JP2003502047A - Fibrin sealant as transfection / transformation vehicle for gene therapy - Google Patents

Abstract

  (57) [Summary] For example, transducing a cell comprising applying a transforming effective amount of a nucleic acid to the cell, applying a fibrin gel to the cell to trap the transforming effective amount of the nucleic acid, and transforming the cell with the nucleic acid. A method for converting is provided. In one aspect, the nucleic acid is applied in a mixture with fibrin or a fibrinogen composition that forms a fibrin gel.

Description

Detailed Description of the Invention

The present application relates to the use of fibrin polymers as vehicles for delivering genetic material to cells or tissues.

Fibrin sealants are used to make solid formulations of polymerized fibrin or other fibrin-related molecules. Typically, the type of polymerization is initially the formation of stable non-covalent associations between fibrin molecules. In many cases, non-covalent associations are complemented by a subsequent covalent cross-linking formed by the action of activated Factor XIII. These solid formulations are often used to stop or reduce fluid leakage (eg, air leakage or blood leakage from the lungs) after injury.

One form of fibrin sealant is one that can polymerize blood products as well as fibrinogen by using recombinant or autologous fibrinogen to remove snake-derived converting enzymes from the process in which the sealant is applied to patients. It overcomes concerns about the safety of the enzyme used to convert to. For example, E
See dwardson et al., US Pat. No. 5,739,288. The preparation of the sealant takes place within minutes from a small amount of blood, and the separation of the converting enzyme allows the enzyme to be separated from the sealant using affinity binding techniques while stabilizing the fibrin in a solubilized form, so that autologous fibrinogen , Edwardso
It is actually useful through the technology of n.

In gene therapy, cells of a particular cell type (eg, hepatocytes, pancreatic cells,
It has been sought to transfect or transform lung cells, muscle cells, white blood cells, etc.) to insert genes to correct genetic defects or otherwise provide useful functions. Such genes include nucleic acid constructs that express antisense RNA to interfere with the expression of a particular mRNA, or one that expresses two complementary strands designed to interfere with the expression of a particular mRNA. The above constructs may be included.

Similarly, nucleic acid-based vaccines are aimed at inducing a percentage of cells to produce an immune response-inducing polypeptide, or to induce an antibody- or cell-based immune response.

When viral vectors are used in gene therapy, tissue specificity can be provided by the cell surface markers that the virus utilizes to achieve entry into the cell. However, this method is only useful if a virus targeting the intended tissue is present and can actually be used as a vector. Moreover, if viral vectors, such as adenovirus, are known to be preferred for gene therapy, their preferred cell targeting machinery may not be appropriate for the desired target cell type. Therefore, additional tools are needed to help increase the specificity for the desired target cell type, and to overcome vector selection for alternative cell targets.

Provided herein are compositions of fibrin sealants that incorporate recombinant vectors for delivery to tissues or cells to which the sealants are polymerized and are typically adherent. The use of such a composition allows the vector to be locally maintained in high concentration in the solid gel produced by the sealant, thereby increasing the efficiency of transfection or transformation of cells.
Moreover, fibrin gels are resistant to many agents used as adjuvants in transfecting or transforming cells. Therefore, such fibrin sealant compositions are used for delivery of vectors to cells or tissues, whether or not the quest for transfection or transformation is related to traditional concepts of gene therapy. Can be done. This method can be used, for example, for desired products (eg, proteins or small molecules resulting from a transformation event).
Can be used to make cells, such as plant cells that produce.

SUMMARY OF THE INVENTION In one embodiment, the invention provides a method of transforming a cell comprising the steps of: applying a transformation effective amount of the nucleic acid to the cell, applying a fibrin gel to the cell. And entrapping an effective amount of the nucleic acid, as well as transforming the cell with the nucleic acid. In one aspect, the nucleic acid is applied in a mixture with a fibrin or fibrinogen composition that forms a fibrin gel.

In another embodiment, the invention provides a method of performing gene therapy comprising the steps of: performing the steps outlined above, implanting the transformed cells in an animal. In one aspect, the cell to which the nucleic acid is applied is a precursor of a more specialized cell type and the above method further comprises the following steps:
Maturating cells into specialized cell types in vitro or in vivo after implantation.

In another embodiment, the present invention provides a method of performing gene therapy comprising the steps of: applying a gene therapy effective amount of a gene therapy effective nucleic acid to a tissue; Applying fibrin gel to the tissue so as to trap the amount of nucleic acid, and transforming the cells of the tissue with the nucleic acid. In one aspect,
The method further includes: surgically exposing the tissue to allow the application step.

In yet another embodiment, the invention provides a method of surgically treating an animal, comprising the steps of: surgically exposing the internal tissue, transforming an effective amount of the nucleic acid into the tissue. Applying, applying a fibrin gel to the tissue so as to trap a transforming effective amount of nucleic acid, and treating the tissue cells with an antibody or cytotoxic T against an antigen or infection by a pathogenic microorganism. Transformation with a nucleic acid containing a peptide that induces a lymphocyte response. In one aspect, the nucleic acid is
Antigens or antibodies containing infections by pathogenic microorganisms that are members of the following genera or peptides that induce cytotoxic T lymphocyte responses include: Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobacterium, Neisseria. , Genus Haemophilus, genus actinomycetes, genus Streptomyces, genus Nocardia, enterobacteria, genus Yersinia, genus Fancisella, genus Pasteurella, genus Moraxella, genus Acinetobacter, genus Erythiperostrix, genus Blanchamella, actinobacillus, Streptococcus, Listeria, Calimatobacterium, Brucella, Bacillus, Clostridium, Treponema, E. coli, Salmonella, Klebsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spiryl Mumps, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia, Mycoplasma, Helicobacter, Saccharomyces, Kluveromyces (Kluve)
Romyces), Candida or Pneumocystis.

In yet another embodiment, the invention provides a kit comprising: (a) either (i) a fibrin monomer, (ii) fibrinogen or another fibrin precursor or (iii) a fibrin analog. A first composition for forming a fibrin gel containing one; (b) (1) reversing the conditions for stabilizing fibrin as a monomer when the first composition conforms to (i). The drug, (2) the first composition is (ii)
An agent that converts a fibrinogen or a fibrin precursor into fibrin if it is in conformity with (3) If the first composition is in conformity with (iii), a fibrin-related molecule that forms a gel with a fibrin analog, A second composition for forming a fibrin gel comprising; and (c) a gene that is separately configured in the third composition or is integrated into the first or second composition. A therapeutically effective amount of the nucleic acid, wherein the first
And the fibrin gel formed from the second composition is effective for trapping nucleic acids in the vicinity of cells or tissues. In one aspect, the nucleic acid is configured with another adjuvant to increase the efficiency with which the nucleic acid transforms or transfects cells.

