JP2002514899A - Method for transducing cells in blood vessels with a recombinant AAV vector - Google Patents

Abstract

  (57) [Summary] Current techniques for expressing recombinant genes in vascular cells after direct gene transfer in vivo are limited by attendant problems or limitations. Furthermore, no effective method for transducing microvascular cells and / or cells involved in the formation of new blood vessels (angiogenesis) has been shown. The present invention provides a method for transducing cells in blood vessels using a recombinant adeno-associated virus (AAV) vector.

Description

DETAILED DESCRIPTION OF THE INVENTION       Methods for transducing cells in blood vessels using recombinant AAV vectorsTECHNICAL FIELD OF THE INVENTION   The present invention relates to gene delivery, and more particularly to recombinant adeno-associated virus (AA V) a method for transducing cells in blood vessels with a vector.Background of the Invention   Blood vessels, the major components of the cardiovascular system, flow from the heart to cells throughout the body. And form a network that allows them to return to the heart. Generally, blood vessels Is composed of three layers, an inner membrane, a middle membrane and an outer membrane. Most healthy movements The intima of the vasculature generally comprises a luminal monolayer of endothelial cells. In the aorta, the intima is flat In diseases such as atherosclerosis, which may contain smooth muscle cells, and in the intima, Can be thickened by smooth muscle cells and inflammatory cells beneath the endothelial monolayer. The inner membrane is It is distinguished from the media by the elastic plate. The media typically contains smooth muscle cells and Around the matrix material. In the aorta, the media is distinguished by elastic fibers It consists of a defined layer of smooth muscle cells. The adventitia forms the outermost layer of the artery wall, It is distinguished from the media by the outer elastic plate. Outer membranes are generally macrophages A loose matrix, including fibroblasts, and other cell types, It is composed of vascular vessels (a network rich in adventitia microvessels).   Microvessels (including arterioles, capillaries, venules, and vascular vessels) Differs from the general structural model outlined above in that layers cannot be well defined . For example, capillaries are monolayers of endothelial cells surrounded by a single layer of smooth muscle cells. But without any well-defined elastic layer.   Angiogenesis is the formation of new blood vessels. Angiogenesis occurs in the vasculature during fetal development. This can also include extensive normal and postnatal pathological processes (eg, wound repair, Tumor formation, and inflammation). [See, for example, Diaz-Flores, 1994. ]. Therefore, many disease processes have important anatomical or pathological features. With abundant microvessels. These microvessels are directly linked to the development of a particular disease. Or may contribute indirectly or may represent benign morphological features. Many places In combination, microvessels and angiogenesis play a central role in the etiology of the disease It is thought that there is. These include, for example, the growth and metastasis of many cancers, diabetes Retinopathy, retinitis, heart failure, arthritis, psoriasis, ischemia, wound healing, hemangiomas and other Abnormal angiogenesis. [See, for example, Diaz-Flores, 1994]. this All tissues and organs, including these cells, support their normal function and viability Requires an extensive network of microvessels to do so. These microvessels are Microvessels may have therapeutic polynuclear properties in tissues, especially because they may have different functions than large vessels. Provides a unique target for delivering nucleotides. In particular, microscopic By altering gene expression in cells of blood vessels, tissues or organs Can be enhanced in a specific direction.   Numerous studies have focused on delivering recombinant genetic material to vascular endothelial cells and smooth muscle cells. Methods for modifying the process of angiogenesis, as well as methods for These studies have obvious limitations, including the following: Low efficiency of gene transfer and restriction of recombinant gene expression in vascular tissue (plasmid DNA, liposome vectors and retroviral vectors); Vector expression (adenovirus vector and liposome A significant immune response to a particular viral vector; And lack of specificity for cells, tissues and organs (in which case the transgene The generalized expression of or reintroduction of ex vivo genetically modified cells is not possible. Could have necessary or detrimental effects). [For example, Clowes, 1994; Geary, 1993; Ge ary, 1994; Lemarchand, 1993; Lim, 1991; Lynch, 1992; Nabel, 1989; Newman, 1995; Ojeifo, 1995; Plautz, 1991; Rome (1), 1994; Rome (2). Schwartz, 1990; and Zwiebel, 1989].   For example, retroviral vectors provide their stable and long-term gene expression. Despite the advantage of having general capabilities, stable proviral Replication of the target cell is required for integration. However, most cells in the arteries The vesicle is generally quiescent. Quiescent cells provide significant and efficient gene transfer Can sometimes be isolated and induced to divide to achieve Often, the method of inducing cells and genetically modifying them ex vivo It requires subsequent transplantation back into the donor host. Liposome vector delivery systems also Limited by inefficient uptake and transient episomal vector expression I have. Adenoviral vectors that are efficient in endothelial cells during infection Episomal Vector Expression and Antigenicity Limit the Efficiency of Duplicate Use More restricted.   Thus, to date, without the above problems or limitations, direct in vivo genetics Research shows efficient and significant expression of recombinant genes in vascular cells after offspring transfer There was no conclusion. In addition, involved in the formation of microvascular cells and / or new blood vessels No effective method for transducing cells to be transformed was shown.                                Summary of the Invention   The present invention provides a method for transducing cells in blood vessels using a recombinant AAV vector. Provide the law. Preferred embodiments of the present invention include:   1. Introduction of recombinant adeno-associated virus (rAAV) vector into individual blood vessels in vivo A method for transducing cells in blood vessels of an individual, comprising the step of transducing.   2. 2. The embodiment of claim 1, wherein the rAAV vector comprises a detectable marker gene. A method for transducing cells in blood vessels.   