U.S. patent application number 11/740200 was filed with the patent office on 2009-08-13 for nucleic acid-containing complex.
This patent application is currently assigned to H. MORI and Y. TABATA. Invention is credited to Kiyoshi Ando, Naoto Fukuyama, Harukazu Iseki, Hirofumi Kasahara, Hidezo Mori, Hiromi Sakamoto, Yasuhiko Tabata, Etsuro Tanaka.
Application Number | 20090203768 11/740200 |
Document ID | / |
Family ID | 26569281 |
Filed Date | 2009-08-13 |
United States Patent
Application |
20090203768 |
Kind Code |
A1 |
Mori; Hidezo ; et
al. |
August 13, 2009 |
NUCLEIC ACID-CONTAINING COMPLEX
Abstract
A nucleic acid-containing complex, containing a nucleic acid and
a biodegradable polymer, especially a positively-charged
water-insoluble biodegradable polymer, is disclosed. The complex
has excellent properties of sustainedly releasing a desired nucleic
acid, especially DNA, to a site in need of a treatment. Since the
complex can be taken up to phagocytes such as macrophages and
delivered specifically to the target site, the function of the
nucleic acid can be exhibited in a target site specific manner, and
thus more specific gene therapy can be achieved. The complex has no
adverse effects, such as occurrence of recombinants or toxicity
which could be caused by using a virus vector such as adenovirus,
or liposome. Thus, the complex is particularly preferable for the
field of gene therapy. Furthermore, the complex enhances the
biological effect of the nucleic acid introduced into the cells,
allowing a gene therapy with a lower dose of nucleic acids.
Inventors: |
Mori; Hidezo; (Tokyo,
JP) ; Tabata; Yasuhiko; (Kyoto, JP) ; Ando;
Kiyoshi; (Kanagawa, JP) ; Tanaka; Etsuro;
(Kanagawa, JP) ; Iseki; Harukazu; (Kanagawa,
JP) ; Sakamoto; Hiromi; (Chiba, JP) ;
Fukuyama; Naoto; (Kanagawa, JP) ; Kasahara;
Hirofumi; (Kanagawa, JP) |
Correspondence
Address: |
FOLEY & LARDNER LLP
P.O. BOX 80278
SAN DIEGO
CA
92138-0278
US
|
Assignee: |
H. MORI and Y. TABATA
|
Family ID: |
26569281 |
Appl. No.: |
11/740200 |
Filed: |
April 25, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10129906 |
Oct 28, 2002 |
7276594 |
|
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PCT/JP00/07882 |
Nov 9, 2000 |
|
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11740200 |
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Current U.S.
Class: |
514/44R ;
424/488; 514/44A |
Current CPC
Class: |
A61K 47/643 20170801;
A61P 35/00 20180101; A61P 9/00 20180101 |
Class at
Publication: |
514/44.R ;
514/44.A; 424/488 |
International
Class: |
A61K 31/7088 20060101
A61K031/7088; A61P 35/00 20060101 A61P035/00; A61K 9/14 20060101
A61K009/14 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 9, 1999 |
JP |
JP 11-318187 |
Sep 13, 2000 |
JP |
JP 2000-278878 |
Claims
1. A nucleic acid-containing complex comprising a nucleic acid and
a positively-charged water-insoluble biodegradable polymer, wherein
said nucleic acid can be released by degradation of the
biodegradable polymer.
2. The nucleic acid-containing complex according to claim 1 wherein
said positively-charged water-insoluble biodegradable polymer
comprises an introduced positively-charged group.
3. The nucleic acid-containing complex of claim 1, wherein said
positively-charged water-insoluble biodegradable polymer comprises
at least one member selected from the group consisting of collagen,
gelatin, chitin, chitosan, hyaluronic acid, alginic acid, starch,
and a derivative thereof.
4. The nucleic acid-containing complex of claim 3, wherein said
derivative comprises an amino derivative.
5. The nucleic acid-containing complex of claim 1, wherein said
positively-charged water-insoluble biodegradable polymer comprises
a crosslinked gelatin which has an introduced positively-charged
group.
6. The nucleic acid-containing complex of claim 1, wherein said
nucleic acid comprises at least one member selected from the group
consisting of a plasmid DNA, an oligonucleotide, and a
double-stranded nucleic acid compound.
7. The nucleic acid-containing complex of claim 6, wherein said
nucleic acid comprises at least one member selected from the group
consisting of vascular endothelial growth factor gene, hepatocyte
growth factor gene, and fibroblast growth factor gene.
8. The nucleic acid-containing complex of claim 7, wherein said
fibroblast growth factor gene comprises SEQ. ID No. 1.
9. A pharmaceutical composition comprising as an active ingredient
a nucleic acid-containing complex comprising a nucleic acid and a
positively-charged water-insoluble biodegradable polymer, wherein
said nucleic acid can be released by degradation of said
biodegradable polymer.
10. The pharmaceutical composition of claim 9, which is used for
gene therapy.
11. The pharmaceutical composition of claim 10, wherein said gene
therapy is achieved by local administration of the gene.
12. A method for controlling a rate of nucleic acid release
comprising: incorporating a nucleic acid into a positively-charged
water-insoluble biodegradable polymer; and allowing the nucleic
acid to be released by degradation of said biodegradable
polymer.
13. The method for controlling a rate of nucleic acid release
according to claim 12 wherein said positively-charged
water-insoluble biodegradable polymer comprises an introduced
positively-charged group.
14. The method of claim 12, wherein said positively-charged
water-insoluble biodegradable polymer comprises at least one member
selected from the group consisting of collagen, gelatin, chitin,
chitosan, hyaluronic acid, alginic acid, starch, and a derivative
thereof.
15. The method of claim 14, wherein said derivative comprises an
amino derivative.
16. The method of claim 12, wherein said positively-charged
water-insoluble biodegradable polymer comprises a crosslinked
gelatin having an introduced positively-charged group.
17. The method of claim 12, wherein said nucleic acid comprises at
least one member selected from the group consisting of a plasmid
DNA, an oligonucleotide, and a double-stranded nucleic acid
compound.
18. The method of claim 17, wherein said nucleic acid comprises at
least one member selected from the group consisting of vascular
endothelial growth factor gene, hepatocyte growth factor gene, and
fibroblast growth factor gene.
19. The method of claim 18, wherein said fibroblast growth factor
gene comprises DNA comprising SEQ. ID No. 1.
20. A method for enhancing the function of a nucleic acid,
comprising: incorporating a nucleic acid into a positively-charged
water-insoluble biodegradable polymer; and allowing the nucleic
acid to be released by degradation of the biodegradable polymer to
exhibit the function of the nucleic acid.
21. The method for enhancing function of a nucleic acid according
to claim 20 wherein said positively-charged water-insoluble
biodegradable polymer comprises an introduced positively-charged
group.
22. The method of claim 20, wherein said positively-charged
water-insoluble biodegradable polymer comprises at least one member
selected from the group consisting of collagen, gelatin, chitin,
chitosan, hyaluronic acid, alginic acid, starch, and a derivative
thereof.
23. The method of claim 22, wherein said derivative comprises an
amino derivative.
24. The method of claim 20, wherein said positively-charged
water-insoluble biodegradable polymer comprises a crosslinked
gelatin having an introduced positively-charged group.
25. The method of claim 20, wherein said nucleic acid comprises at
least one member selected from the group consisting of a plasmid
DNA, an oligonucleotide, and a double-stranded nucleic acid
compound.
26. The method of claim 25, wherein said nucleic acid comprises at
least one member selected from the group consisting of vascular
endothelial growth factor gene, hepatocyte growth factor gene, and
fibroblast growth factor gene.
27. The method of claim 26, wherein said fibroblast growth factor
gene comprises SEQ. ID No. 1.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates to a nucleic acid-containing complex
characterized by containing a nucleic acid, and a
positively-charged water-insoluble biodegradable polymer, and also
relates to a method for controlling the rate of release of the
nucleic acid from the complex.
[0003] The invention is also concerned with phagocytic cells
(hereinafter referred to as phagocytes) comprising a nucleic
acid-containing complex containing a nucleic acid and a
biodegradable polymer. The invention also concerns a drug having
the nucleic acid-containing complex as an active ingredient and
usable for phagocyte-mediated gene therapy.
[0004] 2. Description of the Related Art
[0005] In recent years, as molecular genetic factors of human
diseases have become clear, more and more emphasis has been placed
on studies of gene therapy. Gene therapy is aimed at expressing DNA
at a targeted site or cell. For this therapy, it would be
beneficial to bring the DNA directly to the target site or cell,
transfer it into the target site or cell efficiently, and express
it at the specific site or in the cell functionally. Various
methods have been reported for the effective transfer and
expression of foreign DNA (Fumimaro Takaku, eds. "Idenshichiryo no
Saizensen: Kisogijutu kara Rinsho Oyo made (The Forefront of Gene
Therapy, from Basic Technology to Clinical Applications)",
Experimental Medicine, Vol. 12, No. 15, 1994; Robert E. Sobol and
Kevin J. Scanlon, The Internet Book of Gene Therapy, Appleton &
Lange Stamford, Conn., 1995). They are roughly classified into (1)
physical methods for DNA transfer (microinjection,
electroporation), (2) chemical methods for DNA transfer (calcium
phosphate transfection, DEAE-dextran transfection), and (3)
biological methods (virus vectors, such as retroviruses and
adenoviruses). The existing chemical methods, such as calcium
phosphate transfection, and DEAE-dextran transfection, are
generally low in the efficiency of gene transfer. The physical
methods, such as microinjection and electroporation, may require
special devices, and they are not practical for routine clinical
use. Virus vectors have been expected to find clinical applications
because of their high efficiency of gene transfer. However, these
vectors involve the risk of adverse reactions such as immune
reactions, due to their nature as viruses.
[0006] To overcome the above drawbacks, new technologies have been
developed. Liposome methods incorporate gene into liposomes to
protect the gene from inactivation or degradation. The liposomes
are free from viral DNA, and thereby rule out the possibility that
potentially dangerous recombination events may occur. However,
their potent toxicity to a variety of cell types restricts the use
of liposomes as carriers of DNA. Development of new substances and
means for gene delivery continues even now.