In yet another embodiment, the invention provides a method of performing gene therapy comprising: transforming or transfecting a cell with a nucleic acid to produce a recombinant cell; Implanting in an animal; and applying fibrin gel to trap the recombinant cells at the desired location within the animal. In one aspect, the method further includes enabling surgically exposing and implanting tissue.

Fibrin and Blood Coagulation One mechanism for hemostasis, the prevention of blood loss, is the formation of blood clots. Clot formation in humans depends on thrombin, calcium ions and activated form X
It is produced by a complex reaction cascade with the final step of converting fibrinogen by factor III to form a crosslinked fibrin II polymer (or what is known as an insoluble fibrin II polymer) that is ultimately an insoluble fibrin clot.

Fibrinogen exhibits a plasma protein of about 2-4 g / liter and is a complex protein consisting of three pairs of disulfide-bonded polypeptide chains designated (Aα) ₂ , (Bβ) ₂ and γ _2. Is. "A" and "B" indicate two small amino-terminal peptides known as fibrinopeptide A and fibrinopeptide B, respectively. The six polypeptide chains of fibrinogen consist of one
Within the two linear sequences, the two terminal "D domains" and the central "E domain",
Folds into at least three globular domains. The E domain is believed to include all six N-terminal residues of the polypeptide chain in the fibrinogen molecule.
Each D domain contains C-terminal sequences from one α chain, one β chain and one γ chain.

The formation of insoluble fibrin clots (eg, cross-linked fibrin II polymer) is believed to begin with fibrinogen which is converted by thrombin to fibrin I monomer. This conversion involves a thrombin-mediated cleavage of the 16 amino acid fibrinopeptide A (G1-R16) from each of the two Aα chains of fibrinogen (thus resulting in a novel N-chain having the amino acid sequence G17-P-R-V20-, respectively). Yielding two α-chains with ends). A fibrin I monomer is an intermolecular intercalator between the converted E domain of a fibrin monomer, which now has an available non-covalent binding site, and the D domain of another fibrin I or fibrin II monomer. It is believed that due to the action (ie, non-covalent binding), it may spontaneously polymerize with other fibrin I or fibrin II monomers. Each D domain of the fibrin monomer is either fibrin I or fibrin I
It possesses a polymerization site that can stably interact with the E domain of the I monomer.

Contact of two E domain polymerization sites of one fibrin I monomer with two complementary D domain polymerization sites, each derived from two different fibrin I monomers, results in half-staggered molecular contacts. It is believed to produce linear fibrin fibrils (ie, polymers) that have. The fibrin I polymer so formed is often referred to as soluble fibrin I polymer. This is because the fibrin I polymer is depolymerized and reconverted to the fibrin I monomer by treatment with appropriate chemical means.

The next step in the formation of the fibrin clot involves the conversion of fibrin I monomer to fibrin II monomer. This step involves the thrombin-mediated cleavage of fibrinopeptide B from each of the two Bβ chains of fibrin I. Removal of the 14 amino acid fibrinopeptide B each results in a β chain with the N-terminal sequence of GH-R-. Like the fibrin I monomer, the fibrin II monomer is linked to other fibrin I monomers by virtue of an intermolecular interaction site in the E domain of one fibrin II monomer, which is made available by a cleavage reaction.
It can polymerize spontaneously at the D domain of another fibrin II or fibrin I monomer than another I or fibrin I monomer. Like Fibrin I polymer, Fibrin II polymer is also often referred to as soluble Fibrin II polymer. Because it is depolymerized and reconverted to fibrin II monomer by the use of suitable chemical treatments. The exposure of the β-chain N-terminal sequence in the E domain is important for fibrin clot formation because it facilitates covalent cross-linking of adjacent fibrin II monomers in fibrin II polymer by activated factor XIII. Activated factor XIII can also crosslink fibrin I monomers in fibrin polymers, but the reaction is less efficient due to the presence of fibrinopeptide B on fibrin I. Crosslinked fibrin II polymers are often referred to as insoluble fibrin II polymers because they cannot be depolymerized and reconverted to fibrin II monomers.

In addition to thrombin and Factor XIII, calcium ions are important in the formation of fibrin clots and are believed to have a number of important roles. Calcium ions are essential for the activation of Factor XIII, as calcium ions are believed to be essential for the activation of prothrombin to thrombin, and thrombin activates Factor XIII. Furthermore, the active XIII
The factor is believed to be a calcium-dependent enzyme that is unable to crosslink fibrin polymers in the absence of calcium ions. Calcium ions also bind directly to polymeric fibrin, changing the opacity and physical properties of the fibrin polymeric strands. For a review of the mechanisms of blood aggregation and the components of fibrin clots, see C. M. Jackson, Ann. Rev. Biochem. ，
49: 765-811, 1980, and B. Furie and B.C. C. Fu
See Rie, Cell, 53: 505-518, 1988.

Fibrin Sealants Fibrin sealants are biological adhesives that have the effect of mimicking the aggregation step to form fibrin polymers. The sealant can be designed so that the fibrin monomer is converted into an insoluble fibrin polymer. One type of fibrin sealant uses fibrinogen and consists of two components. 1
The other components are concentrated human fibrinogen, bovine aprotinin and XI
Contains Factor II. The second component contains enzymes that convert fibrinogen to fibrin, such as calcium chloride and thrombin. Application of this type of sealant is generally performed using a twin-barreled syringe that allows simultaneous delivery of both components to the desired fibrin clot formation site. The mixing of the two components at the target site results in a fibrin clot via the series of reactions described above.

The fibrinogen component of this type of fibrin sealant is typically made from pooled human plasma. Fibrinogen can be concentrated from human plasma by cryoprecipitation reactions and precipitation with various reagents such as poly (ethylene glycol), diethyl ether, ethanol, ammonium sulfate or glycine. For a review of this type of fibrin sealant,
M. Brennan, Blood Reviews 5: 240-244, 19
91; W. Gibble and P.M. M. Ness, Transfusion
30: 741-747, 1990; Matras, J .; Oral Max
illofac. Surg. 43: 605-611, 1985 and R.M. Ler
ner and N.N. Binur. J. of Surgical Research
48: 165-181, 1990.