3. The embodiment 1 wherein the rAAV vector comprises a selectable marker gene. A method for transducing cells in blood vessels.   4. Embodiment 2. The vascular cell according to embodiment 1, wherein the rAVV vector comprises a therapeutic gene. A method for transducing.   5. A group consisting of arterioles, capillaries, venules, and adventitia microvessels; Transducing cells in blood vessels according to embodiment 1, which are microvessels selected from: Way.   6. The method according to claim 5, wherein the blood vessel is an adventitia microvessel. How to introduce.   7. Embodiment 1 wherein the blood vessel is a microvessel and the cells are proliferating. The method for transducing cells in a blood vessel according to the above.   8. The cells of claim 1, wherein said cells are primate cells. How to introduce quality.   9. 9. Transducing cells in a blood vessel according to embodiment 8, wherein the cells are human cells. how to.   10. The cells of claim 1, wherein said cells are proliferating cells. How to introduce quality.   11. Embodiment 11. In a blood vessel according to embodiment 10, wherein the cells are proliferating microvascular cells. A method for transducing cells.   12. The cells in blood vessels according to embodiment 1, wherein the cells are microvascular cells. How to transduce.   13. Embodiment 13. In a blood vessel according to embodiment 12, wherein the cell is a microvascular endothelial cell. A method of transducing a cell.   14． Embodiment 1. The embodiment 1 wherein the rAAV vector is introduced into the adventitia of an artery of the individual. A method for transducing cells in a blood vessel as described above.   15. Introduction of recombinant adeno-associated virus (rAAV) vector into the above microvascular cells Transduced microvascular cells produced by   16. A method for transducing cells in blood vessels of an individual according to the method described in Embodiment 4. A method for treating said individual for a disease state, comprising:                             DETAILED DESCRIPTION OF THE INVENTION Definition:   "Polynucleotide" refers to a ribonucleotide or deoxyribonucleotide. Nucleotides of any length, which are either Refers to rimer form. This term refers only to the primary structure of the molecule. Therefore, two Includes single- and double-stranded DNA and double- and single-stranded RNA. Methylation or Also includes modified polynucleotides, such as polynucleotides that have been capped.   "Recombinant" is applied to a polynucleotide, wherein the polynucleotide is Cloning, restriction and / or ligation steps, and naturally occurring polynucleotides Products of various combinations of other procedures that result in constructs that are distinct from nucleotides. Means that   A "vector" is a host cell, either in vitro or in vivo. Refers to a recombinant plasmid or virus containing the polynucleotide to be delivered. The polynucleotide to be delivered contains the coding sequence of interest in gene therapy. I can see.   “Recombinant AAV vector” (or “rAAV vector”) is an AAV inverted terminal repeat. A vector containing one or more polynucleotides of interest flanked by sequences (ITRs). U. One possibility to replicate and package rAAV vectors into infectious virions A successful method is to use the rAAV vector to express the AAV "rep" and "cap" genes and It can be into a host cell infected with a suitable helper virus.   A "gene" is a polynucleotide or polynucleotide containing a sequence that encodes a protein. Refers to part of a nucleotide. In most cases, genes also effectively promote transcription. Preferably, include a promoter operably linked to the coding sequence. Good. As is well known in the art, enhancers may be used to regulate gene activity. , Repressors and other regulatory sequences may also be included. (For example, quoting below (See references).   The terms “polypeptide,” “peptide” and “protein” refer to any length Used interchangeably to refer to a polymer of amino acids. These terms also Post-translationally modified through reactions involving glycosylation, acetylation, and phosphorylation Protein.   AAV "rep" and "cap" genes (replicated and encapsidated Is found in all AAV serotypes tested, It is described in various references cited herein. Typically, rep And the cap gene are found adjacent to each other in the AAV genome and Is generally conserved in the AAV serotype.   The "helper virus" for AAV is wild-type AAV (this is a defective parvovirus Is replicated and packaged by the host cell. Refers to a second virus. Many such helper viruses are common in the art. Have been defined and are similar to adenovirus, herpes virus, and vaccinia Including poxvirus.   "Packaging" as used herein refers to the assembly of the rAAV vector. And a series of subcellular events that result in encapsidation. Therefore, the appropriate vector -If the plasmid is introduced into the packaging cell line under appropriate conditions, It can be replicated and assembled into viral particles.   "Heterologous" is derived from an entity genotypically different from the remaining heterologous entity to be compared. Means something. For example, derived from one cell type and Polynucleotides introduced into different cell types are heterologous polynucleotides . It may encode a heterologous polypeptide when expressed. Similarly, its natural From a coding sequence and operably linked to a different coding sequence. Motors are heterologous promoters.   As used herein, "promoter" refers to an operably linked promoter. Refers to a genomic region that controls transcription of the gene or coding sequence being performed.   "Operably linked" means that the compositions as described are in their intended manner. Means a parallel relationship that can function with The promoter is When controlling transcription of a coding sequence, it is operably linked to the coding sequence. Operable An operably linked promoter is usually in cis configuration with the coding sequence, but not necessarily. It need not be adjacent to the coding sequence.   "Detectable marker gene" specifically detects cells carrying that gene (Eg, distinguished from cells that do not carry the marker gene) Is a gene that Many such marker genes are known in the art . Preferred examples include detectable proteins that encode proteins that appear on the cell surface Marker genes, which allow easy and rapid detection and / or cell Facilitates sorting. For the purpose of illustration, we have detected that The alkaline phosphatase ("AP") gene was used as a viable marker. this Indicates that the cells transduced with the vector carrying the AP gene Allowed to be detected based on the expression of AP.   