[0007] In the field of gene therapy for vascular lesions, the
so-called hydrogel method has also been developed which comprises
adhering a hydrogel to the surface of a catheter to be introduced
into a blood vessel, placing a plasmid gene in the hydrogel, and
directly coating the hydrogel into the blood vessel (Marchall, E.,
Science, 269, 1050-1055, 1995). According to this method, the
plasmid is slowly released from the hydrogel by simple diffusion.
With this method, in general, both the period of slow release is
brief, and this period is difficult to control both with respect to
rate and length. Gene therapy requires that the amount of the
therapeutic gene or the period of its supply be adjusted depending
on the disease to be treated or the status of the disease. Thus,
there is a demand for a method capable of controlling the period of
slow release of the therapeutic gene according to the requirements
of therapy.
[0008] When a foreign gene is to be used in a clinical setting such
as gene therapy, a persistent supply of this gene at a stable level
to the target site is necessary for the functional expression of
the gene.
[0009] Furthermore, studies of gene therapies using antisense
oligonucleic acids have attracted attention in recent years. Means
for supplying such nucleic acids site-specifically and stably in
vivo for controlled periods of time should enhance the efficacy of
such an approach.
SUMMARY OF THE INVENTION
[0010] It is an object of the present invention to provide a
nucleic acid-containing complex which is safe, has a high
efficiency of introducing the nucleic acid into cells, and can
persistently supply a nucleic acid to a target site, along with
providing a method for controlling the rate of release of the
nucleic acid from the nucleic acid-containing complex.
[0011] It is another object of the invention to provide a nucleic
acid-containing complex which is safe, easy to handle, and excels
in site-specific functional expression in vivo, along with a method
which can exhibit the function of nucleic acid specifically to the
target site with the use of the nucleic acid-containing
complex.
[0012] As used herein, the phrase "the function of nucleic acid"
refer to the expression of a gene which is encoded in the nucleic
acid, but it can also refer to nucleic acids which exert a direct
effect. Examples of nucleic acids exerting a direct effect include
antisense nucleotides (Robert E. Sobol and Kevin J. Scanlon, The
internet book of gene therapy, Appleton & Lange Stamford,
Conn., 1995), ribozymes (U.S. Pat. No. 6,127,173), double-stranded
DNA molecules which can function as decoys (Sobol, supra), and
DNA/RNA hybrids (Bartlett et al., (2000) Nat. Biotech 18:615).
[0013] In light of these objectives, the inventors of this
invention conducted extensive studies and obtained findings listed
below. First, by complexing a negatively-charged nucleic acid and a
positively-charged water-insoluble biodegradable polymer to form a
stable complex, degradation of the nucleic acid in vivo can be
suppressed, and the nucleic acid can be released at a sustained
rate at the desired target site or cell. Second, a complex obtained
by complexing a nucleic acid with a biodegradable polymer, like the
nucleic acid-containing complex described herein, is easily taken
into phagocytes which play important roles in the immune system
(first, targeting the complex to phagocytes). The phagocytes taking
up the complex migrate to a target site (second, targeting the
phagocytes to the target site), and therefore the phagocytes
increase the efficacy of the technique by targeting the effect of
the added nucleic acid to the target site. Based on these findings,
the inventors accomplished the present invention.
[0014] In one aspect the invention features a nucleic
acid-containing complex, containing a nucleic acid and a
positively-charged water-insoluble biodegradable polymer, wherein
the nucleic acid can be released via degradation of the
biodegradable polymer.
[0015] In another aspect the invention features a nucleic
acid-containing complex, containing a nucleic acid and a
positively-charged water-insoluble biodegradable polymer having a
positively-charged group added to the polymer.
[0016] In the nucleic acid-containing complex of the invention, the
positively-charged water-insoluble biodegradable polymer contains
at least one member selected from the group consisting of collagen,
gelatin, chitin, chitosan, hyaluronic acid, alginic acid, starch,
and derivatives of any of these substances. Preferably, the
derivatives are amino derivatives.
[0017] In a preferred embodiment, the positively-charged
water-insoluble biodegradable polymer is crosslinked gelatin having
an introduced positively-charged group. In another preferred
embodiment, the nucleic acid is at least one member selected from
the group consisting of a plasmid DNA, an oligonucleotide, and a
double-stranded nucleic acid compound.
[0018] In a preferred embodiment, the nucleic acid encodes a
polypeptide which comprises at least one member selected from the
group consisting of vascular endothelial growth factor gene,
hepatocyte growth factor gene, and fibroblast growth factor gene,
as well as other genes such as kinases, phosphatases, transcription
factors, cytokines, proteases, apoptosis-inducing factors and
apoptosis-retarding factors. More preferably, the DNA comprises a
base sequence described as SEQ. ID No. 1 of the sequence listing
which encodes FGF4/HST1.
[0019] In another aspect the invention features a pharmaceutical
composition comprising the nucleic acid-containing complex of the
instant invention as an active ingredient. In a preferred
embodiment, the pharmaceutical composition is used for gene
therapy, especially where the gene therapy is effected by a local
administration of the gene.
[0020] In yet another aspect the invention features a method for
controlling a rate of nucleic acid release, characterized by
incorporating a nucleic acid into a positively-charged
water-insoluble biodegradable polymer, and releasing the nucleic
acid by degradation of the biodegradable polymer.
[0021] In another aspect the invention features a method for
controlling a rate of nucleic acid release, characterized by
incorporating a nucleic acid into a positively-charged
water-insoluble biodegradable polymer having an introduced
positively-charged group, and releasing the nucleic acid by
degradation of the biodegradable polymer.
[0022] In another aspect the invention features a method for
enhancing functional expression of a nucleic acid, characterized by
incorporating the nucleic acid into a positively-charged
water-insoluble biodegradable polymer, and releasing the nucleic
acid by degradation of the biodegradable polymer to exhibit the
function of the nucleic acid. The invention also features a method
for enhancing functional expression of a nucleic acid,
characterized by incorporating the nucleic acid into a
positively-charged water-insoluble biodegradable polymer having an
introduced positively-charged group, and releasing the nucleic acid
by degradation of the biodegradable polymer to exhibit the function
of the nucleic acid.
[0023] In a preferred embodiment, the positively-charged
water-insoluble biodegradable polymer has at least one member
selected from the group consisting of collagen, gelatin, chitin,
chitosan, hyaluronic acid, alginic acid, starch, and derivatives of
any of these substances (e.g., amino derivatives). Preferably, the
positively-charged water-insoluble biodegradable polymer is
crosslinked gelatin having an introduced positively-charged group.
The nucleic acid is at least one member selected from the group
consisting of a DNA encoding a gene, an oligonucleotide, and a
double-stranded nucleic acid compound. The nucleic acid may encode
vascular endothelial growth factor gene, hepatocyte growth factor
gene, and fibroblast growth factor gene, as well as other genes
such as kinases, phosphatases, transcription factors, cytokines,
proteases, apoptosis-inducing factors and apoptosis-retarding
factors. In a preferred embodiment, a DNA molecule comprising a
base sequence described as SEQ. ID No. 1 of the sequence
listing.
[0024] In yet another aspect the invention features a phagocyte
comprising a nucleic acid-containing complex which contains a
nucleic acid and a biodegradable polymer. In a preferred
embodiment, the biodegradable polymer has at least one member
selected from the group consisting of collagen, gelatin, chitin,
chitosan, hyaluronic acid, alginic acid, starch, and derivatives of
any of these substances.
[0025] In yet another aspect the invention features a method for
exhibiting a function of a nucleic acid at a target site, at least
including the steps of (i) allowing phagocytes to take up a nucleic
acid-containing complex containing the nucleic acid and a
biodegradable polymer, (ii) allowing the taken up nucleic acid to
exert its function or inducing the expression of the nucleic acid
in the phagocytes, and delivering the phagocytes to the target
site. Preferably, the biodegradable polymer is at least one member
selected from the group consisting of collagen, gelatin, chitin,
chitosan, hyaluronic acid, alginic acid, starch, and derivatives of
any of these substances.
[0026] In yet another aspect the invention features a
pharmaceutical composition for gene therapy, containing a nucleic
acid-containing complex as an active ingredient, the nucleic
acid-containing complex containing a nucleic acid and a
biodegradable polymer, wherein the gene therapy at least includes
the steps of (i) allowing phagocytes to take up the nucleic
acid-containing complex containing the nucleic acid and the
biodegradable polymer, (ii) allowing the taken up nucleic acid to
exert its function or inducing the expression of the nucleic acid
in the phagocytes, and (iii) delivering the phagocytes to a target
site. In a preferred embodiment, the biodegradable polymer has at
least one member selected from the group consisting of collagen,
gelatin, chitin, chitosan, hyaluronic acid, alginic acid, starch,
and derivatives of any one of these substances.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 is a schematic view showing ionic bonding of a
three-dimensional lattice of a gelatin hydrogel and plasmid
DNA's.
[0028] FIG. 2 is a graph showing the process of incorporation of
lacZ plasmid into a gelatin hydrogel over time.
[0029] FIG. 3 is a graph showing the results of investigation of in
vivo survival of gelatin hydrogel-conjugated plasmid DNA following
administration into the right femoral muscle of ddY mice. The
survival was confirmed by the residual radioactivity of
radioiodine.
[0030] FIG. 4 shows micrographs demonstrating the expression of
lacZ gene in a rabbit lower limb ischemic model two weeks after
administration of particulate aminated gelatin hydrogel-conjugated
lacZ plasmid. FIG. 4A: Particulate aminated gelatin
hydrogel-conjugated lacZ plasmid (lacZ plasmid: 500 .mu.g). FIG.
4B: Naked lacZ plasmid (500 .mu.g) not conjugated to particulate
aminated gelatin hydrogel. FIG. 4C: Particulate aminated gelatin
hydrogel-conjugated lacZ plasmid (lacZ plasmid: 50 .mu.g).
[0031] FIG. 5 shows micrographs demonstrating the expression of
lacZ gene in a rabbit lower limb ischemic model after
administration of lacZ gene with the use of an adenovirus vector.
FIG. 5A: Three days after administration of lacZ gene. FIG. 5B: Two
weeks after administration of lacZ gene.
[0032] FIGS. 6A-D show radiographs demonstrating vasculogenesis in
a rabbit lower limb ischemic model after administration of
FGF4/HST1 plasmid. FIG. 6A: Particulate aminated gelatin
hydrogel-conjugated FGF4/HST1 plasmid (500 .mu.g). FIG. 6B:
Particulate aminated gelatin hydrogel-conjugated FGF4/HST1 plasmid
(5 .mu.g). FIG. 6C: Particulate aminated gelatin
hydrogel-conjugated lacZ plasmid (500 .mu.g). FIG. 6D: Naked
FGF4/HST1 plasmid (500 .mu.g) not conjugated to particulate
aminated gelatin hydrogel.
