGFG-3B 0.008 100 EGFG-3B2 0.008 60 EGFG-3B3 0.008 40
EGFG-3B4 0.008 20
[0186] The results of dropwise addition are shown in FIG. 10, and
the results of solid coating are shown in FIG. 11. Relative
fluorescence intensities of EGFP in each sample obtained in solid
coating are also shown in FIG. 12. These results show that the
concentration of plasmid DNA affects the transfer efficiency,
specifically the expression level of EGFP and the concentration of
plasmid or DNA are in a positive correlation, and also that the
solid coating is superior to the dropwise addition in transfer
efficiency.
Example 7
Application of Solid Coating to Screening Method
[0187] Ten .mu.M of a phosphorothioate antisense oligonucleotide
(5'-CTCGTAGGCGTTGTAGTTGT-3'; Molecular weight, about 6500; SEQ ID
NO: 2) (5) (Sawaday) having a sequence complementary to a sequence
from 4196 by to 4216 by of fibroblast growth factor HST-1 (FGF4)
gene (described in Proc. Natl. Acad. Sci. USA, 84, 2890-2984
(1987)) was mixed with a 0.05% solution of atelocollagen to form
complexes. The complexes were coated onto the bottom of a 96-well
plate for cell culture, and air-dried to obtain a cell culture
instrument wherein the bottoms were coated with the complex.
Similarly, a phosphorothioate sense oligonucleotide
(5'-GAGCATCCGCAACATCAACA-3'; SEQ ID NO: 3) (1) having the same
sequence as the HST-1 gene, as well as phosphorothioate antisense
oligonucleotides having a sequence (5'-AGTCGCATGCACACAACACA-3'; SEQ
ID NO: 4) (2) as obtained by scrambling the antisense
oligonucleotide sequence, and the three random sequences
(5'-GACCATCGTCGATTCCAGT-3'; SEQ ID NO: 5) (3),
(5'-CATGAACATCCTGAGCATCC-3'; SEQ ID NO: 6) (4), and
(5'-GTTCACGAAGAAAGAAGGCT-3'; SEQ ID NO: 7) (6)) were each complexed
with a collagen to form complexes, which complexes were coated onto
the bottom, and dried. Onto the plate coated with those
oligonucleotides, NEC8 cells in which the proliferation was
promoted by overexpression of HST-1, and HepG2 cells in which the
proliferation was promoted by the gene other than HST-1 were seeded
at 0.5.times.10.sup.5 cells/well, and the cells were cultured for 4
days. After the culture, the inhibition of cell proliferation was
assayed using TetraColor ONE Cell Proliferation Assay Reagent.
Specifically, the sample solution stained was determined
spectrometrically at 650 nm, using the absorbance at 450 nm as
control (FIG. 13).
[0188] As a result, the inhibitory effect of NEC 8 cells
proliferation was observed only in the case of coating of the
complex formed between an antisense oligonucleotide against HST-1
and a collagen (FIG. 13-5). On the other hand, no inhibitory effect
was observed in the case of coating of the complex formed between a
sense oligonucleotide against HST-1 and a collagen onto the bottom
of the plate (FIG. 13-1). Also, no inhibitory effect was observed
in the case of coating of the complex formed between the
phosphorothioate antisense oligonucleotides having the sequence as
scrambling the antisense oligonucleotide sequence (FIG. 13-2) and
the random sequences (FIG. 13-3, 4, 6) and a collagen onto the
bottom of the plate. These results show that it is possible to
screen for a gene responsible for the cell proliferation without
reagents for gene transfer such as cationic liposomes and cationic
lipids, which do not usually exist in living bodies, when an
antisense oligonucleotide complexed with an atelocollagen is coated
onto the solid phase, and cells are cultured thereon. Further,
these results show that the present process enables to examine
sensitively the gene functions even using a small amount of the
cells and the oligonucleotides without noise due to damage to cells
from gene transfer reagents.
Example 8
Storage of Adenovirus Vector Under Air Drying Using
Atelocollagen
Preparation by Solid Coating
[0189] Ten .mu.l of a solution of an adenovirus vector, AdCMVEGFP
(K. Aoki, et al., Mol. Ther. (2000) 1 (6): 555-565) at
1.times.10.sup.8 pfu/ml in a DMEM solution was mixed with 5 .mu.l
of a solution of an atelocollagen in PBS (-). Fifty .mu.l of the
mixture was added to a 24-well culture plate (non-coated) (Corning
Inc.), and the plate was air-dried with a dryer set on the cool
mood at room temperature for 15 minutes to carry out the solid
coating.