A “selectable marker gene” stores the gene in the presence of the corresponding selection agent. Genetics that allow specific selection for or against cells having I am a child. For the purpose of illustration, the antibiotic resistance gene may be As a positive selectable marker gene that allows for positive selection in the presence of Can be used. Various positive and negative selectable markers are known in the art. Yes, some of which are described below.   A "therapeutic polynucleotide" or "therapeutic gene", when transferred to an individual, Nucleotides that can have a prophylactic, healing, or other beneficial effect in this individual Means the array.   "Cytokine" as used herein refers to an intercellular signaling molecule. . The best known cytokines are involved in the regulation of mammalian somatic cells. Many Family of cytokines have both pro- and anti-proliferative effects. Which include, for example, interleukins (eg, For example, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9 (P40), IL-10 , IL-I1, IL-12 and IL-13); CSF-type cytokines (eg, GM-CSF, G-CSF, M -CSF, LIF, EPO, TNF-α and TNF-β); interferons (eg, IFN-α, IFN-β, IFN-γ); cytokines of the TGF-β family (eg, TGF-β1, TGF- β2, TGF-β3, inhibin A, inhibin B, activin A, activin B); Chemotactic factors (eg, NAP-1, MCP-1, MIP-1α, MIP-1β, MIP-2, SISβ, SISδ SISε, PF-4, PBP, γIP-10, MGSA); growth factors (eg, EGF, TGF-α, aFGF , BFGF, KGF, PDGF-A, PDGF-B, PD-ECGF, INS, IGF-I, IGF-II, NGF-β); α type Intercrine cytokines (eg, IL-8, GRO / MGSA, PF-4, PBP / CTAP / βTG, IP-10, MIP-2, KC, 9E3); and β-type intercrine site Cain (eg, MCAF, ACT-2 / PAT 744 / G26, LD-78 / PAT 464, RANTES, G26, 130 9, JE, TCA3, MIP-1α, B, CRG-2). Many other cytokines are also It is also known to those skilled in the art. The sources, characterization, targets and sources of these cytokines And effector activities have been described, and for many cytokines DNA sequences encoding the molecule are also known; for example, Callard & Gearlng ,The Cytokine Facts Book(Academic Press, 1994) and an overview there And / or certain publications cited (these are incorporated by reference herein in their entirety. Involved See).The Cytokine Facts BookReferenced in catalogs like As such, many DNAs and / or proteins encoding such cytokines Protein sequences are also generally similar to GENBANK (DNA) and SWISSPROT (protein). Can be obtained from various sequence databases. Typically, such cytokines are The cloned DNA to be loaded is already available as a plasmid, but the published sequence Synthesizing polynucleotides that encode cytokines based on information It is possible. Polynucleotides encoding cytokines are also known in the art. Obtained using the polymerase chain reaction (PCR) method as described. [example See Mullis, 1987]. A new cytokine that works for a given cell type Detection, purification, and characterization of cytokines, including assays to identify proteins It is also described in many publications as well as in the references referred to herein. . [For example,Lymphokines and Interferons, (Clemens, J.J., et al., IRL Press 1 987); and DeMaeyer, 1988].   As used herein, "transduction" or "transducing" Regardless of the method used to introduce the foreign polynucleotide into the host cell, Is the term. These methods are well known in the art (eg, transfection , Lipofection, viral infection, transformation, and electroporation , As well as non-viral gene delivery techniques). The introduced polynucleotide is May be stable or temporarily maintained in the host cell. Stable maintenance is typically Whether the introduced polynucleotide contains an origin of replication compatible with the host cell, Alternatively, the host cell replicon (eg, extrachromosomal replicon (eg, plus Mid)) or integrate into the cell nucleus or mitochondrial chromosome You need to be.   A “replicon” is a polynucleotide containing an origin of replication, commonly called an ori sequence. Otide is called. This allows replication of the polynucleotide in the appropriate host cell To Examples include target cell replicons into which the desired nucleic acid can be integrated (eg, , Nuclear and mitochondrial chromosomes, and extrachromosomal replicons (eg, Plasmid)).   "Individual" as used herein refers to a mammal, preferably a human.   As used herein, “treatment” or “treatment” refers to preventing an individual from treating this individual. Cells, other reagents, or a combination thereof, that can produce healing or other beneficial effects It refers to administering a combination.   "Gene delivery" refers to the transfer of foreign polynucleotides to cells for gene transfer. Import, targeting, binding, uptake, transport, localization, replicon integration And expression.   "Gene transfer" refers to the introduction of a foreign polynucleotide into a cell, Can include targeting, binding, uptake, transport, localization, and replicon integration However, unlike the expression of later genes, and does not mean this.   "Gene expression" or "expression" refers to gene transcription, translation, and post-translational modifications. Refers to a process.   The “vasculature” or “vascular” is the blood (and lymph) ubiquitous in the mammalian body. Liquid).   "Vascular" refers to any conduit in the mammalian vasculature, arteries, arterioles, capillaries , Venules, veins, sinuses, and vascular vessels.   "Artery" refers to a conduit that passes blood from the heart to various parts of the body. Arterial The wall is typically an outer layer (outer membrane) that is distinguished from the intermediate layer (media) by an outer elastic plate The intermediate layer is distinguished from the inner layer (inner membrane) by the inner elastic plate. The outer membrane is one In general, microvessels (vascular vessels), fibroblasts, and lymphocytes and macrophages A layer of relaxed connective tissue that contains a network of immune cells, such as a page. The media is It contains an annular layer of smooth muscle cells and elastic fibers. The intima, in some cases, It consists of a monolayer of endothelial cells overlying the bleb.   "Microvessel", "microvascular" or "microvasculature" as used herein Is a term that refers to arterioles, capillaries, venules, and adventitia microvessels. Micro blood Tubes generally contain endothelial cells surrounded by one or several layers of smooth muscle cells. Arterioles refer to the branches of microarteries, especially those that are in close proximity to capillaries. Capillaries Connect arterioles and venules and create networks in virtually all organs and tissues Refers to any minute blood vessel that forms. The venules drain blood from the capillary plexus Any small blood vessel that collects and participates in vein formation. "Adventitia microvessels" Movement A microvessel that supplies blood to the adventitia of a large blood vessel such as a pulse. These outer membrane microbloods Vascular networks usually refer to vascular vessels. The adventitia microvessels are the intima and Blood from the lumen of the mother vessel (eg, artery) via small microvessels across the media It is considered to supply liquid. "Microvascular cells" make up the structure of microvessels Refers to cells.   