[0033] FIG. 7 is a micrograph showing lacZ expression in
macrophages in a rabbit arterial wall. Macrophages having expressed
lacZ are present (arrows).
[0034] FIG. 8 shows photographs of halftone images appearing on a
display showing the blood vessel density at baseline (top) and
during vasodilation (adenosine treatment) (bottom), as obtained by
radiation microvascular angiography. The results after
administration of naked VEGF.sub.165 plasmid are presented.
[0035] FIG. 9 is photographs of halftone images appearing on a
display showing the blood vessel density at baseline (top) and
during vasodilation (adenosine treatment) (bottom), as obtained by
radiation microvascular angiography. The results after
administration of gelatin hydrogel-conjugated VEGF.sub.165 plasmid
are presented.
[0036] FIG. 10 shows micrographs demonstrating macrophages having
taken in crosslinked gelatin particles-GFP plasmid. FIG. 10A shows
a phase contrast image, while FIG. 10B shows a fluorescence image
in the same field as in FIG. 10A. Fluorescence is observed in
macrophages, showing that crosslinked gelatin particles-GFP plasmid
has been incorporated into macrophages.
[0037] FIG. 11 shows views demonstrating the results of FACS
analysis of the efficiency of gene transfer into human dendritic
cells (DC). FIG. 11A: The results for DC cultured together with
particulate aminated gelatin hydrogel-conjugated GFP plasmid. FIG.
11B: The results for DC cultured together with naked GFP
plasmid.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0038] The nucleic acid that can be incorporated into the complex
of the invention is not restricted. However, preferred embodiments
are nucleic acids whose introduction brings a therapeutic effect,
and they are selected, as desired, according to the purpose of use
of the complex. In the invention, antisense oligonucleotides for
certain genes, and double-stranded nucleic acid compounds, such as
decoy nucleic acids, can be used preferably in addition to various
DNAs encoding a gene (Sobol, supra). Nucleic acid constructs can
also be delivered to correct point mutations (Bartlett et al.
(2000) Nat. Biotech 18:615). More concretely, nucleic acids which
can be incorporated into the nucleic acid-containing complex of the
invention for gene therapy in the cardiovascular field as well as
gene therapy of cancer are listed in Table 1 along with their
therapeutic aim. However, the invention is not restricted to them,
and the invention is applicable to other clinical fields, as known
by those skilled in the art.
TABLE-US-00001 TABLE 1 Purpose Type of nucleic acid Gene Enhances
Vascular endothelial therapy vasculogenesis and growth factor
(VEGF) in applicable to gene cardio- obstructive Hepatocyte growth
vascular arteriosclerosis and factor (HGF) gene field ischemic
heart Fibroblast growth disease factor (FGF) gene Regenerates
vascular VEGF gene endothelial cells HGF gene and applicable to
Endothelial nitric prevention of oxide synthase gene restenosis
following PGI.sub.2 synthetic gene arterioplasty Regulates cell
cycle Antisense of vascular smooth oligonucleotide to C-myb muscle
cells and Antisense usable for oligonucleotide to PCNA prophylaxis
of gene restenosis following Antisense arterioplasty or
oligonucleotide to cdc-2 bypass operation kinase Usable for Double
stranded nucleic prophylaxis, etc. of acid compound having the
restenosis following same sequence as bypass operation
transcription factor E2F conjugated sequence E2F decoy Applicable
to gene NF.kappa.B decoy therapy for ischemia-reperfusion injury
Gene Suicide gene Herpes simplex therapy transfer virus-thymidine
kinase for gene cancer Tumor suppressor p53 gene gene transfer
Protection of bone Multidrug resistance marrow stem cells (MDR)
gene from anticancer drug Immuno therapy Interleukin 12 gene B7
gene Antisense RNA ras gene
[0039] Preferred examples of the nucleic acids to be incorporated
into the nucleic acid-containing complex of the invention are
vascular endothelial growth factor gene, hepatocyte growth factor
gene, and fibroblast growth factor gene. Other such genes are known
to those skilled in the art including kinases, phosphatases,
transcription factors, cytokines, proteases, apoptosis-inducing
factors and apoptosis-retarding factors. As an example of the
fibroblast growth factor gene, FGF4/HST1 gene having a base
sequence described as SEQ. ID No. 1 of the sequence listing can be
cited.
[0040] The FGF4/HST1 gene was isolated and identified as a gene
having the activity of transforming NIH3T3 cells (hst-1; Proc.
Natl. Acad. Sci. USA, 83:3997-4001, 1986). Then, this gene was
found to have homology to fibroblast growth factor (FGF), and
became the fourth member of the FGF family (FGF4). Currently, 20
members of the FGF family, FGF 1 to 20, are known.
[0041] FGF4/HST1 protein is a secretory protein having a signal
peptide, and has been reported to have the following activities:
Cell growth promotion for fibroblasts and vascular endothelial
cells; vasculogenesis; promotion of growth and differentiation of
megakaryocytes; promotion of secretion of cytokines from
megakaryocytes; promotion of adhesion between megakaryocytes and
endothelial cells; in vitro induction of increases in peripheral
platelet count; alleviation of platelet decreases and shortening of
convalescence by prior administration in thrombopenic models due to
chemotherapy or radiotherapy; and increases in survival rates
following lethal radiation dose by prior administration.
[0042] As the application of the FGF4/HST1 gene to gene therapy, an
attempt has been made to apply an adenovirus vector, into which
this gene has been integrated, to the treatment of chronic stable
angina pectoris due to arteriosclerosis (Collateral
Therapeutics).
[0043] In the instant invention, the nucleic acid is used in a form
in which it is introduced into cells, and can exhibit its function
in the cells. In the case of DNA, for example, it is used as a
plasmid having the DNA located therein so that the DNA will be
transcribed in cells in which it has been introduced, then a
polypeptide encoded by the DNA will be produced, and then the
desired function will be exhibited by the polypeptide. Preferably,
a promoter region, an initiation codon, DNA coding for a protein
having the desired function, a termination codon, and a terminator
region are continuously arranged in the plasmid. Such techniques
and DNA elements are well known to those skilled in the art.
[0044] If desired, two or more nucleic acids can be incorporated
into one plasmid. Also, if desired, two or more nucleic acids may
be separately joined to a water-insoluble biodegradable polymer (as
described below) to form one nucleic acid-containing complex.
[0045] Conveniently, the intended vector can be prepared by
inserting the desired nucleic acid into a plasmid, which is
available in the art, with the use of a suitable restriction enzyme
site. It is also possible to prepare the vector by synthetic means
or semisynthetic means on the basis of the base sequence of the
nucleic acid to be introduced. Such techniques are well known to
those skilled in the art. Techniques such as those set forth in
"Molecular Cloning: A Laboratory Manual", second edition, Cold
Spring Harbor Laboratory, Sambrook, Fritsch & Maniatis, eds.,
1989, which is incorporated herein by reference in its entirety
including any figures, tables or drawings.
[0046] In the invention, "cells" into which the nucleic acid is
introduced are preferably cells in which the functional expression
of the nucleic acid is required, as well as cells having the
feature of taking up substances outside the cell (i.e.,
phagocytosis). In preferred embodiments, these cells are phagocytes
such as macrophages, which are described below. The cells in which
the functional expression of the nucleic acid should be exhibited
are selected variously, for example, depending on the nucleic acid
used (i.e., its function). Examples are myocardial cells, skeletal
muscle cells, and vascular endothelial cells. Phagocytes, such as
monocytes, dendritic cells, macrophages, histiocytes, Kupffer
cells, osteoclasts, synovial A cells, microglial cells, Langerhans'
cells, epithelioid cells, and multinucleate giant cells;
leucocytes; fibroblasts; and certain epithelia cells
(gastrointestinal epithelial cells, renal tubular epithelial cells)
can efficiently take the nucleic acid-containing complex into their
interior by their phagocytosis (first, targeting the nucleic
acid-containing complex to phagocytes), and are favorable in
delivering the nucleic acid to the desired site by their propensity
to migrate in vivo (second, targeting phagocytes to target sites
and cells). Hence, every organ or tissue, such as heart, muscle,
blood vessel, blood, bone marrow, lymphatic tissue, connective
tissue, liver, bone, synovial membrane, nerve, skin, inflammatory
tissue, or cancer tissue, can be affected by gene therapy.
[0047] In the invention, "biodegradable polymer" refers to a
polymer which is hydrolyzed for the first time by the action of a
physiologically active substance present in vivo, for example, an
enzyme. Examples of the biodegradable polymer are polysaccharides,
such as chitin, chitosan, hyaluronic acid, alginic acid, starch,
and pectin, proteins such as gelatin, collagen, fibrin and albumin,
and derivatives of these. A preferred example is gelatin or its
derivative. For the purpose of this specification, "biodegradable
polymer" does not include nucleic acids. In the invention,
"degradation of the biodegradable polymer" refers to the hydrolysis
of the polymer by the action of a physiologically active substance
present in vivo, such as an enzyme, or by its in vivo non-enzymatic
action, as stated above.
[0048] Here, "the derivative" refers to a modified form of the
biodegradable polymer suitable for formation of the nucleic
acid-containing complex, and includes, for example, an amino
derivative having an amino group introduced onto the polymer as
will be described below.
[0049] In the invention, if more controlled release of the nucleic
acid from the nucleic acid-containing complex is desired, the
biodegradable polymer that forms the complex with the nucleic acid
is preferably water-insoluble. Here, the water-insoluble property
refers to the nature of not dissolving in water because of chemical
or physical crosslinking between the molecules. In accordance with
the stipulations of the Japanese Pharmacopoeia, the water-insoluble
property corresponds to "sparingly soluble" to "practically
insoluble".
[0050] The biodegradable polymer is not restricted to a certain set
of molecules, as long as it can form a complex with nucleic acid.
If sustained release is desired, the polymer should preferably be
charged positively so that a stable nucleic acid-containing complex
will be formed. The degree of the positive charge is varied so as
to allow a polyion to form a complex with a normally negatively
charged nucleic acid. The formation of the polyionic complex can be
confirmed by measuring an increase in the turbidity of a mixture
obtained by mixing in water the components present in the
water-soluble state.