Test of Viruses for Stability During Storage
[0190] While the resultant culture plate was left at it was at room
temperature, a human hepatoma cell line, the HepG2 cells were added
thereto at 2.times.10.sup.5 cells/well 1 day, 7 days and 14 days
after the leaving, and each 2 days later, the expression level of
GFP was observed by fluorescence microscopy. As control, a similar
experiment was carried out using a culture plate coated with only
adenovirus, and with only an atelocollagen.
[0191] The results are shown in Table 5.
TABLE-US-00005 TABLE 5 GFP-positive cells ratio 2 days after the
seeding (%) atelo/adeno only adeno only atelo left at r.t. 1 day 64
72 0 left at r.t. 7 days 42 10 or less 0 left at r.t. 14 days r.t.
means room temperature.
[0192] Table 5 shows that the adenovirus vector survived during
storage for even 14 days, suggesting that the solid coating of the
present invention provides a plate for screening that comprises
adenovirus vectors, which could be conveniently distributed on the
market.
Example 9
Gene Transfer Effects of the Solid Coating in a Dose-Dependent
Manner of Adenovirus
[0193] Both equal amounts of a solution of an adenovirus vector
AdCMVEGFP (K. Aoki, et al. Mol. Ther. (2000) 1 (6): 555-565) in
DMEM at 4.times.10.sup.4, 4.times.10.sup.5, 4.times.10.sup.7
pfu/ml, and a 160 .mu.g/ml solution of an atelocollagen were mixed
together. The mixture could be stored at 4.degree. C. for a long
period of time without lowered activity. Fifty .mu.l of the mixture
was added to a 96-well culture plate (non-coated; Corning Inc.),
and the plate was air-dried to carry out the solid coating. The
plate can be stored at least for 2 weeks to 4 weeks without lowered
activity. Embryonic stem cells (ES cells) were seeded on the
resultant culture plate, and three days later, the expression of
GFP was checked with fluorescence microscopy, simultaneously with
determining the fluorescence intensity. As a result, it was found
that the fluorescence intensity of GFP is positively correlated
with the dose of adenovirus (FIG. 14). The results shows that the
present invention should provide a useful tool for high-throughput
screening for ES cells using adenovirus, and a plate for screening,
which is coated with complexes formed by adenovirus and
collagen.
Example 10
Relationship Between the Composition of Complexes Comprising
Plasmid DNAs and the Average Major Axis
[0194] Both equal amounts of an aqueous solution, a 0.1 M phosphate
buffer (PB) or a 0.01 M phosphate buffer (PBS) containing sodium
chloride, each of which contained a plasmid DNA encoding a
fluorescent protein (EGFP) (pCMV-EGFP/pEGFP-N1, 4.7 kbp, Clontech
Co.) at 200 .mu.g/ml and was cooled up to 10.degree. C. or less,
and an aqueous solution containing an atelocollagen (KOKEN CO.,
LTD.) at 0.002 to 0.02% (w/w) cooled up to 10.degree. C. or less
were mixed together, and the mixtures were left as they were
overnight at 10.degree. C. or less so as to prepare a formulation
in a gel form. The gel formulation was penetrated through a filter
having a pore size of 70 .mu.m or 10 .mu.m to prepare a gel
formulation having an ordered size.
[0195] To the formulation, PicoGreen dsDNA Quantitation Reagent
(Molecular Probes) was added to stain pCMV-EGFP, and the major axis
of the complexes were determined by fluorescent microscopy.
Similarly, a plasmid DNA (pCMV-HST-1-IL-2, 6.2 kbp) encoding the
secretory signal peptide of HST-1 and interleukin-2, and an
atelocollagen were mixed together to prepare a formulation in a gel
form, and the major axis of the complexes was determined. Further,
the gel formulation was electrophoresed on agarose gel
(Tris-acetate buffer, 0.8% agarose gel) to separate plasmid DNAs
formed into complexes from those not formed into complexes. The
band of the plasmid DNAs that were not formed into complexes was
stained with ethidium bromide, and the fluorescence intensity was
determined with Fluor-S-Multi Imager (BIO-RAD). Amount of the
plasmid DNAs that were not formed into complexes were determined on
the basis of the standard curve obtained from the fluorescence
intensity of the band of the plasmid DNA with the known
concentration as electrophoresed, and the ratio of the negative
charge of the DNA contained in the complex to the molecular weight
of the collagen. The results are shown in Table 6.