A second, more novel type of fibrin sealant uses compositions that are predominantly composed of fibrin I or fibrin II monomers. See European Patent Application No. 0592242 (published April 1994). These types of sealants include fibrin I monomer or fibrin II monomer or des
The BB fibrin monomer is derived from fibrinogen prior to application of the sealant by the appropriate proteolytic enzyme (eg thrombin, or batroxobin
xobin)). The fibrin monomer is maintained in solubilized form using a suitable buffer. Useful buffers include those with low pH or chaotropic agents. Fibrin I monomer in such a solution,
The fibrin II monomer or desBB fibrin monomer can be converted to a fibrin polymer by mixing with a solution containing a second solution to give a mixture having a state capable of forming a fibrin clot by spontaneous polymerization of the fibrin monomer. .

Fibrin I, fibrin II and desBB fibrin monomer-based sealants have several advantages over fibrinogen-based sealants. Notably, fibrin monomer-based sealants do not contain bovine or human thrombin. The use of such sealants does not introduce foreign proteins into the recipient when the fibrin monomer is produced from an autologous (ie, patient's own) source, resulting in complications resulting from immune reactions and blood-borne infections. You can avoid the danger of. Fibrin monomer-based sealants can be conveniently manufactured. Soluble fibrin polymer can be dissolved using a weakly acidic solution. In some embodiments, the resulting fibrin monomer is lyophilized to a fine powder. Such powders readily redissolve in weak acids and repolymerization can be induced by the addition of alkaline buffer. Alternatively, the powdered fibrin monomer can be dissolved in a chaotropic solution (eg, urea) at high concentration (> 150 mg / ml) and water can be added to induce repolymerization.

[0024] A further advantage of fibrin monomer-based sealants is that they commonly use autologous components so that hepatitis (including hepatitis B and non-A non-B hepatitis) and acquired immunodeficiency virus (AIDS) There is a lower risk of exposure to such blood-borne infectious agents. L. E. Silberstein et al.
Transfusion, 28: 319-321, 1988; Laitak
ari and J. Lutonen, Laryngoscope 99: 974.
-976,1989; and A. Dresdale et al., Annals of
See Trioracic Surgery 40: 385-387, 1985. Diseases caused by such factors may be transmitted by traditional fibrinogen-based sealants. This is because the fibrinogen component is usually produced from pooled human plasma. Furthermore, fibrin-based sealants can avoid the risks associated with the bovine thrombin component of fibrinogen-based sealants. Bovine thrombin formulations may carry bovine spongiform encephalopathy (BSE) as well as mammalian viral pathogens. Bovine thrombin is also a strong antigen, which may cause adverse immunological hyporeactivity in humans. For further discussion of these types of complications associated with fibrinogen-based sealants, see Tayl.
or. Hospital Infection 18 (Appendix A): 141-
146, 1991 and Prusiner et al., Cornell Vet 81:
85-96, 1991.

Recombinant fibrinogen and fibrin have been engineered to produce hepatitis and HI in relatively high yields and in substantially pure form.
Fibrinogen and fibrin monomers can be produced in the absence of pathogenic viruses such as V. Constructing mutants capable of mimicking naturally occurring fibrin variants by exogenous expression of fibrinogen and fibrin chains, as well as patients with these rare genetic defects for the isolation and study of these proteins Can be done without the need for.

Each of the three different polypeptide chains of fibrinogen (Aα, Bβ and γ) are encoded by separate genes. CDNAs for each of these chains have been prepared (Chung et al., Ann. NY Acad. Sci. 408: 44.
9-456, 1983; Rixen et al., Biochemistry 22:32.
37-3244, 1983; Chung et al., Biochemistry 22:
3244-3250, 1983; Chung et al., Biochemistry 2
2: 3250-3256, 1983), expressed in prokaryotes. further,
Each human fibrinogen chain was individually expressed in an expression plasmid (Huang et al., J.
． Biol. Chen 268: 8919-8926, 1993; Roy et al.,
J. Biol. Chem. 267: 23151-23158, 1992; Roy.
Et al., J. Biol. Chem. 266: 4758-4763, 1991) or in combination (Hartwig and Danishefsky, J. Bi.
ol. Chem. 266: 6578-6585, 1991; Huang et al., J. Am.
Biol. Chem. 268: 8919-8926, 1993; Roy et al., 19
91, J. Biol. Chem. , 266: 4758-4763; Redman.
And Samar, US patent application Ser. No. 07 / 663,380 (filed March 1991, Natl. Technology Information Service).
e No. PAT-APPL07663 380INZ available)) introduced and transfected into eukaryotic cells.

Most of the plasmids used in expressing recombinant human fibrinogen are D. Dr. Chung (University of Washington
, Seatle) and is based on the cNA clone (Rixen et al., Biochemistry 22: 3237-3244).
, 1983; Chung et al., Biochemistry 22: 3244-32.
50, 1983; Chung et al., Biochemistry 22: 3250-.
3256, 1983). Expression of recombinant fibrinogen chains was first achieved in E. coli (Bolyard and Lord, Gene 66: 183, 1).
988; Boldard and Lord, Blood, 73: 1202-120.
6, 1989; Lord and Fowlkes, Blood, 73: 166-1.
71, 1989). The individually expressed chains exhibit antigenic similarity to fibrinogen and present thrombin cleavage sites similar to those found in native fibrinogen (Bolyard and Lord, Blood, 73: 1202-
1206, 1989; Lord and Fowlkes, Bloom. 73:16
6-171,1989). Fibrinopeptides A and B can be released from recombinant fibrinogen (Bolyard and Lord, Blood, 73: 1).
202-1206, 1989; Lord and Fowlkes, Blood, 7
3: 166-171, 1989).