[0051] The strong binding (ionic bonding) between the negative
charge of the nucleic acid and the positive charge of the
biodegradable polymer results in the formation of a stable nucleic
acid-containing complex. If a biodegradable polymer which is
neutral or only slightly positively charged is used in the
invention, the polymer may be made cationic by introducing an amino
group or the like therein beforehand. Even in an already
positively-charged biodegradable polymer, a positively-charged
group, such as an amino group, may be introduced. By so doing, the
positive charge of the overall molecule is enhanced, and binding to
the nucleic acid increases, so that a more stable nucleic
acid-containing complex can be formed. The cation-imparting
procedure can be performed by methods known to those skilled in the
art.
[0052] The cation-imparting process is not restricted, as long as
such a process can introduce a functional group which will be
cationic under physiological conditions. A preferred method is to
introduce an amino group or an ammonium group onto a hydroxyl group
or a carboxyl group, already part of the biodegradable polymer,
under mild conditions. An example of the method comprises reacting
the polymer with an alkyldiamine, such as ethylenediamine or
N,N-dimethyl-1,3-diaminopropane, trimethylammonium acetohydrazide,
spermine, spermidine, or diethylamidochloride, with the use of any
of various condensing agents, e.g.,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride,
cyanuric chloride, N,N'-carbodiimidazole, cyanogen bromide, diepoxy
compound, tosyl chloride, a dianhydride compound such as
diethyltriamine-N,N,N',N'',N''-pentanoic acid dianhydride, or
trityl chloride. In a preferred embodiment, the method involves a
reaction with ethylenediamine as it is convenient and
versatile.
[0053] In the invention, the step of "introducing a
positively-charged group" refers to the introduction of a
functional group which makes the biodegradable polymer cationic
under physiological conditions. It means to introduce the
above-mentioned functional group onto the biodegradable
polymer.
[0054] In the invention, moreover, it is preferred to make the
biodegradable polymer water-insoluble by a crosslinking treatment
or other similarly effective treatments for the purpose of enabling
controlled release of nucleic acid. Generally, many biodegradable
polymers are water soluble, and thus the resulting nucleic
acid-containing complex is also water soluble. When this complex is
administered in vivo, the nucleic acid is rapidly released from the
complex, thus it is difficult to obtain stable local and sustained
supply of the nucleic acid. The instant invention uses a
water-insoluble biodegradable polymer, making it possible to
release nucleic acid in a controlled fashion in accordance with the
degradability of the biodegradable polymer in vivo. That is, the
sustained rate of release the nucleic acid can be controlled
according to the degradation of the biodegradable polymer.
Furthermore, the sustained release form permits the increase in the
efficiency of local expression of gene by the nucleic
acid-containing complex.
[0055] In preferred embodiments, the water-insoluble biodegradable
polymer used in the invention is a gelatin hydrogel insolubilized
in water by crosslinking. In more preferred embodiments, a gelatin
hydrogel has a water content of 85%, 88%, 91%, 94%, 95%, 97%, 99%
or more.
[0056] Crosslinking of the biodegradable polymer can be performed
by methods known to those skilled in the art. Examples are methods
using crosslinking agents, heat treatment, and methods using
ultraviolet radiation.
[0057] Preferred crosslinking agents may be selected according to
the type of the biodegradable polymer used. Normally, the following
crosslinking agents are used: formalin, glutaraldehyde, water
soluble carbodiimides
[1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride,
1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide-metho-p-toluenesulfonate,
etc.], epichlorohydrin, and diepoxy compounds [bisepoxydiethylene
glycol, 1,4-bis-(2,3-epoxypropoxy)-butane, etc.]. In the
crosslinking reaction, the concentration of the biodegradable
polymer is 1 to 30% by weight, preferably 5 to 10% by weight, while
the concentration of the crosslinking agent is 10.sup.-3 to 10% by
weight, preferably 10.sup.-2 to 1% by weight. The reaction is
performed for 1 to 48 hours, preferably 12 to 24 hours, at 0 to
40.degree. C., preferably 25 to 30.degree. C.
[0058] Crosslinking of the biodegradable polymer can also be
performed by heat treatment. The method of thermal crosslinking
will be described in the examples below, with gelatin taken as an
example.
[0059] An aqueous solution of gelatin (preferably, about 10% by
weight) is cast into a plastic petri dish, and air dried to obtain
a gelatin film. The film is allowed to stand for 1 to 48 hours,
preferably for 6 to 24 hours, at a temperature of between 110 and
160.degree. C., preferably 120 and 150.degree. C., under reduced
pressure, preferably, of about 10 mmHg, whereby the film is
thermally crosslinked.
[0060] When the gelatin film is to be crosslinked with ultraviolet
rays, the gelatin film is allowed to stand, normally, at 0 to
40.degree. C., and preferably at room temperature, below a
germicidal lamp.
[0061] The gelatin used may be a mixture of gelatins having
different physical properties, such as solubility, stability, and
swelling properties. A mixture of crosslinked gelatins different in
physical properties may also be used.
[0062] The positively-charged water-insoluble biodegradable polymer
incorporated in the nucleic acid-containing complex of the
invention may be incorporated in the complex in the following
manner: the biodegradable polymer having the above-mentioned
characteristics is incorporated alone, or two or more types of the
biodegradable polymers are incorporated as a mixture (simply mixed
to be incorporated in the same nucleic acid-containing complex).
Alternatively, two or more types of the biodegradable polymers may
be chemically bonded beforehand, and then incorporated in the
complex. Any of these embodiments are included in the
invention.
[0063] To chemically bond two or more types of the biodegradable
polymers beforehand, and incorporate the bonding product in the
complex, the respective biodegradable polymers can be separately
made water-insoluble, and then chemically bonded, or can be
chemically bonded, and then made water-insoluble. In preferred
embodiments, the respective biodegradable polymers are first bonded
chemically, and then treated to render them water-insoluble.
[0064] In the invention, the complex containing nucleic acid and a
positively-charged water-insoluble biodegradable polymer can be
prepared easily by mixing the aforementioned nucleic acid and the
aforementioned positively-charged water-insoluble biodegradable
polymer. The ratio between the amounts of the nucleic acid and the
positively-charged water-insoluble biodegradable polymer differs
according to the degree of positive charge of the biodegradable
polymer used. Usually during the mixing, the nucleic acid is used
at a saturating concentration with respect to the biodegradable
polymer.
[0065] In a preferred embodiment for preparing a nucleic
acid-containing complex containing a nucleic acid in a crosslinked
gelatin gel, a crosslinking agent is directly added to an aqueous
solution of gelatin of between 5% to 30% by weight to prepare a
crosslinked gelatin gel. In a more preferred embodiment, an
uncrosslinked gelatin gel is dipped in an aqueous solution of a
crosslinking agent to prepare a crosslinked gelatin gel. The
resulting crosslinked gelatin gel is then directly dipped in a
solution containing nucleic acid. In a preferred embodiment, the
crosslinked gelatin gel is dried, and then swollen again in a
solution containing nucleic acid.
[0066] The strongly negatively charged nucleic acid is ionically
bonded to the positively-charged biodegradable polymer to form a
complex. In such a complex of the instant invention, the nucleic
acid incorporated in the complex is characterized by being released
from the complex by degradation of the biodegradable polymer by the
action of an enzyme, or other physiological processes. FIG. 1 shows
a schematic view of the complex, with DNA taken as an example of
the nucleic acid.
[0067] In complexing the nucleic acid, other components may be
added, if desired, for purposes such as stability of the resulting
nucleic acid-containing complex, sustained release of the nucleic
acid, and functional expression of the released nucleic acid.
Examples of these other components are aminosugars or their
macromolecular compounds or chitosan oligomers, basic amino acids
or their oligomers or macromolecular compounds, and basic polymers
such as polyallylamine, polydiethylaminoethylacrylamide, and
polyethyleneimine. Furthermore, ligand proteins capable of binding
to receptors expressed in an organ specific fashion, or antibodies
directed specifically to selected targets are added, thereby making
possible the delivery of the nucleic acid-containing complex to the
desired site, and eventually, the localized release of the nucleic
acid. Such ligands and/or antibodies are well known to one skilled
in the art.
[0068] The invention also relates to a method for controlling a
rate of nucleic acid release, characterized by incorporating a
nucleic acid into a positively-charged water-insoluble
biodegradable polymer, and allowing the release of the nucleic acid
in a physiological setting.
[0069] As the nucleic acid and the positively-charged
water-insoluble biodegradable polymer used in the method for
controlling a rate of nucleic acid release according to the
invention, all those biodegradable polymers and nucleic acids named
herein may be used, as well as others known to one those skilled in
the art.
[0070] The nucleic acid incorporated in the complex is slowly
released from the complex as the water-insoluble biodegradable
polymer is degraded in vivo (in other words, the, degradation rate
determines the rate of release). This release is preferably
effected only through the mediation of the biodegradable polymer,
because that would be able to control the rate of release more
reproducible. The rate of release is closely related to the
strength of bonding between the nucleic acid and the biodegradable
polymer in the complex, along with the stability of the complex, in
addition to the degree of biodegradability of the biodegradable
polymer used (which in turn can depend on the water content of the
biodegradable polymer). The rate of release can also be controlled
by the balance between the positive charge and the negative charge
in the complex. Usually, the higher the positive charge of the
biodegradable polymer used, the more the nucleic acid in the
resulting nucleic acid-containing complex is retained. Thus, a
biodegradable polymer with a higher positive charge is superior in
terms of the controlled release of the nucleic acid via degradation
of the water-insoluble biodegradable polymer. If the positive
charge of the biodegradable polymer is insufficient to provide
controlled release, an amino group or similar substituent is
further introduced into the polymer to make the polymer cationic,
thereby increasing its positive charge.