TABLE-US-00006 TABLE 6 Average major axis of complexes Number of
(.mu.m) C.C. N.M per After After 70 .mu.m After 10 .mu.m Sample
Plasmid DNA Solv. (%) C.M mixing filter filter 10-1 pCMV-EGFP Water
0.001 2406 9.1 8.9 <3.4 10-2 pCMV-EGFP Water 0.005 742 37 26
10-3 pCMV-EGFP Water 0.008 699 6.6 10-4 pCMV-EGFP Water 0.01 299 53
44 35 10-5 pCMV-EGFP Water 0.02 356 17 10-6 pCMV-EGFP Water 0.05 76
122 105 10-7 pCMV-EGFP Water 0.1 66 303 174 10-8 pCMV-EGFP PB 0.001
448 <3.4 <3.4 10-9 pCMV-EGFP PB 0.01 152 7.7 10-10 pCMV-EGFP
PB 0.1 97 22 14 10-11 pCMV-EGFP PBS 0.01 300 13 9.2 10-12 pCMV-EGFP
PBS 0.1 97 22 15 10-13 pCMV-HST-1- Water 0.001 4228 6 6.1 <3.4
IL-2 10-14 pCMV-HST-1- Water 0.005 1122 20 9.8 IL-2 10-15
pCMV-HST-1- Water 0.008 426 6.1 IL-2 10-16 pCMV-HST-1- Water 0.01
324 20 13 27 IL-2 10-17 pCMV-HST-1- Water 0.02 360 16 IL-2 10-18
pCMV-HST-1- Water 0.05 95 151 201 IL-2 10-19 pCMV-HST-1- Water 0.1
66 256 201 IL-2 10-20 pCMV-HST-1- PB 0.001 701 6.1 <3.4 IL-2
10-21 pCMV-HST-1- PB 0.01 262 20 IL-2 10-22 pCMV-HST-1- PB 0.1 97
25 9.4 IL-2 10-23 pCMV-HST-1- PBS 0.01 315 9.8 IL-2 10-24
pCMV-HST-1- PBS 0.1 97 29 IL-2
In the table, "Solv." means solvent. "C.C. (%)" means collagen
concentration (%). "Number of N.M per C.M" means number of
nucleotide monomer per collagen molecule. "After 70 .mu.m filter"
means after penetrating through a filter having a pore size of 70
.mu.m. "After 10 .mu.m filter" means after penetrating through a
filter having a pore size of 10 .mu.m.
[0196] In both cases of pCMV-EGFP and pCMV-HST-1-IL-2, a water
without salts was used as a solvent to obtain complexes wherein the
average major axis is 122 .mu.m or more, when collagen
concentration was 0.05% or more. When collagen concentration was
0.005% to 0.02%, complexes having an average major axis of 6.1
.mu.m to 53 .mu.m were obtained, and when collagen concentration
was 0.001%, complexes having an average major axis of 10 .mu.m or
less were obtained. The results show that the form or the shape of
a complex can be controlled by adjusting the concentration of a
plasmid DNA and a collagen to be mixed, when a water without salts
is used as a solvent.
[0197] The results are consistent with those as obtained in Example
2 wherein pCAHST-1 was used to prepare complexes. The plasmid DNAs
as used therein were different each other in size as pCAHST-1 is
7.9 kbp, pCMV-EGFP is 4.7 kbp, and pCMV-HST-1-IL-2 is 6.2 kbp,
showing that sizes of plasmid DNAs never affect the form or the
shape of complexes to be formed.
[0198] On the other hand, 0.1 M phosphate buffer and 0.01 M
phosphate buffer containing sodium chloride were used as a solvent
to obtain complexes having an average major axis of 3.4 .mu.m to 29
.mu.m and no complexes having a 100 .mu.m or more in size, when
collagen concentration was 0.001% to 0.1%. It was believed that
this was caused by the salts, which inhibited the formation of
collagen association bodies, showing that when complexes are
prepared in the presence of an agent that inhibits the formation of
collagen association body, then complexes having an average major
axis of 100 .mu.m or less can be prepared irrespective of collagen
concentration.
[0199] Further, when the gel formulation as obtained by mixing a
plasmid DNA and a collagen was penetrated through a filter having a
pore size of 70 .mu.m or 10 .mu.m, complexes having a smaller
average major axis and having an ordered size could be
prepared.