Eukaryotic cells carrying the appropriate expression plasmids encoding the individual fibrinogen chains synthesize the encoded fibrinogen chains and dimer chain molecules (eg,
It has been shown to result in intracellular formation of Aα ₂ , Bβ ₂ or γ ₂ dimers) (Roy et al., J. Biol. Chem., 265: 6389-6393,199).
0; Zhang and Redman, J .; Biol. Chem. 267: 217
27-21732, 1992). Furthermore, when a suitable plasmid containing the genes encoding all three human fibrinogen chains is transferred into the same cell, not only all three chains are expressed, but also the polypeptide chains associate in pairs, Intact fibrinogen is secreted into the surrounding medium (Roy et al., J. Biol.
． Chem. 266: 4758-4763, 1991; Hartwig and Danishefsky, J. Am. Biol. Chem. 266: 6578-658
5, 1991). Like native fibrinogen, secreted recombinant fibrinogen is composed of three pairs of non-identical polypeptide chains and is functional in the formation of fibrin polymers.

Fibrinogen is naturally synthesized by the liver and megakaryocytes and transformed hepatocytes maintained in culture can continue to synthesize and secrete fibrinogen (Otto et al., J. Cell. Biol. 105: 106.
7-1072, 1987; Yu et al., Thromb. Res. 46: 281-29
3, 1987; Alving et al., Arch. Biochem. Biophys.
217: 19, 1982). One such cell line is HepG
2 cells (Dr Knowles and Aden, Wister Ins
Title, Philadelphia). This strain has an excess of A over the Bb chain.
The α- and γ chains are synthesized, which results in a non-productive Aα- and γ-chain dimer complex (eg, Aα2γ2). Introduction of an additional expression vector encoding the Bβ chain formed a trimer complex (AαBβγ) that adopted correct folding and intrachain disulfide bond patterns (Roy et al., J. Biol. Ch.
em. 265: 6389-6393, 1990). The mechanism of this folding is unknown, and ancillary proteins and enzymes (ancillary prot.
eins and enzymes) (Roy et al.,
J. Biol. Chem. 267: 23151-23158, 1992). These studies not only demonstrate correct transcription of Bβ cDNA, but also excess B
It was demonstrated that the β chain increases the assembly and secretion of intact fibrinogen.

In HepG2 cells, the AαBβγ trimer complex associates in pairs to form an intact fibrinogen molecule, which is glycosylated and actively secreted from the cell (Huang et al., J. Biol. Chem. 268: 8919-
8926, 1993). In fact, only correctly assembled fibrinogen molecules are secreted. Thus, HepG2 cells have synthetic and secretory machinery for the assembly of fibrinogen.

In the subsequent experiment, a cDNA plasmid encoding the fibrinogen chain was
It was introduced into eukaryotic cells, which normally do not synthesize fibrinogen. In these experiments, functional fibrinogen was successfully produced, indicating that the factors required for fibrinogen assembly and secretion are not unique to liver-derived cells such as HepG2. Eukaryotic cells known to be capable of assembling and secreting recombinant fibrinogen include baby hamster kidney cells (BHK), COS cells and Chinese hamster ovary cells (CH
O) (Roy et al., J. Biol. Chem. 266: 4758-4).
763, 1991; Hartwig and Danishefsky, J. Am. Bio
l. Chem. 266: 6578-6585, 1991; Farrell et al., B.
iochemistry 30: 9414-9420, 1991).

Intact, functional fibrinogen, secreted by stably transformed eukaryotic cells, results in the accumulation of fibrinogen levels of about 1-2 μg / ml. Methods are known to increase the production of recombinant proteins from transfected cells such as CHO cells, so that expression levels can reach 1000-fold above basal secretory levels.

Further description of methods for recombinantly producing fibrin-related molecules can be found in PCT /
It can be found in US 95/05527.

Gene Therapy Over the last two decades, lessons learned by scientists who have been actively considering how to introduce genetic material into the appropriate target tissues to overcome hereditary disorders, the effort first anticipated. Was much more complicated than that. Some of the goals that are needed to satisfy the creation of good gene therapy tools include:
(1) efficient transduction of target cells, (2) long-term gene expression, (3) lack of damaging immune response to vector or transduced cells, and (4)
No toxicity exists. Samulski et al., "Adeno-associat"
ed Virtual Vectors "Development of Hum
an Gene Therapy, Cold Spring Harbor L
See Laboratory Press, 1998, pages 131-172, which is incorporated herein by reference with all references to gene therapy. All of the above goals (especially the first three) identify areas that create a substantial barrier to effective gene therapy. Usually, the vectors transduce only a percentage of the cells to which they have been applied. Often, the transgene is maintained on the episome and, therefore, often does not stably integrate and maintain the genetic element. Furthermore, integration into chromosomal DNA is often dependent on cell division, thus limiting the extent of target cells to replicating tissues. Viral vectors often carry nucleic acids encoding proteins that elicit immunity and thus have a cause for the destruction of transduced cells. Certain viral vectors overcome some of these problems, but at least risk is associated otherwise. For example, the non-replicating human immunodeficiency virus is genetically engineered for use as a gene therapy vector that allows the integration of genetic material into genomic DNA. Such a vector must carry the genetic tools to facilitate integration into the genome, but not the infection (in this case AIDS).
The gene product that causes the infectious particle must be sufficiently deleted so that there is no opportunity for a recombination event to create the infectious particle. Naldini et al., "L
initial Vectors "Development of Hum"
an Gene Therapy, Cold Spring Harbor L
See Laboratory Press, 1998, pages 47-60.

The good news is that all of these problems are now very well recognized and viral vectors used in gene therapy have been modified to address these problems. Furthermore, gene therapy can be performed without the use of viral vectors. Also, in other genetic transformations, the problems of toxicity and immune response do not appear to some extent. In nucleic acid-based vaccines, the immune response is desirable, for example, because it may be a process in which the expression of the transforming gene is attenuated and, consequently, the production of immunostimulants over an extended period of time.

Viral vectors have also been genetically engineered to alter target cell preference, eg, by binding or incorporating antibodies. For example, Valses
ia-Wittmann et al. modified the cell surface binding properties of avian leukemia virus (J. Virol. 68: 4609-4619, 1994). Erythropoietin, which naturally binds to cognate receptors, has been incorporated into the Moloney murine leukemia virus (Mo-MLV) (Kasahara et al., Scien).
266: 1373-1376, 1994). Tumor-targeting single chain antibodies have been incorporated into splenic necrosis virus (Chu and Dornburg, J. Vi.
roll. 69: 2659-2663, 1995). The HIV envelope protein has been incorporated into a murine leukemia virus vector (Mammamo et al.,
J. Virol. 71: 3341-3345, 1997). Such targeting methods involving adenovirus vectors are described by Reynolds and Curiel.
, "Strategies to Adapt Adenoviral Vec
tors for Gene Therapy Applications:
Targeting and Integration ”Development
nt of Human Gene Therapy, Cold Spring
g Harbor Laboratory Press, 1998, 111-
P. 130).