The "endothelium" is generally aligned with the heart and blood vessels, and the cavities of the blood vessels of the lymphatic system. (Ie, "endothelial cells"). As used herein As such, blood vessels such as arteries can contain both endothelial cells and smooth muscle cells, They are distinguishable in terms of origin, function, and attributes such as cell surface markers. Noh. For example, generally, endothelial cells derived from embryonic ectoderm are cells that constitute the endothelium. And provide a non-thrombogenic surface and include vFW as exemplified below. For example, many well-known cell surface markers can be used to quickly distinguish. Generally, embryonic mesoderm-derived smooth muscle cells have structural and contractile mechanisms against the vessel wall. Α-actin that provides the ability to provide a non-thrombogenic surface For the purpose of illustration, including, for example, obtain.   “Proliferating” or “proliferation” is a term that refers to growth due to cell proliferation. Blood vessel new Birth occurs in the vasculature during fetal development and also involves a wide range of normal and postnatal pathology. New blood vessels that also occur in processes (eg, wound repair, tumor formation, and inflammation) Is the formation.References   The practice of the present invention will not be limited to molecular biology, microbiology within the art unless otherwise indicated. Utilizes conventional techniques of recombinant DNA, and immunology. Such techniques are described in the literature Well explained. For example, see the following references:   Describes surgical delivery and logistics that can be used in the methods of the present invention. Further references include:   Cell types found in blood vessels and vasculature structures that can be used in the methods of the invention Further references which describe: W. Bloom & D. Fawcett,A Textboo k of Histology , 10th edition, (W.B.Saunders Co. 1975).   Further references describing AAV vectors that can be used in the methods of the invention are: Including: Bibliography of quoted text   All patents, patent applications, and publications mentioned herein, both supra and And both below) are incorporated herein by reference.Detailed Description of the Preferred Embodiment   The described invention transduces cells in blood vessels using a recombinant AAV vector For how to. In vivo transduction of cells in blood vessels can be in vivo or It presents a problem not encountered in transducing other cells in vitro.   AAV vectors are commonly used in a few recombinant viral vector systems, It has been shown to be useful as a transposable element and, therefore, potentially human Very important for gene therapy. AAV vectors are used for various ex vivo Genes can be transferred and expressed with high frequency in cell lines (Carter, 1992; Ega n, 1992; Flotte, 1992; Flotte (1), 1993; Flotte (2), 1993; Kaplitt, 1994; Kotin Muzyczka, 1992; and Walsh, 1992). However, rAA in animal models There were only a few studies of transduction in vivo with the V vector ( See, for example, Flotte (2), 1993; Kaplitt, 1994; and Kotin, 1994). A AV is a DNA parvovirus often found in association with human adenovirus infection. Ruth. AAV has not been shown to cause disease in humans, Not a transformed or oncogenic virus. In contrast, other proposed Viral systems (eg, retrovirus, adenovirus, herpes virus, Or pox virus) is the disease-causing virus. In fact, 85% Adults are considered antigen-positive for one of the four AAV serotypes I have. AAV is also replication deficient, and helper virus such as adenovirus Possibly such as ills, herpes virus, or in some cases, vaccinia Can replicate only in the nucleus of cells co-infected with auxin virus. AAV is a proper help If present, in virtually any cell line of human, monkey, or rodent origin, It is believed that (albeit with different efficiencies) it can infect and replicate. In addition, AAV is particularly important for humans, where most cells are ultimately differentiated and do not divide. Has clear advantages over retroviruses in tissues such as airway epithelium . Because AAV does not require active cell division for gene transfer is there. Therefore, the AAV vector is the CFTR gene for cystic fibrosis, Can transduce quiescent cells, and recently transduce monkey lung epithelium in vivo (See, eg, Flotte, 1994). these Research recently transitioned to a human trial approved by the NIH Recombinant DNA Advisory Committee. Was. While AAV can transduce quiescent cells, it is most useful in primary fibroblast cultures. Recent studies have shown that AAV vectors selectively transduce cells in S phase. Suggest (see, for example, Russell, 1994). AAV General Reviews are, for example, Carter, 1990; Berns, 1990; Muzyczka, 1992; and Kotin, 199. 4 can be found.   AAV can be modified to create a vector for delivery of a heterologous gene. Preferably, such an AAV vector has no wild-type coding sequence and It cannot replicate even in the presence of Ruper virus. Generally, the modification method is reversed So that only terminal repeats (requiring vector replication in cis) remain The wild-type AAV coding sequence (rep and cap). Genetics of interest Offspring can be inserted between the viral terminal inverted repeats and then packaged You.   The general principles of AAV vector constructs have been reviewed recently. (For example, Carte r, 1992; Kotin, 1994; and Muzyczka, 1992). A portion of the AAV coding sequence with heterologous DNA to create a vector Thereby, it can be constructed in an AAV recombinant plasmid. Vector Plasmi In the terminal, the terminal (ITR) portion of the AAV sequence is cis-critical for some functions This can be achieved by excision from plasmids after transfection, Vector replication and integration and rescue from the host cell genome including. This vector is then used to transfer the vector plasmid into the cells. Packaged in AAV particles to produce AAV transducing virus by The cells may be (1) adenovirus or herpes virus Infected by the appropriate helper virus and (2) in trans, AAV replication And may provide capsid forming functions (these functions are provided by the vector plasmid). Because it was deleted in the construction). Some recent publications include high titer recombinants Methods for producing AAV vectors have been described (eg, Flotte (1) 1995 ; Trempe, 1995; and Allen, 1995).   