[0200] FIG. 15 shows the relationship between the number of
collagen bound to one molecule of plasmid DNA and the average major
axis of the complexes. In both cases of pCMV-EGFP and
pCMV-HST-1-IL-2 as used in a water without salts, it was found that
the average major axis of complexes trends to extend as increasing
in the number of collagen bound to plasmid DNA. On the other hand,
when 0.1 M phosphate buffer and 0.01 M phosphate buffer containing
sodium chloride were used as a solvent to prepare complexes, such
trend as above was not found, and even in the condition that
collagen molecules having an average major axis of 100 .mu.m or
more was complexed with a plasmid DNA in case of the use of a water
as a solvent, the average major axis did not exceed 50 .mu.m. It is
understood that this is caused by extension reaction along the
major axis of collagen association body, of which formation is
developed by the formation of complexes of a plasmid DNA and a
collagen as observed in the case that a water is a solvent as
mentioned above. On the other hand, in case of the presence of
salts, it is understood that collagen association body formed by
attaching a collagen molecule with a plasmid DNA does not extend
along the major axis well, and therefore a lot of fine association
bodies attach to a plasmid DNA to form a structure. Collagens have
been known to form fine association bodies at appropriate salt
concentrations. Thus, it can be considered that a partial amount of
collagens has already formed with a plasmid DNA into fine bodies
before mixing with a plasmid DNA, and the fine collagen association
bodies are attached to a plasmid DNA to form complexes. This means
that complexes having an average major axis of 50 .mu.m or less can
be prepared by mixing a plasmid DNA and a collagen that has been
formed into fine association bodies.
[0201] For the general analysis of those results regardless of size
of plasmid DNAs, the number of nucleotide monomer of a plasmid DNA
per one collagen molecule in complexes was counted. FIG. 16 shows
the relationship between the number of nucleotide monomer of
plasmid DNAs per one collagen molecule, and the average major axis
of the complexes. It was found that the average major axis of
complexes trends to extend as decreasing in the number of
nucleotide monomer of plasmid DNAs.
[0202] Taken together FIG. 16 and Table 6, the followings are
concluded. When the number of nucleotide monomer is 95 or less, the
complexes having an average major axis of 120 .mu.m or more were
obtained. This means that the decrease in the number of nucleotide
monomer per one collagen molecule represents the increase in the
number of collagen molecules relative to the plasmid DNA in the
complexes, and thus complexes having an average major axis of 120
.mu.m or less can be prepared by keeping the number of nucleotide
monomer of plasmid DNAs per one collagen molecule to be 96 or more.
On the other hand, minimum unit of the complexes is a complex
formed by one molecule of collagen and one molecule of plasmid DNA.
One molecule of collagen and many plasmid DNA hardly form complexes
since plasmid DNAs repel each other by their negative charge and
undergo steric hindrance. Accordingly, the maximum number of
nucleotide monomer per one collagen molecule is obtained by forming
a complex with each one molecule of collagen and plasmid DNA, and
is defined by the number of nucleotide monomer comprised in a
plasmid DNA.
Example 11
[0203] Relationship Between the Composition and the Average Major
Axis of the Complex comprising an Oligonucleotide
[0204] An equal amount of an aqueous solution, or a 0.1 M phosphate
buffer (PB), each of which contained a phosphorothioate antisense
oligonucleotide (.sup.5'-CTCGTAGGCGTTGTAGTTGT-3'; Molecular weight,
about 6500; SEQ ID NO: 8) (Espec Inc.) having a sequence
complementary to a sequence from 4196 by to 4216 by of fibroblast
growth factor HST-1 (FGF4) gene (described in Proc. Natl. Acad.
Sci. USA, 84, 2890-2984 (1987)) in a concentration of 2.0 .mu.M to
40.0 .mu.M, and was cooled up to 10.degree. C. or less, and an
aqueous solution containing an atelocollagen (KOKEN CO., LTD.) at
0.002 to 3.0%(w/w) cooled up to 10.degree. C. or less, 0.1 M
phosphate buffer were each mixed together at 10.degree. C. or less,
and the mixtures were left as they were overnight at 10.degree. C.
or less so as to prepare formulations in a gel form.
Oligonucleotide amounts that were complexed with a collagen in the
gel formulations were determined as follows. The gel formulations
were centrifuged at 150,000 rotations per hour at 5.degree. C. to
precipitate the complexes, and the amounts of the oligonucleotides
released in the supernatant were determined by HPLC (column:
Puresil C18 5 (Waters), mobile phase: a gradient of 24.5% to 35%
acetonitrile over 35 minute in 0.1 M ammonium acetate containing 5
mM tetrabutyl ammonium and 1mM EDTA, flow rate: 1 ml/min, detection
wavelength: 260 nm). Single-Stranded nucleic acid fluorescence
staining regent YOYO (Molecular Probes) was added to the resultant
formulations to stain the oligonucleotides, which were observed by
fluorescent microscopy to measure the major axis of the complexes.
The results are shown in Table 7.