[0071] The invention also provides phagocytes comprising a nucleic
acid-containing complex which contains a nucleic acid and a
biodegradable polymer. This is based on the new finding that such a
complex is readily taken into phagocytes which play important roles
in the immune system (first, targeting of the complex at
phagocytes), and the phagocytes are carried to a target site based
on their normal, in vivo migration (second, targeting of the
phagocytes at the target site), thus making it possible to exhibit
the function of the nucleic acid readily and stably at the target
site. The phagocytes used in the invention are not restricted, as
long as they are cells exhibiting phagocytosis and which migrate to
lesions (e.g., inflammatory sites, cancer tissue, etc.). For
example, macrophages and monocytes are preferred examples of such
cells. Also, dendritic cells, histiocytes, Kupffer cells,
osteoclasts, synovial A cells, microglial cells, Langerhans' cells,
epithelioid cells, and multinucleate giant cells are also
preferred, even though their migration is minimal. The nucleic
acid-containing complex containing the nucleic acid and the
biodegradable polymer is readily taken into the phagocytes by the
phagocytosis. The uptake of the nucleic acid-containing complex by
the phagocytes can be performed in situ or in vitro according to
the purpose of use.
[0072] As the nucleic acid and the biodegradable polymer
incorporated into the nucleic acid-containing complex used, all
those biodegradable polymers and nucleic acids listed herein may be
used. The charge properties and solubility of the biodegradable
polymer are not restricted. From the viewpoints of the ease of
uptake by phagocytes, the uptake efficiency and the rate of uptake,
the biodegradable polymer is preferably water-insoluble. From the
viewpoint that firm bonding to the nucleic acid is desirable for
reliable binding of the nucleic acid, the biodegradable polymer is
preferably positively-charged. The terms "water-insoluble" and
positively-charged are intended to have the same meanings as
described earlier.
[0073] The phagocytes incorporating the nucleic acid-containing
complex which contains the nucleic acid and the biodegradable
polymer can be used for experimental purposes, and can also be used
preferably as drugs. That is, phagocytes incorporating the nucleic
acid-containing complex are prepared in vitro, and then the
resulting phagocytes are administered in vivo, whereby desired
genetic information from the nucleic acid can be expressed at the
target site by making use of the phagocytes' characteristic
accumulation at a lesion or other diseased site.
[0074] The invention also provides a method for exhibiting a
function of a nucleic acid in a target site-specific manner,
including at least the steps of (i) allowing phagocytes to take up
the nucleic acid-containing complex containing the nucleic acid and
a biodegradable polymer, (ii) inducing expression of the nucleic
acid in the phagocytes, and (iii) delivering the phagocytes to the
target site. Each of the steps will be described below.
(i) The Step of Allowing Phagocytes to Take Up a Nucleic
Acid-Containing Complex Containing a Nucleic Acid and a
Biodegradable Polymer:
[0075] This step, as has been described earlier, is achieved by the
incorporation of the nucleic acid-containing complex into
phagocytes by a phagocytic action inherent in the phagocytes. This
step can be performed by mixing the nucleic acid-containing complex
and phagocytes beforehand in vitro, or by administering the nucleic
acid-containing complex in vivo and utilizing its uptake by
phagocytes in vivo. The mixing ratio of the nucleic acid-containing
complex to phagocytes in vitro is not restricted, and may be any
ratio at which phagocytes can take in the nucleic acid-containing
complex. Normally, the step is achieved by adding the nucleic
acid-containing complex in an excess amount. The administration of
the nucleic acid-containing complex in vivo, or the administration
of in vitro prepared phagocytes under in vivo conditions can be
performed in accordance with the mode of administration of the
nucleic acid-containing complex and drug of the invention
(described below). Such nucleic acid-containing complex phagocytes
when prepared in vitro can also be considered a drug for the
purposes of the instant invention. When administering the
phagocytes of the invention which comprise the nucleic
acid-containing complex into a living organism, it is necessary to
perform administration while maintaining the viability and activity
of the phagocytes. Methods complying with bone marrow
transplantation or immunotherapy can be adopted, and are known to
those skilled in the art. Preferably, typical examples of the
methods for administration are as follows: (a) administration into
a lesion or neighboring tissue; (b) administration into a body
cavity (pericardial cavity, thoracic cavity, abdominal cavity,
cerebrospinal cavity); (c) administration into a blood vessel or
lymphatic tissue governing the lesion; (d) Administration into a
blood vessel or dermal, adipose or skeletal muscle tissue apart
from the lesion. Any of these administration methods can be
expected to take effect at the site of lesion, rather than at the
site of administration, because of the inherent migratory capacity
of phagocytes.
(ii) The Step of Inducing Expression of the Nucleic Acid in
Phagocytes:
[0076] This step is performed using a technique which is known to
those skilled in the art. That is, the step is performed by
incorporating the nucleic acid in a manner in which the nucleic
acid can display its function in phagocytes when introduced into
these cells. In preferred embodiments, the function of the
incorporated nucleic acid is the expression of a gene encoded by
the nucleic acid. In other preferred embodiments, the function of
the nucleic acid may be by direct action of the nucleic acid
incorporated in the complex. In the instant invention, the nucleic
acid is taken into phagocytes as a nucleic acid-containing complex
having the nucleic acid complexed with the biodegradable polymer.
Hence, the sustained release of the nucleic acid controlled by the
biodegradability of the nucleic acid-containing complex increases
the efficiency of introduction of the nucleic acid into the cells,
and promotes the expression or other function of the nucleic acid
in the cells.
(iii) The Step of Delivering Phagocytes to the Target Site:
[0077] This step is performed easily and safely by the migration of
phagocytes. Preferably, phagocytes are selected according to the
desired target site. If targeting at cancer tissue or inflammatory
tissue is desired, for example, macrophages, epithelioid cells, and
multinucleate giant cells are preferably used. Monocytes are
preferably used for blood targets; dendritic cells for targets in
bone marrow and lymphatic tissue; histiocytes for targets in
connective tissue; Kupffer cells for targets in the liver;
osteoclasts for targets in the bone; synovial A cells for targets
in the synovium; microglial cells for targets in the nervous
system; and Langerhans' cells for targets in the skin. Furthermore,
the administration of the phagocytes to the surfaces of various
organs enables the nucleic acid (carried in the phagocytes) to be
transported deep in the target organ.
[0078] The invention also provides a novel drug for gene therapy
which utilizes the uptake of a nucleic acid-containing complex
based on the phagocytosis of phagocytes, and which utilizes the
delivery of the complex to the target site based on the inherent
migratory capacity of the phagocytes, as detailed above. This drug
has a nucleic acid-containing complex containing a nucleic acid and
a biodegradable polymer as an active ingredient. The intended range
of the components of the complex are described above and below.
[0079] The nucleic acid-containing complex of the invention can be
administered in vivo by a variety of methods. For persistent and
local release of the nucleic acid at the desired particular site,
parenteral administration is particularly preferred. A drug
containing the nucleic acid-containing complex of the invention as
an active ingredient can be prepared by mixing the complex, if
necessary, with pharmaceutically acceptable carriers (stabilizer,
preservative, solubilizer, pH regulator, viscosity-increasing
agent, etc.). These carriers are known to those skilled in the art.
Various additives for adjusting the sustained release effect may
further be incorporated and are known to those skilled in the
art.
[0080] The drug having the nucleic acid-containing complex of the
invention as the active ingredient also includes two or more types
of nucleic acid-containing complexes containing different kinds of
nucleic acids. Such a drug which has a plurality of therapeutic
purposes is particularly useful in the field of gene therapy which
has become diversified.
[0081] The nucleic acid-containing complex of the invention can be
pharmaceutically manufactured in various forms according to the
intended purpose. Examples of the forms are solid and semisolid
preparations in the form of granule, cylinder or prism, sheet,
disc, paste, etc., or injections such as suspensions and emulsions.
Preferred are solid preparations having an excellent sustained
release effect at the desired particular site, and preferred for
local administration. For instance, the nucleic acid-containing
complex of the invention prepared in a sheet form is suitable for
fixing to the inner wall of a local blood vessel. More concretely,
a method is available in which the sheet-shaped nucleic
acid-containing complex is wound about a stent for arterioplasty,
the stent is inserted into an appropriate blood vessel ramus by
means of a catheter, and a balloon is inflated in the local blood
vessel to fix the nucleic acid-containing complex to the inner wall
of the blood vessel. This method enables gene transfer into a blood
vessel wall at a site where the complex is fixed, and gene therapy
for the region peripheral to the site of fixing. Preferred
embodiments include gene therapy of cancer, such as
anti-vasculogenesis therapy, or gene therapy of a circulatory
disorder, such as vasculogenesis therapy.
[0082] The injections of the nucleic acid-containing complex can be
administered intramuscularly, into adipose tissue, subcutaneously,
intradermally, intravenously, into the lymphatic vessel, into the
lymph node, intra-arterially, into a body cavity (pericardial
cavity, thoracic cavity, abdominal cavity, cerebrospinal cavity),
or into the bone marrow. The intramuscular administration is
preferred. Direct administration into diseased tissue is also
possible.
[0083] In the case of solid and semisolid preparations, the
following methods are cited as examples: The preparation is
directly embedded at a site where release of the nucleic acid is
expected; A pasty preparation is injected by a syringe; A granular
preparation is injected as a parenteral suspension; A catheter is
inserted in a percutaneous, transluminal manner, and the complex
adhered to a stent is self-retained in the blood vessel via the
catheter; and fine particles of the complex (particle size: about
several microns to about 15 microns) are injected through a
catheter, and localized at a site where release of the nucleic acid
is expected. These methods and others are known to those skilled in
the art.
[0084] In pharmaceutically manufacturing the complex of the
invention, it is further desirable to subject it to a sterilization
step such as sterile filtration.
[0085] According to the invention, the dose administered in an
animal, especially human, varies with various factors, such as the
desired nucleic acid, the biodegradable polymer used, the mode of
administration, and the particular site to be treated. However, the
dose should be an amount sufficient to bring a therapeutic
response. Such doses are known to those skilled in the art.
[0086] The nucleic acid-containing complex of the invention is
applied, preferably, to gene therapy. Diseases to which the complex
is applicable differ according to the type of the nucleic acid
incorporated in the complex. Examples of the diseases are diseases
in the cardiovascular field, such as peripheral arterial diseases,
coronary arterial diseases, and diseases causing lesions, e.g.,
restenosis following arteriodilating operation. Other examples are
cancer (malignant melanoma, intracranial tumor, metastatic
malignant tumor, breast cancer, etc.), infections (HIV, etc.), and
monogenic diseases (cystic fibrosis, chronic granulomatous disease,
.alpha..sub.1-antitrypsin deficiency, Gaucher disease, etc.).
[0087] In preferred embodiments, when a fibroblast growth factor
gene, especially, DNA comprising a base sequence described as SEQ.