In the method of the present invention, the recombinant AAV vector preparation comprises an AAV ITR region and a receptor. Transcript operably linked to any gene of interest to be transduced into Pient cells Can include a promoter, such as a detectable gene, a selectable gene, And / or therapeutic genes. For illustrative purposes, the following specific examples are provided. Thus, the present inventors have determined that human placental alkaline phosphatase (AP) is a detectable residue. Biography Used as offspring. However, based on the teachings herein, one or more detectable Genes, selectable genes, and / or other recombinant AAV vectors containing therapeutic genes. It will be apparent to those skilled in the art that the reactor can be easily used. So, for example, The method of the present invention comprises the steps of (instead of detectable and / or selectable genes) (In addition to or in addition to) the rAAV vector containing the therapeutic gene, which Used to alter the activity of the recipient cells The vesicles and / or their progeny may have beneficial effects in individuals receiving such cells. Have fruit. For purposes of illustration, a representative example is a cell and / or progeny thereof. Enhances the level of beneficial proteins or other factors in And / or reduces the levels of harmful proteins or other factors in their progeny Provides less or resistance to cytotoxicity or other harmful factors Transduction of cells in blood vessels with a therapeutic gene.   As another basic illustrative example, the present invention provides a method for encoding a selected protein. Or proteins involved in the secretion of other factors from the cell and / or its progeny Transduce cells in blood vessels with the gene (s) encoding the protein Can be used for This selected protein or other factor Has a beneficial effect on the individual.   As yet another illustrative example, the present invention relates to cells and cells in a recipient individual. And / or a polynucleotide that affects the interaction between its progeny and other cells, It can be used to transduce cells in blood vessels with the gene (s). For illustrative purposes, a therapeutic gene is a transduced cell and / or progeny thereof. In some cases, after some examples, it may have some effect on activation by other cells, Somewhat resistant to chemotherapeutic agents or infectious agents (eg, virus or Toxic reagents such as chemotherapeutics).   Thus, as will be appreciated by those skilled in the art, the present invention provides It can be used to "deliver" to cells in the vasculature of animals, especially humans. Of the present invention Some examples of specific therapeutic strategies to gain benefits and recombinant gene delivery The ability to transduce cells in all blood vessels, especially microtubules, is summarized below. These further examples are provided for the purpose of further illustrating the experimental application of the present invention. It is. Numerous other genes can be transmitted using the methods of the invention, as will be apparent to those skilled in the art. Can be reached. Thus, for example, using the methods of the present invention, a large number of Increase the cell proliferation of duct cells (eg, promote angiogenesis) and / or Cell proliferation in target tissues or organs supplied by transduced microvessels It can be expressed in the vasculature, especially in microvessels, to increase multiplication. like this Genes include, for example, human growth factor, plasma-derived growth factor (PDGF), vascular endothelium Growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor-β (TGF-β), and epidermal growth factor (EGF) (eg, Andree, 1994; Betsholtz, 1986; Bringstock, 1990; Conn, 1990; Derynck, 1985; Houck, 1991; It. oh, 1990; Johnsson, 1984; and Kondo, 1995). Many like this Gene sequences for various human growth factors are known and are incorporated herein by reference. Cloned into an rAAV vector using standard cloning techniques described in Or could be expressed thereby. In addition, various other cytokines are described above. And the known gene sequences of such cytokines have been cloned as well. And can be expressed by an rAAV vector using the methods of the invention (eg, Callard, R. And Gearing, A.,The Cytokine Facts Book, (Academic Pr ess 1994)).   Conversely, a large number of genes inhibit microvascular cell proliferation and / or Cell proliferation in target tissues or organs supplied by transduced microvessels Can be expressed in the vasculature, especially in microvasculature. For example, Growth factors have specific cell surface receptors, so each growth factor Combine to initiate their growth promoting effect. Many growth factors and above For other cytokines, corresponding receptors have also been identified (eg, For example, Callard, R.A. And Gearing, A.,The Cytokine Facts Book, (Academic Press 1994)). Gene arrangements for many such receptors The columns are known and, for example, bind growth factors without initiating a growth-promoting signal Can be modified to create a mutant receptor. This is also a variant This could be achieved using a soluble (eg, secreted) form of the putter. So, like this Various mutant receptors as antagonists of each of these growth factors. I The expression of such mutant receptors in cells in blood vessels, especially microvessels Supplies organs or tissues, and these cells and / or their organs and tissues Cell growth can be inhibited.   Numerous other inhibitors of cell growth to be delivered are identified using the methods of the invention. Can be delivered to blood vessels, especially microvessels, for example, the non-reactive retinoblastoma (Rb) gene product Includes phosphorylated forms, which bind to specific cellular transcription factors and Inhibit cycle progression (eg, Chang, 1995; Lee, 1987; and Robbins, 1990). checking). Also, for example, aortic cell proliferation can occur in the vasculature of the parental vasculature. Epithelial cell nitric oxide synthase (which is a nitric oxide, an inhibitor of vascular smooth muscle cell proliferation) (Synthesizing inhibitors) can be inhibited by overexpressing a gene . Thus, the method of the present invention is useful for atherosclerosis and arterial remodeling It can be used to reduce stenosis (eg, Geller, 1993; Janssens, 1992; and And Von Der Leyen, 1995). In addition, many other genes are It can be expressed in blood vessels, especially microvessels, to promote death. Further below As described, such a "suicide" gene in transduced microvascular cells Expression is induced by the death of the transduced cells and a marker via the "bystander" effect. Results in the death of adjacent cells in the target organ or tissue. Such genes include, for example, One example is herpes simplex virus thymidine kinase, which helps cells Encodes a protein that renders it susceptible to the ruth accessory drug ganciclovir (eg, See, for example, Guzman, 1994). In addition, cells in blood vessels, especially microvascular cells Cell death (apoptosis) in the apoptosis pathway By overexpressing the p53 tumor suppressor gene or other genes in mice (See, eg, Lamb, 1986).   