Development of Human Gene Therapy,
Cold Spring Harbor Laboratory Press,
A wide variety of viral vectors have been selected and engineered for gene therapy, as reviewed in 1998. Moreover, nucleic acids can be successfully delivered without the use of viral vectors. For example, an initially developed method of increasing transfection efficiency was to use calcium phosphate precipitated nucleic acids. The transfection ability of nucleic acids is determined by DEAE dextran (Veh
eri et al., Virology 27: 434-436, 1965), polylysine (Wu et al., J. Biol. Chem. 266: 14338-14342, 199).
1), cationic peptides (Wadhwa et al., Bioconjugate Ch.
em. 8: 81-88, 1997, and Niidome et al., J. Am. Biol.
Chem. 272: 15307-15312, 1997), polyethyleneimine (Boussiff et al., Proc. Natl. Acad. Sci. USA 92).
: 7297-7301, 1995), glucaramide (glucaramide)
) Based polyamino polymers (Goldman et al., Nat. Biotechn).
ol. 15: 462-466, 1997), polyamidoamine dendrimer (D
ielinska et al., Biochim. Biophys. Acta 1353:
180-190, 1997) and tightly packed with polycationic polymers such as Other polymers useful as enhancers for nucleic acid uptake include erodable microspheres (Mat).
hiowitz et al., Nature 386: 410-412, 1997) and polyvinylpyrrolidone (Mumper et al., Pharm. Res. 13: 701-7).
09, 1996). Other enhancers include cationic liposomes that incorporate nucleic acids (Felgner et al., 1987; Fe.
lgner and Ringold, 1989). Such liposomes (or "lipoplexes") are believed to insert nucleic acids into target cells by a membrane fusion mechanism. An example of many of the cationic lipid formulations currently available is DOTMA (N [1- (2,3-dioleyloxy) propyl]-
N, N, N-trimethylammonium) (Felgner et al., “Synt
"heavy Delivery Systems" Development o
f Human Gene Therapy, Cold Spring Har
bor Laboratory Press, 1998, pp. 241-260)
. Other such cationic lipid formulations include Lipofectin ^™ (cationic lipid N- [1- (2,3-dioleyloxy) propyl] -N,
1: 1 (w / w) liposome preparation of N, N-trimethylammonium chloride (DOTMA) and dioleylphosphatidylethanolamine (DOPE))
, LipofectAMINE ^™ (2,3-dioleyloxy-N- [2- (spermine-carboxamido) ethyl] -N, N-dimethyl-1-propaneaminium trifluoro, which is a polycationic lipid in membrane-filtered water. 3: 1 (w / w) liposome formulation of acetate (DOSPA) and the neutral lipid dioleylphosphatidylethanolamine (DOPE)), and Lipo
fectACE ^™ (1: 2.5 (w / w) liposome preparation of dimethyldioctadecyl ammonium bromide (DDAB), which is a cationic lipid, and dioleylphosphatidylethanolamine (DOPE) in membrane-filtered water. (These are all Life Technologies, Rockvi
Ile, from MD). Furthermore, gene transfer can be achieved without the use of such adjuvants. Targeting techniques that bind or attach targeting molecules to the nucleic acid or nucleic acid complex used for transfection can also be utilized (Cotton and Wagner, "Receptor-me."
divided Gene Delivery Strategies ”Dev
element of Human Gene Therapy, Cold
Spring Harbor Laboratory Press, 1998,
Pp. 261-277).

As one of ordinary skill in the art will recognize, nucleic acids that have been found for introduction into cells often are readily detectable in their own right, in addition to the moiety that retains the primary genetic property of interest. It includes a moiety that encodes a substance that is capable of producing such a molecule. This "reporter" molecule serves as a surrogate to detect or predict success in introducing the major genetic characteristic. If the cells in culture are transformed, a portion of the nucleic acid may encode the substance required for the cells to survive despite the appropriate challenge.

Non-virus mediated techniques that seek to express some nucleic acids typically
Although single-stranded nucleic acid is used, the nucleic acid can be single-stranded or double-stranded.

Preparation of the Preferred Sealant Composition First, blood is collected and plasma is isolated. An enzyme that converts fibrinogen to fibrin is added to plasma. When converting fibrinogen to fibrin,
It is preferable to pay attention to prevent the formation of cross-linked via the first XIII ^a factor of transaminase activity between fibrin molecules. This means that heavy metals (eg mercury), thiomersal {[(o-carboxyphenyl) thio] ethyl mercury sodium salt}, inhibitory antibodies or calcium chelators (
Calcium may be performed by a number of techniques, including use of the XIII ^a factor inhibitors such as at a) because essential cofactors for this enzyme. Examples of calcium chelators include, but are not limited to, EGTA (ethylene glycol bis- (2-aminoethyl ether) tetra-acetic acid) and the like. For example, the converting enzyme is batroxobin, which is a proteinase from the snake venom of snakes of the American genus Bothrops.
“Btx”), which is used at a concentration of about 0.1 μg / ml to about 100 μg / ml, preferably at a concentration of about 0.5 μg / ml to about 50 μg / ml.

Other proteinases of suitable specificity can also be used. The snake venom proteinase is particularly suitable and includes Acrystrodon Actus (Agkist).
rodon acutus, Acristodon contortrix contortrix (Agkistrodon contortrix contortor)
trix), Acristrodon Harris Pallas (Agkistrodon h)
alys pallas), Acrystrodon (Calloselasma), Rhodosoma
), Bottrops asper, Bottrops atrox, Botrops insularis, Bottrops jararaka (B)
Othrops jararaca, Botrops Mougeni (Bothr)
Ops Moojeni, Lachesis muta
ta muta), Crotalus adam
anteus), Crotalus durisus terificus (Crotalus)
Durissus terrificus, Trimereslus flavorviridis, Terimeresus gramineus, and venoms that are not limited to these but not limited to Bitis gabonica, such as Bittis gabonica.

