A nucleic acid-complex, containing a nucleic acid and a positively charged, water-insoluble biodegradable polymer, is disclosed. The complex has properties of sustainedly releasing a desired nucleic acid, especially DNA, to a site. The complex can be taken up by phagocytes such as macrophages and delivered to a target site, allowing the function of the nucleic acid to be exhibited in a target specific manner.

SUMMARY OF THE INVENTION

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.

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.

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).

In light of these objectives, the inventors of this invention conducted extensive studies and obtained findings listed below. Fist, 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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 (see Original Patent) 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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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".

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.

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.

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.

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.

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.

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.

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.

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-to luenesulfonate, 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.

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.

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.

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.

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.

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.

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.

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.

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.

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 (see Original Patent) shows a schematic view of the complex, with DNA taken as an example of the nucleic acid.

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.

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.

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.

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.

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.

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.

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.

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:

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:

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:

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.

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.

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.

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.

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.

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.

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.

In pharmaceutically manufacturing the complex of the invention, it is further desirable to subject it to a sterilization step such as sterile filtration.

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.

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.).

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.

Claim 1 of 2 Claims

1. A nucleic acid-containing complex comprising a nucleic acid and a positively-charged water-insoluble biodegradable polymer, wherein said positively-charged water-insoluble biodegradable polymer is crosslinked prior to complexation with said nucleic acid; wherein said nucleic acid can be released by degradation of said biodegradable polymer; wherein said nucleic acid is a fibroblast growth factor gene comprising SEQ ID NO:1.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.