TABLE-US-00007 Average major Number of Number of axis of Sample
Solv. O.C. (.mu.M) C.C (%) O.M per C.M N.M per C.M complexes
(.mu.m) 11-1 water 1.0 0.05 0.6 12 32 11-2 water 5.0 0.05 3 60 13
11-3 water 10.0 0.05 4.4 88 12 11-4 water 20.0 0.05 5.2 104 17 11-5
water 10.0 0.01 6.3 126 32 11-6 water 10.0 0.1 2.9 58 20 11-7 PB
1.0 0.05 0.5 10 25 11-8 PB 5.0 0.05 1.2 24 29 11-9 PB 10.0 0.05 1.4
28 35 11-10 PB 20.0 0.05 1.3 26 36 11-11 PB 10.0 0.01 3.3 66 23
11-12 PB 10.0 0.1 1.3 26 33 11-13 PB 10.0 0.5 0.5 10 46 11-14 PB
10.0 1.0 0.3 6 49 11-15 PB 10.0 1.5 0.2 4 54
In the table, "Solv." means solvent. "O.C.(.mu.M) means
oligonucleotide concentration (.mu.M). " "C.C. (%)" means collagen
concentration (%). "Number of O.M per C.M" means number of
oligonucleotide molecule per one collagen molecule. "Number of N.M
per C.M" means number of nucleotide monomer per collagen
molecule.
[0205] Similarly to Example 10 wherein plasmid DNAs were used,
complexes were obtained by mixing oligonucleotides and collagens.
In both cases of use of a water without salts and 0.1 M phosphate
buffer as a solvent, there were no significant change in major axis
of the complexes between the differences in the concentrations of
collagen and oligonucleotide, which was different from the case of
plasmid DNAs. This is believed to be caused by the fact that
oligonucleotides are smaller than plasmid DNAs in molecular size,
and thus the formation of collagen association bodies is
inhibited.
[0206] FIG. 17 shows the relationship between the molecular number
ratio of oligonucleotide to collagen at the time of the mixture and
the number of oligonucleotide bound to one molecule of collagen in
the complex. It was found that the number of oligonucleotides bound
to one collagen molecule in the formed complexes trends to increase
as increasing in the number of oligonucleotides relative to the
number of collagen molecule at the time of mixing in both cases
that a water and the phosphate buffer were used as a solvent. The
maximum number of oligonucleotide molecule bound to one collagen
molecule varies depending on kinds of solvent, and it is 6
molecules in case of water, whereas it is 3 molecules in case of
the phosphate buffer.
[0207] Since molecular weight of a collagen (300,000) is even
larger than that of an oligonucleotide (about 6,500), and the form
or the shape of complexes depends solely on the number of collagen
molecule forming into complexes, the numbers of collagen molecule
in complexes that have the same major axis are approximately the
same. Accordingly, even for the complexes having the same major
axis, the number of oligonucleotide molecule comprised in complexes
varies depending on the number of oligonucleotide bound to one
collagen molecule. Because individual complex contacts with a cell
to allow oligonucleotides to be transferred into the cell,
oligonucleotide transfer is more efficient when a complex that
contains a high content of oligonucleotide molecules is used.
Accordingly, a complex wherein the number of oligonucleotide
molecule per one collagen molecule is increased is desired, and
generally speaking, a complex wherein the number of nucleotide
monomer bound to one collagen molecule is increased is
preferred.
Example 12
[0208] Cell Culture Instrument Coated with a Complex Comprising an
Oligonucleotide
[0209] Fifty .mu.l of the gel formulation as prepared in Example 11
(11-3, the composition of the formulation of Example 7) was added
dropwise to the bottom of a 96-well microplate, and dried by
directly spraying a cool air (room temperature, 2 hours) or by
placing in a desicator with silica gel (room temperature, 2 days)
so as to prepare a cell culture instrument, of which the cell
culture surface is coated with a complex. One hundred .mu.l of 0.01
M phosphate buffer (PBS) that had been adjusted to be isotonic with
living bodies by supplementing sodium chloride was added to the
wells of the instrument, and the pipetting was slightly conducted
immediately for sampling. Further, 100 .mu.l of 0.01 M phosphate
buffer (PBS) that had been adjusted to be isotonic with living
bodies by supplementing sodium chloride was added to the wells of
the instrument, and the instrument was left overnight at 37.degree.
C., followed by slightly pipetting for sampling. To 10 .mu.l of the
resultant sample, 2 .mu.l of Single-Stranded nucleic acid
fluorescence staining regent YOYO (Molecular Probes) was added to
stain the oligonucleotides, and the complexes that released form
the surface of the instrument were observed by fluorescent
microscopy. The fluorescence micrograph is shown in FIG. 18. The
complexes having a major axis of 50 .mu.l or less were observed in
all of the samples. This means that exposure on PBS allows to
release complexes from the surface of a cell culture instrument.