The fibrinogen converting enzyme is preferably linked to a converting enzyme binding partner used in the affinity method to reduce the concentration of the enzyme in the formulation. For example, the converting enzyme binding partner is biotin, a member of the biotin-avidin binding pair, which is a pair of molecules that bind with very high affinity. The amino acid sequence of avidin is shown in Dayhoff, Atlas of Prot.
ein Sequence, Volume 5, National Biomedical
Research Foundation, Washington, DC, 1
972 (DeLange and Huang, J. Biol. Chem. 246:
698-709, 1971), and the amino acid sequence of streptavidin is Argarana et al., Nucl. Acid Res.
14: 1871-1882, 1986. For example,
Nucleic acid sequences are available: (1) For avidin, chicken mRNA, Ge
ne Bank registration number X05343 (Gore et al., Nucl. Acid Re
s. 15: 3595-3606, 1987), (2) chicken, white leghorn species, avidin gene, Gene Bank accession number L27818, (3) S.
trep. Streptavidin from Avidinii, Gene Bank Accession No. X03591 (Argarana et al., Nucl. Acid Res. 14).
: 1871-1882, 1986), (4) Strep. Synthetic gene for streptavidin from avidinii, Gene Bank accession number A0
0743 (Edwards, W089 / 03422) and (5) Gene.
Bank accession number X65082, a synthetic gene for streptavidin (T
homson et al., Gene 136: 243-246, 1993).

Preferably, avidin and streptavidin are used in the tetrameric form, although monomers can also be used. Other binding pairs that bind with high affinity include antibodies that are specific for polypeptides or other molecules, any polypeptide for which a specific antibody is available or can be prepared, phenyl oxide. Thioredoxin that binds arsine (Examples of expression vectors include Invitrog
, thioredoxin fusion protein vector pTrxFus available from En, Carlsbad, CA), poly-His sequences that bind divalent cations such as nickel II (expression vectors include, for example, Invitrog
pThioHis vectors A, B and C available from En),
Examples include glutathione-S-transferase vectors that bind glutathione (eg, vectors available from Pharmacia Biotech, Piscataway, NJ). Given the description herein of polypeptide expression systems and known antibody production methods, those of skill in the art can utilize such antibody production methods. For the antibody production method, see, eg, Ausubel et al., Sh.
ort Protocols in Molecular Biology, J
See Ohn Wiley & Sons, New York, 1992. Very high affinity binding properties are very convenient, but not essential. Any affinity that can be used in the affinity binding method can be used in the present invention to reduce the concentration of converting enzyme in the formulation. If, in the affinity method, only antibodies to the converting enzyme are used, this aspect of the invention does not require a conjugated converting enzyme binding partner, since the enzyme itself contains the converting enzyme binding partner.

Unless the process is designed to prevent the polymerization of fibrin monomers during the enzymatic conversion of fibrinogen to fibrin, the fibrin formed will polymerize into fibrin polymers and form fibrin clots. After isolation of the solid
Fibrin monomer is recovered from the fibrin clot. The fibrin monomer is recovered, for example, by adding a solubilizer to the fibrin clot. Examples of such a solubilizer include an acid solution such as an aqueous solution having a pH of about 5 or less, or a chaotropic agent such as urea, sodium bromide, guanidine hydrochloride, potassium cyanide, potassium iodide or potassium bromide. Can be mentioned. The solubilizer may be used near the minimum concentration effective to maintain the fibrin monomer (ie, a fibrin solubilizing effective amount). A number of conditions for forming fibrin monomers are described in Edwardson et al., European Patent Application EP 592,242.

A solid substance having a second binding partner, which is a binding partner complementary to the converting enzyme binding partner, may then be added to the fibrin monomer formulation and subsequently found in the formulation. It binds a converting enzyme that may not. Then, according to the protocol used, solids which may contain solid material or which may contain any residual fibrin clot material are removed, for example by filtration or centrifugation.

The treated material is stored, for example, in liquid form in the frozen state at about 4 ° C. or lower, or in dried form, such as lyophilized. Lyophilizates are formed by standard methods. These lyophilizates are generally reconstituted in purified water or buffered aqueous solutions. For fibrin monomers, lyophilizates can generally be reconstituted using the same solution composition as the solubilizer used in the previous process. Alternatively, if the user wishes to polymerize fibrin during reconstitution, then (a)
An aqueous solution is utilized that either lacks the solubilizer or (b) is capable of reversing any of the solubilization conditions performed in lyophilization.

As illustrated, the fibrin monomer and the non-enzymatic polymerization agent can be mixed together to form a fibrin sealant (ie, a clot). The polymerization agent is any reagent effective to reverse the conditions that prevent the polymerization of fibrin monomers. For example, the fibrin monomer may be an acidic solution (eg, 0.2M sodium acetate,
When in a pH 4 solution), the polymerizing agent may be, but is not limited to, a basic solution such as: HEPES (N- [2-hydroxyethyl) piperazine-N '-[ Ethanesulfonic acid]), sodium hydroxide, potassium hydroxide, calcium hydroxide, bicarbonate buffers such as sodium bicarbonate and potassium bicarbonate, trimetallic citrate, acetate and sulfate. Preferred alkaline buffers include: carbonic acid / bicarbonate, glycine, bishydroxyethylaminoethanesulfonic acid (BES), hydroxyethylpiperazinepropanesulfonic acid (EPPS), tricine, morpholinopropanesulfonic acid (
MOPS), trishydroxymethylaminoethanesulfonic acid (TES), cyclohexylaminoethanesulfonic acid (CHES), trishydroxymethylaminoethanesulfonic acid (TES). The amount of alkaline buffer utilized should be sufficient to allow the polymerization of fibrin. It is preferable to mix about 1 part of the alkaline buffer with about 10 parts to about 1 part of the composition containing the fibrin monomer. Even more preferred is such a ratio of about 9: 1.
The preferred ratio depends on the buffer, its concentration and pH, and the desired concentration of fibrin polymer. If an acidic pH is used as the solubilizer, the solubilization of fibrin can occur in the presence of calcium ions, eg at a concentration of about 20 mM.

Incorporation of Nucleic Acids into Fibrin Gels In one preferred embodiment, three aqueous formulations are mixed to initiate the rapid clot formation process. These formulations can be, for example, fibrin monomer formulations, compositions containing nucleic acids for transforming or transfecting cells ("transformation compositions" or "TCs") and non-enzymatic polymerization agents. Alternatively, in another example, the formulation is fibrinogen, fibrinogen converting enzyme and TC. The resulting gel-forming mixture is allowed to remain in a variable state (pl
In order to remain iaable), the sealant mixture is typically formed either during the process in which the sealant is applied to the recipient surface or a few minutes before application. Usually, the sealant mixture is in a convenient variable state for about 30 seconds or less.