ID No. 1 of the sequence listing, is used as a nucleic acid
incorporated in the complex, it can be applied to various diseases
against which the physiological activity of the aforementioned
FGF4/HST1 protein is therapeutically effective.
EXAMPLES
[0088] Examples and Experimental Examples will be offered to
illustrate the invention in more detail, but they in no way limit
the invention.
Example 1
(1) Preparation of Aminated Gelatin
[0089] 1 Gram of gelatin having an isoelectric point of 9.0 (Nitta
Gelatin Company) was dissolved in 50 ml of 0.1 M phosphate buffer
(PB, pH 5.0). Then, ethylenediamine (molecular weight: 60.1) in an
amount of 50 mols, per mol of the carboxyl groups of the gelatin
(molecular weight: 10,000, carboxyl group content: 95 mols/gelatin
molecule), was added to the solution, and
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(molecular weight: 191.7) in an amount of 50 mols per mol of the
carboxyl groups was further added. The resulting mixed solution was
stirred for 12 hours at 37.degree. C. Upon completion of the
reaction, the reaction mixture was dialyzed against water for 2
days, and lyophilized to obtain aminated gelatin. Colorimetric
quantitative analysis for amino groups with the use of sodium
trinitrobenzenesulfonate showed that 56% of the carboxyl groups of
the gelatin were aminated.
(2) Preparation of Particulate Aminated Gelatin Hydrogel
[0090] An aqueous solution (0.2 ml) of 100 mg/ml of the aminated
gelatin that had been pretreated to 40.degree. C. was charged into
5 ml of an olive oil. The mixture was emulsified by touch mixing
for 1 minute at 40.degree. C. The emulsified mixture was further
ultrasonically emulsified for 40 seconds, followed by rapid cooling
on ice. Acetone (1.43 ml) was added, and the mixture was
centrifuged (5,000 rpm, 5 min, 4.degree. C.) to recover the
uncrosslinked particles. The resulting particles were subjected to
centrifugal washing (5,000 rpm, 5 min, 4.degree. C.) using acetone.
Then, the resulting particles were suspended in an aqueous solution
mixture of 30 .mu.l of an aqueous solution of 25% (w/v) of
glutaraldehyde and 20 ml of an aqueous solution of 0.1% (w/v) of
Tween 80. The suspension was stirred for 40 hours at 4.degree. C.
to crosslink the gelatin particles chemically. Then, the resulting
particles were recovered by centrifugation (5,000 rpm, 5 min,
4.degree. C.). Then, in order to block the unreacted aldehyde
groups, the particles were dispersed in a 0.1 (w/v) % Tween 80
aqueous solution (20 ml) of 100 mM glycine, and the dispersion was
stirred for 1 hour at 37.degree. C. After completion of the
reaction, the reaction mixture was centrifugally washed with a 0.1
(w/v) % Tween 80 aqueous solution (two times) and with distilled
water (three times) to recover the particles. After thorough
rinsing with acetone, the particles were air dried. The sizes of
the dried particles, and the particles swollen in PB (pH 7.0) for
12 hours at 37.degree. C. were measured from their
photomicrographs. Based on the ratio of their sizes, the water
content was evaluated as 98.6% by volume (particulate aminated
gelatin hydrogel). The average size of the resulting particles in
dry state was 100 .mu.m (200 .mu.m in swollen state).
(3) Preparation of DNA Conjugated to Particulate Aminated Gelatin
Hydrogel
[0091] DNA used was lacZ expression plasmid DNA obtained by
inserting lacZ gene of Escherichia coli into the EcoRI site of a
pCAGGS expression vector having the promoter CAG (chicken
.beta.-actin promoter; Gene, 108:193-200, 1991) which has high
activity particularly in the muscle (the plasmid will be called
pCAGGS-lacZ, or simply referred to as lacZ plasmid; furnished by
Miyazaki Laboratory, Molecular Defense Medicine, Osaka University
Faculty of Medicine). This DNA was radiolabeled with TlCl.sub.3.
That is, 5 .mu.l of an Na.sup.125I, solution (740 MBq/ml in 0.1N
NaOH aqueous solution, NEN Research Products, DuPont) was mixed
with a 0.3 mM Na.sub.2SO.sub.3 aqueous solution (2 .mu.l), and the
mixture was allowed to stand for 30 minutes at 25.degree. C. To the
mixture, 5 .mu.l of a 0.1M CH.sub.3COONa-40 mM CH.sub.3COOH mixed
solution (pH 5.0) having 5 .mu.g DNA dissolved therein was added.
Further, 0.3 ml of a 0.2M CH.sub.3COONa-1.0 mM CH.sub.3COOH mixed
solution (pH 4.0) having 0.3 mg TlCl.sub.3 dissolved therein was
added. Then, the resulting mixture was allowed to stand for 40
minutes at 60.degree. C. Then, 0.1 ml of an aqueous solution of 0.1
mM Na.sub.2SO.sub.3 was added to the mixed solution, and a 0.1 mM
NaCl-50 mM tris-hydrochloric acid solution (pH 7.0) containing 0.9
ml of 1 mM EDTA was further added. The resulting mixture was heated
for 30 minutes at 60.degree. C. Upon completion of the reaction,
the resulting solution was cooled, and subjected to a PD-10 gel
chromatography column (Amersham Pharmacia Biotech) to separate
radioiodinated DNA and free radioiodine. Then, 10 .mu.l of an
aqueous solution of the radioiodinated DNA was added dropwise to
the lyophilized particulate aminated gelatin hydrogel, and the
system was allowed to stand for 1 hour at 25.degree. C. During this
period, the particles were impregnated with the aqueous solution to
obtain a radioiodinated DNA-containing complex.
Example 2
[0092] An aqueous solution (end concentration: 10.7 mM) of
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was
added to an aqueous solution of gelatin (end concentration: 5% by
weight). Then, the mixture was poured into a plastic petri dish
measuring 15 cm.times.15 cm, and held at 4.degree. C. for 24 hours
to carry out a crosslinking reaction. The gelatin used was an
alkali-treated gelatin having an isoelectric point of 4.9. After
the reaction was completed, the crosslinked gelatin gel was removed
from the petri dish to obtain a gelatin sheet of 200 .mu.m in
thickness. The resulting sheet was washed thoroughly with water,
and then lyophilized. The dry crosslinked gelatin sheet was allowed
to stand for 1 hour in an aqueous solution of radioiodinated DNA
(lacZ plasmid) prepared in the same manner as in Example 1 to
obtain a radioiodinated DNA-containing crosslinked gelatin
sheet.
Experimental Example 1
Incorporation of Gene into Particulate Aminated Gelatin
Hydrogel
[0093] It was confirmed that nucleic acid was taken into a
positively-charged water-insoluble biodegradable polymer.
[0094] An aqueous solution containing lacZ plasmid in a
concentration of 500 .mu.g/ml, and the particulate aminated gelatin
hydrogel prepared in Example 1-(2) were mixed, and the lacZ plasmid
concentration in the aqueous solution was measured over time by the
ultraviolet absorbance method (wavelength: 260 nm). As a control,
water (containing no lacZ plasmid) was used. The results are shown
in FIG. 2.
[0095] The lacZ plasmid in the aqueous solution was rapidly
incorporated into the particulate aminated gelatin hydrogel within
1 hour (as measured by a decrease in the lacZ plasmid concentration
in the aqueous solution). The decline in the lacZ plasmid
concentration in the aqueous solution persisted until 24 hours
later, demonstrating that the lacZ plasmid continued to be adsorbed
to the gelatin hydrogel.
Referential Experimental Example 1
Biodegradability of Particulate Aminated Gelatin Hydrogel
[0096] The particulate aminated gelatin hydrogel, a constituent of
the nucleic acid-containing complex of the invention, was confirmed
to be biodegradable.
[0097] An anhydrous benzene solution of .sup.125I-Bolton-Hunter
reagent (NEN-120X, 147 MBq/ml in anhydrous benzene, NEN Research
Products, DuPont) was taken in an amount of 0.1 ml into a test
tube. Benzene was evaporated by nitrogen gas bubbling. To the test
tube, 5 ml of distilled water, in which the particulate aminated
gelatin hydrogel prepared in Example 1-(2) was dispersed to a
concentration of 10 mg/ml, was added. The mixture was stirred for
12 hours at 4.degree. C. to radioiodinate the gelatin hydrogel. The
resulting radioiodinated particulate aminated gelatin hydrogel was
centrifugally washed (5,000 rpm, 5 min, 4.degree. C.) with
distilled water to remove the .sup.125I-Bolton-Hunter reagent
taking no part in labeling. Finally, the concentration of the
particles was adjusted to 0.5 mg/ml with the use of 0.1M phosphate
buffered saline (PBS, pH 7.0).
[0098] The resulting radioiodinated particulate aminated gelatin
hydrogel was administered into the right femoral muscle of ddy mice
(female, 6-week-old, purchased from Shimizu Experimental Material
Company) at a dose of 0.1 ml per mouse. After a lapse of a
predetermined time, the right femoral muscle was resected, and
measured for radioactivity. The measured radioactivity was compared
with the initial radioactivity administered, to evaluate the
residual radioactivity. The experiments were conducted in 3 mice
under each of respective experimental conditions. The particulate
aminated gelatin hydrogel was degraded over time in vivo, and thus
was confirmed to be biodegradable.
Experimental Example 2
Evaluation of In Vivo Survival of DNA
[0099] A radioiodinated particulate aminated gelatin hydrogel
prepared in the same manner as in Referential Experimental Example
1, and the particulate aminated gelatin hydrogel impregnated with
radioiodinated DNA prepared in Example 1 (3) were each dispersed in
0.1 ml of PBS. Then, the dispersion was administered into the right
femoral muscle of ddy mice (female, 6-week-old, purchased from
Shimizu Experimental Material Company). As a control, 90 .mu.l of
PBS was added to 10 .mu.l of an aqueous solution of radioiodinated
DNA, and then the mixture was administered into the right femoral
muscle of mice. The number of the experimental animals was three
for each of the experimental conditions, and the residual
radioactivity was expressed as the mean value.+-.standard
deviation. The results are shown in FIG. 3.