The form or the shape of the released complexes was not changed
between before and after the coating onto the surface of the
instrument irrespective of drying method, and condition of PBS
exposure. This shows that the complexes are retained on the surface
of a cell culture instrument with keeping their form or shape
irrespective of drying method, and that the form or the shape of
the complexes maintains in a temperature condition of 37.degree. C.
that is usually used in cell culture.
Example 13
[0210] Cell Culture Instrument Coated with a Complex Comprising a
Plasmid DNA
[0211] Fifty .mu.l of the gel formulation as prepared in Example 10
(10-3, having the composition that was found efficient in transfer
efficiency in Example 6) was added dropwise to the bottom of a
96-well microplate, and dried by directly spraying a cool air (room
temperature, 2 hours) or by placing in a desicator with silica gel
(room temperature, 2 days) so as to prepare a cell culture
instrument, of which the cell culture surface is coated with a
complex. One hundred .mu.l of 0.01 M phosphate buffer (PBS) that
had been adjusted to be isotonic with living bodies by
supplementing sodium chloride was added to the wells of the
instrument, and the pipetting was slightly conducted immediately
for sampling. Further, 100 .mu.l of 0.01 M phosphate buffer (PBS)
that had been adjusted to be isotonic with living bodies by
supplementing sodium chloride was added to the wells of the
instrument, and the instrument was left overnight at 37.degree. C.,
followed by slightly pipetting for sampling. To 10 .mu.l of the
resultant sample, 2 .mu.l of Single-Stranded nucleic acid
fluorescence staining regent YOYO (Molecular Probes) was added to
stain the oligonucleotides, and the complexes that released form
the surface of the instrument were observed by fluorescent
microscopy. The fluorescence micrograph is shown in FIG. 19. The
complexes having a major axis of 100 .mu.l or less were observed in
all of the samples. This means that exposure on PBS allows to
release complexes from the surface of a cell culture instrument.
The form or the shape of the released complexes was not changed
between before and after the coating onto the surface of the
instrument irrespective of drying method, and condition of PBS
exposure. This shows that the complexes are retained on the surface
of a cell culture instrument with keeping their form or shape
irrespective of drying method, and that the form or the shape of
the complexes maintains in a temperature condition of 37.degree. C.
that is usually used in cell culture.
Example 14
[0212] Gene Transfer with a Cell Culture Instrument Coated with a
Complex
[0213] Three hundreds .mu.l of the gel formulations as prepared in
Example 10, 10-1, 10-4, and 10-7, comprising pCMV-EGFP were added
to a 6-well cell culture microplate, and dried by spraying a cool
air so as to prepare a cell culture instrument onto which a complex
comprising pCMV-EGFP was coated. As control, 300 .mu.l of an
aqueous solution comprising pCMV-EGFP at 100 .mu.m/ml was added to
a plate, and dried similarly. Into each well, 7.5.times.10.sup.4
cells of 239 cells were seeded, and a DMEM medium containing 10%
FBS was added thereto, followed by culturing at 37.degree. C. From
the start of the culture, the medium was replaced with a fresh
medium every 4 or 5 days. eleven days after the seeding, the cells
were observed by fluorescent microscopy, and the number of the
cells expressing GFP was counted to estimate transfer
efficiency.
[0214] Similarly, 300 .mu.l or 500 .mu.l of the gel formulations as
prepared in Example 10, 10-13 to 22, and 24, comprising
pCMV-HST-1-IL-2 were added to a 6-well cell culture microplate, and
dried by spraying a cool air so as to prepare a cell culture
instrument onto which a complex comprising pCMV-HST-1-IL-2 was
coated. As control, 300 .mu.l or 500 .mu.l of an aqueous solution
comprising pCMV-HST-1-IL-2 at 100 m/ml, PB solution or PBS solution
were added to a plate, and dried similarly.
1) Transfer of Plasmid DNA into 239 Cells
[0215] Into each well wherein 300 .mu.l of the gel formulation was
coated, 7.5.times.10.sup.4 cells of 239 cells were seeded, and a
DMEM medium containing 10% FBS was added thereto, followed by
culturing at 37.degree. C. for 10-13 to -19. Eight days after the
cell seeding, the medium was replaced, and IL-2 concentration in
the medium sampled 11 days later was determined by ELISA
(Quamtikine human IL-2 (R&D Systems)). For 10-20 to -22, a DMEM
medium was added to the plate, and cultured overnight at 37.degree.
C., after which on the following day the medium was replaced with a
DMEM medium containing 10% FBS, and then cultured at 37.degree. C.