In another preferred embodiment, the three are combined by spraying and mixing them. A suitable spray head is US Pat. No. 5,605,5
No. 41, No. 5,376,079 and No. 5,520,658 and P
It is described in CT application 97/20585. If the spray head utilized is designed to spray only one solution, additional spray heads can be aligned to deliver the other solution to the delivery site. For example, if a spray head delivers two concentric spray solutions or suspensions and uses a gas atomizing port to create and mix streams, a second such spray head delivers a third solution. Can be used for.

Instead of the three types, TC can be incorporated into one of the other two formulations, if appropriate.

Alternatively, TC can be mixed with the sealant after the polymerization process has begun, while the composition is in the variable state. Alternatively, TC can be applied to cells or tissues and a sealant applied to lock the sealant in place. Such subsequently applied sealant is preferably applied at the same time or shortly after the process of polymerizing the sealant is initiated.

Other types of fibrin sealants, other than those detailed above, useful in the present invention are described, for example, in Wadstrom, US Pat. No. 5,631,011, Co.
chrum, US Pat. No. 5,510,102, Pines et al., US Pat.
330, 974, Matras, J .; Oral Maxillofacial
Surgery 13: 605-611, 1985, and Brennan,
Blood Reviews 5: 240-244, 1991. Another device for spraying fibrin sealant is, for example, Avoy.
, U.S. Pat. No. 4,902,281.

[0053] If the various aspects fluid is used as fibrin source, in many cases it is desirable to isolate using fibrin cofactor, such as factor XIII or XIII, ^a Factor and thrombin. When using a purification technique that isolates fibrin via the reversible formation of fibrin polymers, the fibrin polymers have an affinity for many such cofactors, so the isolated products are It is considered to retain the factor. In some cases, the non-fibrin material is separated from the fibrin polymer by limiting the degree to which the fibrin polymer is compressed in a series of methods described herein, for example, to ensure that a sufficient amount of cofactors are co-isolated. It is desirable to limit the amount washed out.

The present invention can be used to treat any animal having a fibrin-based system for controlling bleeding, but is preferably used to treat mammals, most preferably humans.

Definitions The following terms should have the individual meanings set out below for the purposes of this application.

Cellular Progenitors of More Specific Cell Types Progenitor cells are cells that are typically referred to as “stem” cells or “pluripotent” cells, which differentiate into more specialized cells. Have the ability to, but have not yet differentiated.

Fibrin One of the many fibrinogen derivatives capable of polymerizing to form a precipitate of fibrin polymer [eg fibrin I (ie desAA-fibrin), fibrin II (ie desAADesBB fibrin) or desBB]. Fibrin]. Derivatives are typically made by cleaving A or B fibrinopeptides from fibrinogen.

Fibrin Analogs One form of fibrin monomer is a genetically engineered version of fibrin, or “fibrin analog,” which does not self-polymerize but polymerizes with another fibrin-related molecule such as fibrinogen. Such genetically engineered fibrin has been described by Cederholm-Williams et al., “Reco
mbinant Fibrin Chains, Fibrin and Fib
rin-Homologs ", PCT application number PCT / US95 / 05527 (
(Filed May 2, 1995).

Fibrin Chain Precursor A precursor of a fibrin α or β chain that contains an N-terminal peptide that can be cleaved to yield an effective fibrin chain when polymerizing fibrin.

Fibrin Clot Forming Effective Amount An effective amount of clot forming fibrin is the amount or concentration (when in liquid form) of fibrin (eg, fibrin monomer) that forms sufficient clot material to be utilized as a fibrin sealant. Is.

Fibrin Monomer Fibrin Monomer refers to a polymer that is retained in liquid form and that is present in the presence of a polymerization inhibitor such as, for example, acidic pH or a chaotropic agent, or in a fully dehydrated or frozen form. It is a fibrin that prevents clot formation by being maintained in a preventative form.

Fibrin Polymer The fibrin molecules interact non-covalently to form a polymer referred to herein as a “fibrin polymer” when the conditions are not to prevent the polymerization of fibrin monomers. , When sufficient mass is achieved, forms a visible adherent precipitate with clot-like properties. By the action of the XIII ^a factor, fibrin polymer is covalently crosslinked. Before crosslinking action of the XIII ^a factor, fibrin polymers may be reversibly converted to fibrin monomer. It is believed that fibrin polymers can be reversibly converted to fibrin monomers even if some such initial cross-linking occurs.

Fibrin Precursors Precursors of fibrin contain one or more fibrin chain precursors that undergo processing to produce a form of fibrin that polymerizes with the corresponding fibrin molecule. The fibrin precursor contains one or more leader peptides above its constituent chains. The leader peptide is processed in vivo or in vitro to produce fibrin from the fibrin precursor.

Gene Therapy As used herein, “gene therapy” includes any intervention in an animal, preferably a mammal, more preferably a human, including: (i) on the cells of the animal Transiently or stably expressing recombinant nucleic acid (as RNA or protein), or (ii) causing changes (eg, insertions) in the cell's genome that alter the cellular gene expression pattern. . Thus, gene therapy includes the use of nucleic acid-based vaccines.

High Affinity Binding High affinity binding between a first substrate and a second substrate means that the first or second substrate is an affinity ligand for the isolation of the other substrate. A binding that has sufficient affinity to be used as Typically, high affinity binding is reflected in an association constant of about 10 ⁵ M ⁻¹ or higher, preferably 10 ⁶ M ⁻¹ or higher, even more preferably 10 ⁷ M ⁻¹ or higher.

Or (or or) the conjunction "or (or or)" means at least one of the described alternatives that is associated with or applicable to a given context, And it contains the meaning of the connective word that connects two or more alternatives described. In other words, "or / or" unless the context indicates otherwise.
Includes any meaning of "and / or" and / or (or).