[0100] The residual radioactivity of the gelatin particles
gradually decreased over time, thus showing that the particles were
degraded in vivo, in other words, they were biodegradable
(-.cndot.-). The residual radioactivity of DNA impregnated into the
gelatin hydrogel also decreased with the passage of time, and its
profile over time was the same as that of the residual
radioactivity of the particulate aminated gelatin hydrogel itself
(-.degree.-). This is proof that DNA was released in a sustained
manner from the particles as the particles were degraded. When DNA
was administered as an aqueous solution, the residual radioactivity
rapidly decreased (-.DELTA.-). This finding shows that DNA was
rapidly excreted or metabolized without being retained at the site
of administration.
Experimental Example 3
Gene Expression of Particulate Aminated Gelatin Hydrogel Conjugated
DNA
[0101] A rabbit's femoral artery was removed to prepare a lower
limb ischemic model. Ten days later, lacZ plasmid was administered
into the muscle of the ischemic site. At 2 weeks after
administration, the muscular tissue of the ischemic part was
stained for LacZ to investigate the expression of the gene. The
results are shown in FIGS. 4A to 4C.
[0102] The investigations were carried out for the particulate
aminated gelatin hydrogel (diameter 200 .mu.m when swollen)
conjugated lacZ plasmid prepared in Example 1 (3) (500 .mu.g as the
amount of lacZ plasmid: FIG. 4A), and naked lacZ plasmid not
conjugated to the particulate aminated gelatin hydrogel (500 .mu.g,
control: FIG. 4B). The investigation was also conducted for the
administration of the particulate aminated gelatin hydrogel
conjugated lacZ plasmid with the amount of the lacZ plasmid being
decreased to 1/10 (50 .mu.g as the amount of lacZ plasmid: FIG.
4C).
[0103] Staining of lacZ was performed in the following manner:
1. Fix the sample in a 37% formaldehyde-25% glutaraldehyde mixed
solution for 5 minutes at 4.degree. C. 2. Wash with PBS (3 times).
3. Stain with a staining solution (1 mg/ml X-gal, 5 mM potassium
hexacyanoferrate(III), 5 mM potassium hexacyanoferrate(II), 1M
magnesium chloride).
[0104] Compared with the administration of lacZ plasmid alone, lacZ
plasmid bound to the particulate aminated gelatin hydrogel showed
more extensive expression of the gene (comparison of A with B).
[0105] The lacZ plasmid bound to the particulate aminated gelatin
hydrogel also showed significant expression of the gene even when
its dose was decreased to 1/10.
Referential Experimental Example 2
Gene Expression Using Adenovirus Vector
[0106] Gene transfer using an adenovirus vector was investigated in
a rabbit's lower limb ischemic model in the same manner as in
Experimental Example 3.
[0107] An adenovirus vector incorporating 1.times.10.sup.9 pfu of
lacZ gene (furnished by Saito Laboratory, The Institute of Medical
Science, The University of Tokyo) was administered. Three days
after administration, the expression of lacZ gene was stronger than
by the administration of lacZ plasmid alone and the administration
of lacZ plasmid bound to the particulate aminated gelatin hydrogel
(FIG. 5A). However, the gene expression significantly declined at 2
weeks after administration (FIG. 5B).
Experimental Example 4
Vasculogenesis in Rabbit's Lower Limb Ischemic Model
[0108] Vasculogenesis by the administration of fibroblast growth
factor FGF4/HST1 plasmid was investigated in a rabbit lower limb
ischemic model as in Experimental Example 3.
[0109] Particulate aminated gelatin hydrogel conjugated FGF4/HST1
plasmid was prepared in the same manner as in Example 1, except
that the nucleic acid used was FGF4/HST1 plasmid (500 .mu.g or 5
.mu.g). As the plasmid, there was used FGF4/HST1 expression plasmid
DNA (hereinafter referred to simply as FGF4/HST1 plasmid) obtained
by inserting FGF4/HST1 gene (SEQ. ID No. 1 of the sequence listing)
into the HindIII site of pRc/CMV2 vector (INVITORGEN).
[0110] As controls, naked FGF4/HST1 plasmid (500 .mu.g), and lacZ
plasmid (500 .mu.g) conjugated to the same gelatin hydrogel were
used.
[0111] Ten days after preparation of the lower limb ischemic model,
the various plasmids of the above embodiments were each
intramuscularly injected into the ischemic site. At 2 weeks after
administration, angiography of neogenetic blood vessels
(radiography) was performed. The results are shown in FIGS. 6A to
6D.
[0112] FIGS. 6A and 6B show the results for the administration of
the particulate aminated gelatin hydrogel-conjugated FGF4/HST1
plasmid (amount of FGF4/HST1 plasmid: 500 .mu.g in FIG. 6A, 5 .mu.g
in FIG. 6B). FIG. 6C show the results for the administration of the
particulate aminated gelatin hydrogel-conjugated lacZ plasmid (500
.mu.g, control). FIG. 6D show the results for the administration of
the naked FGF4/HST1 plasmid (500 .mu.g, control). The
administration of FGF4/HST1 plasmid conjugated to the particulate
aminated gelatin hydrogel resulted in the new growth of more blood
vessels than the administration of the plasmid in its naked form.
Thus, the functional expression of FGF4/HST1 plasmid was found to
be enhanced (comparisons of FIG. 6A with FIGS. 6C and 6D). This
effect was observed even when its dose was decreased to 1/100 (FIG.
6B). Furthermore, it became possible for FGF4/HST1 plasmid to
function locally with high efficiency when administered in a form
conjugated to the gelatin hydrogel, rather than in a naked
form.
[0113] Experiments using VEGF.sub.165 plasmid (500 .mu.g) instead
of FGF4/HST1 plasmid gave similar results. VEGF.sub.165 plasmid was
provided by Prof. Shibuya of the University of Tokyo. This plasmid
is obtained by inserting VEGF165 gene into the XhoI site of a
pCAGGS expression vector (Shibuya M, Adv. Cancer Res., 67, 281-316,
1995).
Experimental Example 5
Gene Transfer to Rabbit Arterial Wall
[0114] (1) lacZ Plasmid
[0115] The thin sheet of dry crosslinked gelatin prepared in
Experimental Example 2 was mounted on a stainless stent (Synthesis;
Cardio Vascular Dynamics, Inc.). A lacZ plasmid solution (5 to 10
.mu.g/.mu.l) was added dropwise to the thin sheet of crosslinked
gelatin, with the dropwise addition being repeated for 2 hours to
avoid drying (total amount added dropwise: 50 to 100 .mu.l). By
this measure, the crosslinked gelatin and the lacZ plasmid were
conjugated. The stent coated with this crosslinked gelatin was
implanted into a rabbit's iliac artery. Five days later, the iliac
artery was withdrawn, embedded in Tissue-Tek(R) O.C.T. Compound,
and fixed in liquid nitrogen. This sample was stored at -80.degree.
C. until use. Slices were prepared from the fixed sample, and
subjected to hematoxylin-eosin (HE) staining, DAB
(diaminobenzenezidene), and Xgal staining. the results are shown in
FIG. 7.
[0116] In the layer outward of the vascular smooth muscle tunica,
the expression of lacZ was observed, confirming the uptake of the
plasmid by macrophages and the expression of the plasmid.
(2) VEGF165 Plasmid
[0117] In the same manner as described in (1) above, a solution of
1 to 2 .mu.g/.mu.l of VEGF165 plasmid was added dropwise in an
amount of 50 to 100 .mu.l to the thin sheet of crosslinked gelatin.
The expression of this plasmid was confirmed by
immunohistochemistry staining (anti-human VEGF IgG fraction
(Austral Biologicals) was used as a primary antibody).
Experimental Example 6
Study of Maturity of Neogenetic Vasoganglia by Radiation
Microvascular Angiography
(1) Administration of Plasmid
[0118] An aqueous solution containing 500 .mu.g of naked
VEGF.sub.165 plasmid unconjugated to crosslinked gelatin was
administered visually into a diseased lower limb femoral muscle of
a rabbit having lower limb ischemia (as in Experimental Example
3).
[0119] Separately, physiological saline containing 500 .mu.g of
VEGF.sub.165 plasmid conjugated to 4 mg of particulate crosslinked
gelatin having a diameter of about 200 .mu.m was administered
visually into a diseased lower limb femoral muscle of a rabbit
having lower limb ischemia.
(2) Adenosine Loading
[0120] Adenosine was administered to the abdominal aorta at a dose
of 100 .mu.g/kg/min.
(3) Measurements and Results
[0121] The maturity of neogenetic vasoganglia was measured in the
naked VEGF.sub.165 plasmid group and the gelatin hydrogel
conjugated VEGF.sub.165 plasmid group by radiation microvascular
angiography (space resolution 25 microns). The results of the naked
VEGF.sub.165 plasmid treatment are shown in FIG. 8, while the
results of the gelatin hydrogel conjugated VEGF.sub.165 plasmid
treatment are shown in FIG. 9. In the naked VEGF.sub.165 plasmid
treated group, the blood vessel density following adenosine loading
(during vasodilation) decreased compared with the baseline (no
loading of adenosine). In the gelatin hydrogel conjugated
VEGF.sub.165 plasmid treated group, the blood vessel density
following adenosine loading clearly increased. These findings show
that in the gelatin hydrogel conjugated VEGF.sub.165 plasmid group,
the bloodstream at the baseline was controlled to a low level, and
vasodilation reserve capacity was mobilized during adenosine
loading. In the naked VEGF.sub.165 plasmid group, on the other
hand, the bloodstream at the baseline was not controlled to a low
level, and there was no vasodilation reserve capacity to be
mobilized during adenosine loading. Maturity of the blood vessel
system, such as vascular smooth muscle and neural or humoral
regulation mechanism in neogenetic blood vessels, is indispensable
to the control of the bloodstream and vasodilation during adenosine
loading. The administration of the gelatin hydrogel conjugated
VEGF.sub.165 plasmid has been found to be able to achieve these
states.
Referential Example 3
Uptake of Nucleic Acid-Containing Complex by Macrophages
[0122] Macrophages incorporating a nucleic acid-containing complex
were prepared in vitro. Thioglycolate was injected
intraperitoneally into mice, and macrophages were taken 4 days
later. Separately, 100 .mu.g of 2 mg green fluorescence protein
(GFP) plasmid (Prasher D C et al. Gene, 111, 229-233, 1992; Heim R
et al. Nature, 373, 663-664, 1995) was conjugated to the
crosslinked gelatin particles prepared in Example 1. The plasmid
was prepared by inserting cDNA for GFP (purchased from Clontech)
into the multicloning site of HIV-CS vector. The crosslinked
gelatin particles-GFP plasmid was mixed with the macrophages
collected, and the mixture was cultured in PBS for 4 days at
33.degree. C. After four days of culture, the fluorescence of GFP
was checked with a fluorescence microscope and a fluorescence
activated cell sorter (FACS). The results are shown in FIG. 10.