Eight days and 11 days after the cell seeding, the medium was
replaced, and IL-2 concentration in the medium sampled 15 days
later was determined by ELISA. Further, for 10-20, 21, and 24, a
DMEM medium containing 10% FBS was added to the plate, and the
plate was cultured at 37.degree. C. IL-2 concentration in the
medium sampled 8 days after the cell seeding was determined by
ELISA.
2) Transfer of Plasmid DNA into NIH3T3 Cells
[0216] Into the well wherein 500 .mu.l of 10-14 to -17 was coated,
5.times.10.sup.4 cells of NIH3T3cells were seeded, and a DMEM
medium containing 10% FBS was added thereto, followed by culturing
at 37.degree. C. Eight days after the cell seeding, the medium was
sampled, and IL-2 concentration therein was determined by
ELISA.
[0217] The results as obtained in above 1) and 29 are summarized in
Tables 8 and 9. When complexed with a collagen and coated onto a
cell culture instrument, both pCMV-EGFP and pCMV-HST-1-IL-2 could
be efficiently transferred into 293 cells and NIH3T3 cells. The
result shows that the effect of the complexes of the present
invention on facilitation of gene transfer is not affected by the
kinds of plasmid DNA, species of the cells, and the presence or the
absence of serum in the culture. Further, in case of plasmid DNAs,
it has been found that coating of the complex having 1112 or less
of the nucleotide monomer per one collagen molecule, and having an
average major axis of 151 .mu.m or less, provided superior transfer
efficiency and gene expression.
TABLE-US-00008 TABLE 8 Number of nucleotide monomer per collagen
Average major axis Sample molecule of complexes (.mu.m) transfer
efficiency Control 0.0028 10-1 2406 9.1 0.0760 10-4 299 53 0.0729
10-7 66 303 0.0374
TABLE-US-00009 TABLE 9 Number of Presence nucleotide Average IL-2
or monomer major axis of concentration in Cell Absence per collagen
complexes media Sample species Solvent of FBS molecule (.mu.m)
(pg/ml) Control 293 water Presence 5.3 10-13 293 water Presence
4228 6 7.3 10-14 293 water Presence 1122 20 14.8 10-15 293 water
Presence 426 6.1 19.3 10-16 293 water Presence 324 20 6.3 10-17 293
water Presence 360 16 18.2 10-18 293 water Presence 95 151 8.4
10-19 293 water Presence 66 256 5.3 Control 293 PB Absence 23.55
10-20 293 PB Absence 701 6.1 72.93 10-21 293 PB Absence 262 20
264.61 10-22 293 PB Absence 97 25 86.22 Control 293 PB Presence
3.35 10-20 293 PB Presence 701 6.1 21.01 10-21 293 PB Presence 262
20 86.68 Control 293 PBS Presence 1.07 10-23 293 PBS Presence 315
9.8 15.26 Control NIH3T3 water Presence 0 10-14 NIH3T3 water
Presence 1122 20 0.79 10-15 NIH3T3 water Presence 426 6.1 6.06
10-16 NIH3T3 water Presence 324 20 4.05 10-17 NIH3T3 water Presence
360 16 2.78
Example 15
[0218] Gene Transfer into Cells by Addition of Complexes
[0219] Into a 6-well plate, 5.times.10.sup.4 cells of 239 cells
were seeded, and cultured in the presence of a DMEM medium
containing 10% FBS at 37.degree. C. Three days after the seeding,
300 .mu.l of the gel formulations as prepared in Example 10, 10-1,
10-4, 10-7, 10-8, 10-9, and 10-10, comprising pCMV-EGFP, and a
water or a PB solution comprising pCMV-EGFP at 100 .mu.g/ml as
control were each added thereto, and the cells were cultured in the
presence of a DMEM medium containing 10% FBS at 37.degree. C. On
the following day, the media were replaced with a fresh medium, and
13 days later for 10-1, 10-4, and 10-7, or 5 days later for 10-8,
10-9, and 10-10, the cells were observed by fluorescent microscopy,
followed by counting the number of the cells expressing GFP.
[0220] Similarly, 7.5.times.10.sup.4 cells of 239 cells were seeded
into a 6-well plate, and cultured in the presence of a DMEM medium
containing 10% FBS at 37.degree. C. Two days after the seeding, 300
.mu.l of the gel formulations as prepared in Example 10, 10-20 and
10-21, comprising pCMV-HST-1-IL-2, and a PB solution comprising
pCMV-HST-1-IL-2 at 100 .mu.g/ml as control were each added thereto,
and the cells were cultured in the presence of a DMEM medium
containing 10% FBS at 37.degree. C. On the following day, the media
were replaced with a fresh medium, and 5 days later, the media were
sampled, and IL-2 concentration therein were determined by ELISA
(Quamtikine human IL-2 (R&D Systems)).