Polynucleotide or nucleic acid The term polynucleotide or nucleic acid (herein “polynucleotide”) refers to
In general, it means any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotide” includes, but is not limited to, single-stranded and double-stranded DNA, single-stranded and double-stranded regions or single-stranded, double-stranded and triple-stranded regions. DNA that is a mixture, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, 1
Mention may be made of hybrid molecules comprising DNA and RNA which may be double-stranded or more typically double-stranded or triple-stranded regions, or a mixture of single-stranded and double-stranded regions. Further, "polynucleotide" as used herein refers to RNA or DN.
It means a triple-stranded region containing both A or RNA and DNA. One of the molecules in the triple helix region is often an oligonucleotide. As used herein, the term "polynucleotide" also includes a DNA or RNA as described above that contains one or more modified bases. Thus, a DNA or RNA having a backbone that has been modified for stability or for other reasons is a "polynucleotide" as that term is intended herein. Further, as the term is used herein, DNA or RNA containing unusual bases such as inosine, or modified bases such as tritylated bases (here only two examples are given. Is a polynucleotide. It will be appreciated that a great variety of modifications can be made to DNA and RNA that serve many useful purposes known to those of skill in the art. As used herein, the term "polynucleotide" refers to such chemically, enzymatically, or metabolically modified forms of the polynucleotide, as well as chemical forms of the DNA and RNA characteristic viruses. And cells (including, for example, single cells or composite cells). “Polynucleotide” also includes short polynucleotides, often referred to as oligonucleotides.

Transformed cell (1) A polypeptide or RNA, which is not expressed by the cell when the nucleic acid is not introduced, is expressed in the cell in a constant amount, transiently or stably, or (2) A) A cell is transformed when the nucleic acid has been recombinantly introduced into the cell or its ancestors so as to transiently or stably interfere with the translation or transcription of the nucleic acid found in the cell. Has been done.

[0069]   Transformation composition   A transformation composition is a composition containing a gene therapeutically effective amount of a nucleic acid.

All publications and references cited herein, including but not limited to patents and patent applications, are individually incorporated by reference. As specifically and individually described as referred to herein, it is incorporated by reference in its entirety. The patent applications to which this application claims priority are also incorporated herein by reference in their entirety, as described above for publications and references.

Although the present invention has been described with emphasis on preferred embodiments, the preferred devices and methods can be modified, and that the invention is different from those specifically described herein. It will be apparent to those skilled in the art that it is intended to be implemented in. Accordingly, this invention includes all modifications that come within the spirit and scope of the invention as defined by the appended claims.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification symbol FI theme code (reference) A61K 39/395 A61K 47/42 47/42 48/00 48/00 C12N 15/00 AF term (reference) 4B024 AA01 BA31 CA02 DA03 GA11 HA17 4C076 AA09 BB32 CC26 EE41A FF68 4C084 AA02 AA13 BA35 MA28 NA10 NA11 4C085 AA13 BA07 BA09 BA10 BA16 BA18 BA22 BA24 BA26 BA27 BB23 EE01 EE06

Claims (12)

[Claims] 1. A method comprising applying a transforming effective amount of a nucleic acid to a cell, applying fibrin gel to the cell so as to trap a transforming effective amount of the nucleic acid, and transforming the cell with the nucleic acid. , A method of transforming cells. 2. The method of claim 1, wherein the nucleic acid is applied in a mixture with a fibrin or fibrinogen composition which forms a fibrin gel. 3. A method for performing gene therapy, which comprises performing the step according to claim 1 and transplanting the transformed cell into an animal. 4. The cell to which the nucleic acid is applied is a precursor of a more specialized cell type, and the method matures the cell to a specialized cell type in vitro or in vivo at a post-implantation step. The method of claim 3, further comprising: 5. A step of applying a transforming effective amount of a gene therapy effective nucleic acid to a tissue, a step of applying a fibrin gel to the tissue so as to trap the transforming effective amount of a nucleic acid, and cells of the tissue. A method of performing gene therapy, which comprises: 6. The method of claim 5, further comprising surgically exposing the tissue to allow the applying step. 7. Surgically exposing the internal tissue, applying a transforming effective amount of the nucleic acid to the tissue, applying fibrin gel to the tissue to trap the transforming effective amount of the nucleic acid, A method of performing surgery on an animal, comprising transforming cells of a tissue with said nucleic acid, wherein said nucleic acid encodes an antigen or an antibody against infection by a pathogenic microorganism or a cytotoxic T A method comprising a peptide that induces a lymphocyte response. 8. The nucleic acid encodes an antigen, or Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes, Streptomyces, Nocardia. , Enterobacteria, genus Yersinia, genus Fancisella, genus Pasteurella, genus Moraxella, genus Acinetobacter, genus Eridiperostrix, genus Blanchamera, actinovatilus, genus Streptococcus, genus Listeria, genus Calimatobacterium, Brucella, Bacillus, Clostridium, Treponema, Escherichia coli, Salmonella, Klebsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirylum, Campylobacter, Shigella , Legionella, pseudo Antibodies or cytotoxicity against infection by pathogenic microorganisms that are members of the genus Solanum, Aeromonas, Rickettsia, Chlamydia, Borrelia, Mycoplasma, Helicobacter, Saccharomyces, Kluveromyces, Candida or Pneumocystis. 8. The method of claim 7, which comprises a peptide that induces a sex T lymphocyte response. 9. A first composition for forming a fibrin gel comprising any one of (a) (i) fibrin monomer, (ii) fibrinogen or another fibrin precursor or (iii) fibrin analog. (B) (1) an agent that reverses the conditions for stabilizing fibrin as a monomer when the first composition conforms to (i), (2) the first composition is (ii)
An agent that converts a fibrinogen or a fibrin precursor into fibrin if it is in conformity with (3) If the first composition is in conformity with (iii), a fibrin-related molecule that forms a gel with a fibrin analog, A second composition for forming a fibrin gel comprising: (c) a gene that is separately configured in the third composition or is integrated into the first or second composition A therapeutically effective amount of a nucleic acid, wherein the fibrin gel formed from the first and second compositions is effective to trap the nucleic acid in the vicinity of cells or tissues. kit. 10. The kit of claim 9, wherein the nucleic acid is configured with another adjuvant to increase the efficiency of transforming or transfecting the cells. 11. A method of transforming or transfecting a cell with a nucleic acid to produce a recombinant cell, transplanting the recombinant cell into an animal, and applying a fibrin gel to direct the recombinant cell into the desired cell in the animal. Trapping in place, a method of performing gene therapy. 12. The method of claim 11, further comprising surgically exposing the tissue to allow the implanting and applying steps.