Fluorescence was observed in the macrophages, confirming the uptake
of the crosslinked gelatin particles-GFP. Similar results were
confirmed in experiments using the crosslinked gelatin particles
immersed in an eosin staining solution (confirmation of eosin
particles in macrophages).
Experimental Example 7
Gene Transfer to Dendritic Cells and Induction of CTL to Particular
Antigen
[0123] Dendritic cells (DC) separated from human peripheral blood
were administered GFP plasmid, and examined for antigen presenting
ability.
[0124] Particulate aminated gelatin hydrogel conjugated GFP plasmid
was prepared in the same manner as in Example 1, except that the
nucleic acid used was GFP plasmid (described earlier; 20
.mu.g).
[0125] A CD 14 positive fraction was obtained from monocytes of
healthy subjects, and added together with GM-CSF and IL-4 to pooled
human serum/RPMI medium to induce DC. After a lapse of 4 to 6 days,
the particulate aminated gelatin hydrogel conjugated GFP plasmid
was added to DC. The mixture was allowed to stand for 60 minutes,
and the gelatin hydrogel was removed, then the cells were further
cultured for 3 days. The DC after culture were stained with CD1a-PE
and CD83-PE as DC markers, and the efficiency of gene transfer to
the DC was investigated by FACS analysis. The results are shown in
FIGS. 11A and 11B. As a control, naked GFP plasmid (20 .mu.g) was
used. The gene transfer efficiency was 77%.
[0126] A similarly high gene transfer efficiency was obtained when
WT1 plasmid (Call K M, et al. Cell, 60, 509-520, 1990) was used
instead of GFP plasmid.
[0127] Whether DC having the WT1 gene transferred thereto has the
ability of cell presentation for a gene product was measured by CTL
induction capacity. T lymphocytes of the same healthy subjects
having induced DC were co-cultured with DC which carried the WT1
gene to obtain activated T lymphocytes. These cells were labeled
with .sup.51Cr to form target cells. Using these target cells, CTL
assay was performed. CTL was induced to the transferred WT1,
showing the antigen presenting capability of the WT1 gene
transformed DC.
[0128] According to the invention, as described above, a stable
firm binding between the negative charge of a nucleic acid and the
positive charge of a biodegradable polymer leads to the formation
of a complex. Release of the nucleic acid is controlled by the in
vivo degradation of the biodegradable polymer. Thus, more accurate
control of the rate of release can be achieved, in comparison with
slow release by simple diffusion as observed with conventional slow
release preparations, and transient release of nucleic acid by the
use of a water soluble biodegradable polymer. Moreover, a sustained
release over a longer time is improved.
[0129] With the nucleic acid-containing complex of the invention,
this complex is insoluble, and the nucleic acid is protected from
degradation in vivo. Thus, the nucleic acid can be maintained in a
sufficiently active state until the nucleic acid arrives at a site
where its function either by expression of a gene or by direct
action of the enclosed nucleic acid should be exhibited. Thus, the
local expression and/or action of the nucleic acid can be achieved.
That is, gene therapy restricted to the site requiring treatment
can be achieved.
[0130] Furthermore, the rate of release of the nucleic acid is
controlled by the type of the biodegradable polymer used, and the
balance between the positive charge and the negative charge. Thus,
the rate of release is constant, and particular attention need not
be paid to its shape in formulating the complex.
[0131] Releasing a nucleic acid locally, consistently and
persistently for a long period is particularly useful in the field
of gene therapy. As the desired gene can be administered for a long
term to a site or a living organism requiring therapy, there will
be an increased need for adjusting the timing of gene transfer, and
the timing of introducing a nucleic acid most preferred for gene
expression and functional expression.
[0132] According to the invention, the desired effect can be
expected with the amount of nucleic acid which is about 1/10 to
1/100 of the usual dose. That is, the invention acts to enhance the
effective activity of the nucleic acid. This action is preferred
from the aspects that the gene may be administered at a low dose,
and the collateral effect of the gene on other sites than the
desired site can be diminished.
[0133] Another embodiment of the nucleic acid-containing complex of
the invention is to provide a new method capable of achieving
target site-specific functional expression of nucleic acid by use
of the phagocytosis of phagocytes such as macrophages, and the
migration of the phagocytes. This method makes safer and easier
gene therapy possible.
Sequence CWU 1
1
113149DNAHomo sapiens 1gcactgctcc tcagagtccc agctccagcc gcgcgctttc
cgcccggctc gccgctccat 60gcagccgggg tagagcccgg cgcccggggg ccccgtcgct
tgcctcccgc acctcctcgg 120ttgcgcactc ctgcccgagg tcggccgtgc
gctcccgcgg gacgccacag gcgcagctct 180gccccccagc ttcccgggcg
cactgaccgc ctgaccgacg cacggccctc gggccgggat 240gtcggggccc
gggacggccg cggtagcgct gctcccggcg gtcctgctgg ccttgctggc
300gccctgggcg ggccgagggg gcgccgccgc acccactgca cccaacggca
cgctggaggc 360cgagctggag cgccgctggg agagcctggt ggcgctctcg
ttggcgcgcc tgccggtggc 420agcgcagccc aaggaggcgg ccgtccagag
cggcgccggc gactacctgc tgggcatcaa 480gcggctgcgg cggctctact
gcaacgtggg catcggcttc cacctccagg cgctccccga 540cggccgcatc
ggcggcgcgc acgcggacac ccgcgacagc ctgctggagc tctcgcccgt
600ggagcggggc gtggtgagca tcttcggcgt ggccagccgg ttcttcgtgg
ccatgagcag 660caagggcaag ctctatggct cgcccttctt caccgatgag
tgcacgttca aggagattct 720ccttcccaac aactacaacg cctacgagtc
ctacaagtac cccggcatgt tcatcgccct 780gagcaagaat gggaagacca
agaaggggaa ccgagtgtcg cccaccatga aggtcaccca 840cttcctcccc
aggctgtgac cctccagagg acccttgcct cagcctcggg aagcccctgg
900gagggcagtg ccgagagtca ccttggtgca ctttcttcgg atgaagagtt
taatgcaaga 960gtaggtgtaa gatatttaaa ttaattattt aaatgtgtat
atattgccac caaattattt 1020atagttctgc gggtgtgttt tttaattttc
tggggggaaa aaaagacaaa acaaaaaacc 1080aactctgact tttctggtgc
aacagtggag aatcttacca ttggatttct ttaacttgtc 1140aaaagttgtc
acgagtgtgc tgctattctg tgttttaaaa aaaggtgaca ttggattccg
1200atgtcatccc ctgtagtatg gcgtggagca tctctgtctg gaaaggcccg
cctgaggctt 1260gggcagccag ttcagggagc tcccaggctt ggctctcggc
tagcatcctc agaggcccac 1320tccctttgtg ccctgttgct attaatcggg
acatatcggt ttacttcggg tacagaaagt 1380gcggtgttga agtcctcgct
gccactctgt ttttagatct gccaagactg acctttgaac 1440tttcctgtag
tcaatcttcc tcgatctacc agatgggaga gacccttgga caactttata
1500aactcctgtt tgcctttttt ggatcagcga cagcccccat cgctgtgact
attggggaaa 1560agacgaagct ctttcataaa ttccatggag aggaatcaat
atcccactgg aaggctagaa 1620atggacaaga tagtgtattt gcaatcacaa
acaaaaccct agtgatgaaa aataatttgt 1680gatggcagat gcttctgatg
gtgtgataga atatgttttt gaaaacaaac catcgaaccc 1740cccgccccac
ccccaaaacg ggcttccctg tgtttaggga gctttgggct agaactagct
1800acgattttta ggtgaaatgt ccttgtaatt gtacaaagca cttggtgcag
tgtttgcgtg 1860gagcagcctg ctgctttctg atgcattccc tgtttaagtg
cgtttaacat ctacctcaca 1920agccctgaaa ccccaggcaa aacccacaga
aagctcatac ccggtgcagg agtttgccat 1980cccaagtggc tttttttcca
tatgtagcca aaaaggattg cagatagcgt cggtgcgtcc 2040cattcgaacc
ttgtcacgtt tgagctatct ttaccctgtg atttactttt agtaagggtg
2100atcatggtga aaatatttgc agacagctgt tacagtacac tatatggtca
ccaagtaacc 2160ttatattttt ctttatatat tttacaaatg taacccctgt
cattgaagca accgtggaag 2220aggcagggtc ggtgatgttt aaaaaaagtt
ccgaggtgat ggcaaacatt taattttaat 2280gaatgacttt ttagagttta
tacaaaatga ccttagcttg ctaccagaaa tgctccgaat 2340gtttcgtcaa
gactttaata ctctcctagg atgtttctga actgtctccc gaattaactt
2400tatgggagtc tacagacagc aagactggaa aatctgattg gagtttttgt
ctttcacatt 2460ccttttgaaa actctttgtt cgaatgcaaa tcatcgactt
aaaatactat tcttaaccaa 2520ggcctggaag aaagaagaca cttgcaaagc
cgctaagaca ggaccacaca tcttaaactg 2580ctgttcctac catgcactaa
actgttttta agttttaaac cacaccctag gctccaggag 2640tgttcaggaa
agatggtgtt tgtaggtctc catgctgttt ggcgttgggg ggtgtggagg
2700gatcatccgt cgactttctg aattttaatg tattcactta gtaacaaacc
atgattgtct 2760taaatgcctt aaattattat gagatttctt gtctcagagc
ccaatcagat tgtcaggaat 2820taacatgtgt taggtttgat cacccttgac
cacttcttat agatatttct tcaacaaatc 2880atgtgtgatg cctgtaggaa
cacaactgta cctttaaaat attgttttca tattgctgtg 2940atggggattc
gaggttcctg tatgtgccac tgttttcaga atctgtagtt ttatacaggt
3000gccgaccctc gttgtgatgt atgtgctgtg cacattgaca tgctgaccga
caatgataag 3060cgtttatcgt gtataaaaag acaccactgg actggatgta
cacaactggg aaaggaatta 3120aaagctatta aaattgtgcc ttgaaatgc 3149
* * * * *