The rAAV vector may also include one or more detectable markers. Various of this Such markers are known and, for illustrative purposes, bacterial β-galactosidase (l acZ) gene; human placental alkaline phosphatase ("AP") gene (single or Multiple). It is used as a reporter molecule both in vitro and in vivo It encodes various cell surface markers.   The rAAV vector may also include one or more selectable markers. As above For applications involving gene therapy, the rAAV vector may be It may also be an advantage to include a "suicide" gene that allows for the selective elimination of . Suicide genes are a type of negative selectable marker gene, The host cell is inhibited or eliminated in the presence of the corresponding selection agent. This Such suicide genes, if necessary or desired, can thereby selectively host cells. (Eg, Lupton, 1991; and Lupton, 1994). See).   Recombinant AAV vectors also contain polynucleotides that do not encode proteins. I can see. This involves, for example, translation of normal mRNA by forming a duplex with it. Encoding an antisense mRNA that can be used to block DNA And ribozyme-encoding polynucleotides (RNA Catalyst).   The introduction of the rAAV vector by the method of the present invention may also be useful and well-known in the art. It may involve the use of a number of delivery techniques, both surgical and non-surgical. This Such delivery techniques include, for example, vascular catheterization, cannulation, Insertion (cannulization), injection, inhalation, rubbing, topical, oral, transdermal, intraarterial , Intravenous, and / or intraperitoneal administration. Vectors can also be It can be introduced by means of a vascular graft (PTFE and Dacron (da cron)), heart valves, endovascular stents, endovascular paving, and other non-vascular prostheses Including organs. General information on delivery, frequency, composition, and dosage range of vector solutions Specific techniques can be found in the references as cited herein.   As described herein, the method of the present invention provides that vascular and / or vascularization is In a number of disease states that play a role, it can be completely beneficial. For example, certain The ability to transduce cells in a tissue or organ's blood vessels, especially microvessels, is Locally alter the manifestation and locally enhance tissues or organs in a positive way To be able to Furthermore, gene expression in cells in blood vessels, It can be modified to inhibit or enhance. Limiting angiogenic potential using the method of the present invention Or the many clinical conditions associated with angiogenesis whose ability to eliminate can be very beneficial A situation exists. For purposes of illustration, benign and bad to support growth and metastasis Both sexual neoplasms typically require a large blood supply. Therefore, blood Inhibition of angiogenesis can be used to inhibit or eliminate these neoplasms. Thing In fact, the performance of many tumors can be directly correlated to the degree of microvascular content (eg, See Baillie, 1995). Therefore, a common approach to treating neoplasms Roaches (including surgical resection, radiation, and chemotherapy) are Or have the potential for significant side effects, the method of the present invention To target the tumor's own blood supply, and in a minimally invasive manner, It can be used to promote its elimination without permanent side effects.   Similarly, there are a number of other diseases where excessive blood supply is critical, and Thus, this is a microvascular cell involved in angiogenesis using the gene delivery method of the present invention. Can be beneficial. Examples are psoriasis; rheumatoid joints Inflammation; eye diseases such as diabetic retinopathy and retinitis; and separately or in association with each other Have congenital or acquired vascular malformations composed of arteries, veins, or lymph vessels But not limited thereto.   In addition, there are numerous clinical situations where it may be advantageous to promote angiogenesis . By way of example, cardiac ischemia may result in insufficient blood and oxygen flow to the heart Is an example of a system that is characterized by and is beneficial by the application of the present invention. For illustrative purposes, the methods of the present invention can be used to cannulate a coronary artery supplying an ischemic area. And insert the rAAV vector (eg, to transform microvascular cells in that region). (Including polynucleotides that can introduce and modify its growth) Delivery to localized microvasculature around the ischemic area to guide the growth to the ischemic area obtain.   Similarly, there are a number of other diseases where lack of blood supply is critical, and This, in turn, stimulates cells involved in angiogenesis using the gene delivery method of the present invention. May be beneficial. Examples include brain voids such as those occurring during an embolic stroke Blood, certain ischemic renal disorders such as renal parenchymal disease, and skin ulcers such as apical ulcers Ulcers, but are not limited thereto.   In addition to regulating angiogenesis, organs or tissues that allow gene expression The ability to transduce blood vessels, especially cells in microvessels, necessarily implies the microvessels themselves. Used to directly affect tissues or organs supplied without any action Can be For illustrative purposes, a coronary artery blocked by atherosclerosis When reconstructing such vascular tissue, the vestiges may indicate recurrent obstacles (eg, (Restenosis) frequently occurs. Following coronary angioplasty or atherectomy 30-60% of the treated arteries develop restenosis. For example, vascular smooth muscle cell proliferation Book to transduce cells in the vessels of the vessels of the affected arteries with an inhibitor of By using the method of the invention, atheromatous movement following such arterial reconstruction Pulse sclerosis, scarring and subsequent restenosis may be reduced.   