[0221] The results are summarized in Tables 10, 11 and 12. When
complexed with a collagen and added to cells, both pCMV-EGFP and
pCMV-HST-1-IL-2 could be efficiently transferred into the cells. It
has been found that in case of the complexes having an average
major axis of 53 .mu.m or less as shown in the present Example, the
addition of the complex having 2406 or less, and further 701 or
less of the nucleotide monomer per one collagen molecule, provided
superior transfer efficiency and gene expression.
TABLE-US-00010 TABLE 10 Number of nucleotide Average major monomer
per collagen axis of complexes transfer Sample Solvent molecule
(.mu.m) efficiency Control water 0.0012 10-1 water 2406 9.1 0.0231
10-4 water 299 53 0.0247 10-7 water 66 303 0.0041
TABLE-US-00011 TABLE 11 Average Number of nucleotide major axis
Number monomer per collagen of complexes of EGFP- Sample Solvent
molecule (.mu.m) expressing cells Control PB 100 10-8 PB 448
<3.4 128 10-9 PB 152 7.7 1200 10-10 PB 97 22 12000
TABLE-US-00012 TABLE 12 Average IL-2 Number of nucleotide major
axis concentration monomer per collagen of complexes in Sample
Solvent molecule (.mu.m) media (pg/ml) Control PB 0 10-20 PB 701
6.1 8.98 10-21 PB 262 20 76.81 10-22 PB 97 25 120.74
INDUSTRIAL APPLICABILITY
[0222] According to the complexes of the present invention, it is
possible to preserve and stabilize desired nucleic acids by
complexing with collagens or collagen derivatives, 2) to transfer
efficiently desired nucleic acids into cells when administered to
living bodies, and 3) to express and inhibit desired nucleic acids
without reagents for gene transfer. The complexes of the present
invention can be applied to various aspects since the complexes of
the invention have advantages 1) that DNAs, antisense
oligonucleotides and virus vectors can be preserved and stabilized,
2) that the complexes can be stored during a long period of time on
plates dried where the complexes are coated, 3) that genes can be
expressed without reagents for gene transfer when cells are seeded
onto the plates, 4) that kinds of subjects of nucleic acids and
methods for immobilization are not limited to specific ones, and 5)
that the duration time of the expression in case of plasmid DNAs is
excellently extended.
[0223] According to the present invention, it is possible to
readily examine the functions of genes or proteins, of which the
expressions are inhibited by antisense oligonucleotides by
determining the proliferation or the morphology of the cells, or
the expression levels of cytokines or receptors. Not only antisense
oligonucleotides, but also other materials can be complexed with
collagens to form complexes, which in turn can be coated onto
plates; in case of ribozymes, it is possible to carry out a
screening similar to that of antisense oligonucleotides; and
further in case of plasmid DNAs or adenoviruses, it is possible to
examine the functions of genes or proteins by determining the
changes of cells by the expression of certain genes. Collagens or
collagen derivatives comprised in the complexes of the present
invention have been known to affect no cells, and the screening
using the complexes of the present invention makes it possible to
carry out the examinations of gene functions that seldom avoid the
unspecific affections to cells, which would be induced by
conventional reagents for gene transfer.
Sequence CWU 1
1
8120DNAArtificial SequenceDescription of Artificial Sequence
antisense oligomer 1ctcgtaggcg ttgtagttgt 20220DNAArtificial
SequenceDescription of Artificial Sequence phosphorothioate
antisense oligomer 2ctcgtaggcg ttgtagttgt 20320DNAArtificial
SequenceDescription of Artificial Sequence phosphorothioate sense
oligomer 3gagcatccgc aacatcaaca 20420DNAArtificial
SequenceDescription of Artificial Sequence phosphorothioate
scramble oligomer 4agtcgcatgc acacaacaca 20519DNAArtificial
SequenceDescription of Artificial Sequence phosphorothioate random
oligomer 5gaccatcgtc gattccagt 19620DNAArtificial
SequenceDescription of Artificial Sequence phosphorothioate random
oligomer 6catgaacatc ctgagcatcc 20720DNAArtificial
SequenceDescription of Artificial Sequence phosphorothioate random
oligomer 7gttcacgaag aaagaaggct 20820DNAArtificial
SequenceDescription of Artificial Sequence phosphorothioate
antisense oligomer 8ctcgtaggcg ttgtagttgt 20
* * * * *