The examples provided below are provided as further guidance for those skilled in the art, and And is not to be construed as limiting the invention in any manner.                                  Example Example 1 rAAV Preparation of preparation   For purposes of illustrating the present invention, we have prepared several rAAV vectors. did. For a number of primary explant cultures and in vivo studies described below, the inventors Used an adeno-associated virus reference vector called "ACAPSN". this Is operably linked to the AAV ITR sequence, human placental alkaline phosphatase (AP) cDNA. Ligated human cytomegalovirus (CMV) promoter; coli transposon Operably linked to Tn5 neomycin phosphotransferase (neo) gene Includes simian virus 40 promoter, and synthetic polyadenylation site.   ACAPSN Vector construction   The construction of the ACAPSN vector was as follows: ITR sequence and plasmid bone AAV-CFTR (AAV2 nucleotides 1-145 containing the left hand ITR, cystic fibers Transmembrane regulator (CFTR) cDNA nucleotides 133 to 4573, based on mouse β-globin Synthetic polyadenylation signals (see, eg, Flotte (1), 1993), And pBR322 inserted into the plasmid backbone of nucleotides 2295 (Ndel) to 4284 (Aat2) AAV2 containing the right-handed ITR (including nucleotides 4490-4680). simply As stated, the AAV-CFTR vector was digested with Xho1 and SnaB1, and The rasmid backbone was gel isolated. Xho1-Sna1 flags containing part of the CMV promoter (Nucleotides 671--464) (see, eg, Boshart, 1985). Gel isolated and ligated to the ITR plasmid backbone fragment from AAV-CFTR, “PAAV-CMV (SnaB1)” was generated. Next, S containing the synthetic polyadenylation signal The pe1 to Sna1 fragment was inserted into Spe1 / SnaB1-digested pAAV-CMV (SnaB1), and “pA AV-CMV (Spe1) -spA "was generated. pAAV-CMV (Spe1) -spA vector is a Includes motor nucleotides -671--584. Next, E. coli neomycin gene Placental Alkaline Phosphatase Linked to the Simian Virus 40 Promoter That Drives The cDNA sequence was converted from LAPSN (see, for example, Lynch, 1992) to Spe1-Nhe1. Isolated as a fragment and inserted into pAAV-CMV (Spe1) -spA (linearized with Spe1) Then, "pAAV-APSN" was prepared. S containing CMV promoter nucleotides -585 to +71 The pe1-Nhe1 fragment was inserted into the Spe1 linearized pAAV-APSN and the vector `` ACAPSN '' Generated.   AAV-CMV-AP Vector construction   We also have a secondary adeno-associated virus reference vector ("AAV-CMV-AP"). Was used in their primary explant culture studies. Construction of vector AAV-CMV-AP AAV vector pTRF46 (see, eg, Flotte (1), 1993) And restriction endonuclease digestion with Hind3 and Asp718, and the ITR sequence Contains a synthetic polyadenylation site (and contains the plasmid backbone from pBR322) The fragment was gel isolated. Neomycin phosphotransferase gene ( neo) is engineered with the Kozak consensus eukaryotic translation initiation sequence and is 5 'of the neo sequence. PCR amplification using primers that provide a Hind3 site at the end and an Asp718 site at the 3 'end. Width. Hind3 / Asp718 digested neoPCR product ligated to pTRF46-derived fragment Then, "pAAVneoBR" was generated. PB at the Bg12 site adjacent to the ITR with AAV and neo sequences Excised from the plasmid backbone derived from R322, the multiple cloning site is Bg12 linker Subcloned into the Bluescript vector replaced with Produced. The Sal1 fragment containing the human placental alkaline phosphatase cDNA sequence Isolated from the retroviral vector DAP (see, for example, Fields-Berry, 1992). See). Perform a two base pair fill and linearize the fragment with BamH1 It was then inserted into pAAVneo, partially filled to receive the insert. Got The intermediate construct "pAAVneoAP" was digested with Hind3 to remove the neo sequence. Rebuild Ligation was performed to prepare “pAAVAP”. Two base pair fills with CMV immediate early promoter Spe1 to Nhe1 fragment containing the sequence (see, eg, Boshart, 1985). Nucleotides -583- + 71), then linearized with Hind3 and partially Inserted into filled pAAVAP. The resulting construct was the vector “AAV-CMV-AP”. Was.   rAAV Particle packaging   Large-scale packaging of rAAV particles was performed as previously described (eg, Flotte (1), 1995; and Flotte (2), 1995). These packages Equal amounts of packaging plasmid pRS5 and rAAV vector (Ie either ACAPSN or AAV-CMV-AP) with the helper virus Ad 293 cells infected at 5 (m.o.i = 5) were co-transfected. 72 hours at 37 ° C After incubation, cells were harvested and lysed. Virus particles Purify by a cesium gradient, fractionate and permeate against Ringer's balanced salt solution. Was analyzed. The vector preparation is then heat treated to remove any residual adenovirus. Killed. Although the potential for adenovirus contamination is thought to be minimal, The product was published in the U.S. Department of Health and Human Services, Publication No. (CDC) 93-8395, entitled "Biosafety in Mic. robiological and Biomedical Laboratories '' and `` NIH Guidelines for Res earch Involving Recombinant DNA Molecules, '' Federal Register 1994, 59:34 Blo suitable for adenovirus (class 2 pathogen) as summarized in 496-34547 Operated using safety Level 2 precautionary measures.                              Example 2 (a)         AAV-CMV-AP In primary human explant cultures using vectors Transduction of smooth muscle cells   Delivery of rAAV vectors to various cell types typically found in blood vessels To study, we used a human primary explant using the AAV-CMV-AP vector. Transduction of smooth muscle cells in culture was examined.   Human smooth muscle cells are enzymatically purified using procedures as described in the art. Derived from biopsy samples by digestion. As described in Example 6 below Primary explant cultures using immunocytochemical staining for α-actin Were characterized and confirmed to contain predominantly smooth muscle cells. AAV-CMV-AP vector Twenty-four hours after exposure to the medium, the culture is half confluent (50% of the dish). Cover the cells 1x10^FiveCells were seeded at a density of 12 wells. cell For 24 hours in a total volume of 1 ml complete medium (containing a final concentration of 10% fetal bovine serum) Exposed to 10 μl of AAV-CMV-AP vector. Smooth muscle cells, AAV-CMV-AP vector -Transduced with three independent preparations. Transfer the vector preparation from the DNA genome Approximately 10 per ml as assessed by lot blot analysis.⁹To contain the particles of did. Thus, transduction was performed at approximately 100 particles per cell. 24:00 virus At the end of the period of exposure between cells and remove the cells in fresh medium for an additional 24 hours. Cultured for hours.   Cells are harvested using trypsin and 2.5 × 10^FourCytospin cells (cy tospin) on a glass slide. Air dry slides and then Cells were fixed in 0.5% glutaraldehyde in PBS. Cells are replaced with 3 changes of PBS Wash and heat treat at 65 ° C for 30 minutes to remove any endogenous alkaline phosphatase. It also inactivates the protease activity. Cells are harvested using 5-bromo-4-chloro-3-indolyl phosphate. (X-phos, 0.1 mg / ml, Boehringer Mannheim) and nitro-blue tetrazo Lithium (NBT, 1 mg / ml, Boehringer Mannheim, Buffer 3 [100 mM Tris, 100 mM Na Cl, 50mM MgCl_Two-pH 9.5] for 3 hours at room temperature in the dark, , Resulting in dark purple / black staining of cells expressing alkaline phosphatase. Then Rinse slides in PBS and cover slip using aqueous mounting medium Did. The percentage of cells that express alkaline phosphatase is determined by the Count cells under a light microscope in at least two fields using a microscope Was measured. The average transduction frequency from three independent infections is about 5. 37%.                              Example 2 (b) AAV-CMV-AP In primary rat explant cultures using vectors Transduction of smooth muscle cells   To further study the delivery of rAAV vectors to various species, we studied the AA Smooth muscle cell traits in primary rat explant cultures using the V-CMV-AP vector The introduction was also considered.   Rat smooth muscle cell cultures were purified from Fischer 344 rat aorta by enzymatic digestion. Induce and use immunocytochemical staining for α-actin as described above. It was confirmed that it mainly contained smooth muscle cells [see, for example, Lynch, 1992. When]. Exactly as described in Example 2 (a) for human smooth muscle cell transduction. As described, cells were transduced with the AAV-CMV-AP vector at 100 particles per cell. The average frequency of the three transductions was about 0.032%.   Rodent (rat) smooth muscle cells were tested in primate (human and macaque) vascular cells. It was transduced 100 times less frequently than the vesicle type. Human adeno-associated virus-derived virus Reduced sensitivity of rodent cells to transduction in Can show species specificity.                              Example 2 (c) AAV-CMV-AP In rabbit primary explant cultures using vectors Transduction of smooth muscle cells   As part of our work, we use the AAV-CMV-AP vector. Transduction of smooth muscle cells in primary rabbit explant cultures was also studied. Husa Giant smooth muscle cell cultures are derived from rabbit aorta by enzymatic digestion as described above , Characterize, and use immunocytochemical staining for α-actin , Mainly containing smooth muscle cells. Cells as described in Example 2 (a) Was transduced with a single preparation of the AAV-CMV-AP vector at 100 particles per cell. Human and rat smooth muscle cells were also transduced for comparative purposes. Rabbit smooth muscle cell Is the frequency of about 10.26% or the frequency of human smooth muscle cells in the same study (about 5.4 (1%). Rat smooth muscle cells are again characterized by an order of magnitude lower frequency. Introduced (about 0.016%).                              Example 2 (d) ACAPSN In macaque primary explant cultures using vectors Transduction of smooth muscle cells   Next, we used a monkey primary explant culture using the ACAPSN vector. Transduction of smooth muscle cells was also studied. Smooth muscle cell culture, as described above, Induced and characterized from Macaca fascicularis biopsy samples by enzymatic digestion And immunocytochemical staining for α-actin, mainly It was confirmed that it contained cells. Except for using about 1000 particles per cell, The ACAPSN vector was described as described for human smooth muscle cell transduction in Example 2 (a). And transduced. The average frequency of transduction was about 13.19%.                              Example 2 (e) ACAPSN In primary human explant cultures using vectors Transduction of umbilical vein endothelial cells   Further delivery of rAAV vectors into cell types typically found in blood vessels In order to study in recent years, we have studied primary human explant cultures using the ACAPSN vector. Transduction of endothelial cells in nutrients was also examined. Primary human umbilical vein endothelial cells Following standard protocols for the preparation of umbilical vein endothelial cell explant cultures, Derived from opsi samples [see, eg, Jaffe, 1973]. First explant The explant culture was characterized and described as von Bilb as described in Example 6 below. Includes mainly endothelial cells by immunocytochemical staining for runt factor (vWF) It was confirmed. Cells were prepared as in Example 2 (a) except that approximately 1000 particles were used per cell. ) In ACAPSN vector as described for human smooth muscle cell transduction Quality introduced. The average transduction frequency was about 4.56%.Example 2 (f) ACAPSN In primary human explant cultures using vectors Transduction of microvascular endothelial cells   In addition, we have found that human primary explant cultures using the ACAPSN vector The transduction of microvascular endothelial cells in the cells was also studied. Human microvascular endothelial cells Enzymatic digestion and removal of contaminating fibroblasts as described in the art. On fibronectin-coated plastic cultureware to remove Derived from the omentum (adipose tissue) using serial plating. First explant Cultures were characterized and immunocytochemical staining for vWF as described above. The color confirmed that it mainly contained endothelial cells. About 1000 particles per cell Except that cells were described for human smooth muscle cell transduction in Example 2 (a). Transduced with ACAPSN vector as described. The average transduction frequency is about 12.64% there were.                              Example 2 (g) ACAPSN In macaque primary explant cultures using vectors Transduction of microvascular endothelial cells   The present inventors have investigated microscopic studies in monkey primary explant cultures using the ACAPSN vector. Transduction of vascular endothelial cells was also studied. The macaque microvascular endothelial cells Derived from the omentum, characterized and immunocytochemical staining for vWF It was used to confirm that it mainly contained endothelial cells. About 1000 particles per cell Except that cells were described for human smooth muscle cell transduction in Example 2 (a). Transduced with ACAPSN vector as described. The average transduction frequency is about 4.93%. Was.   Summary of primary explant culture studies   As noted above, we typically find in blood vessels from a variety of different species. To delivered cells (eg, smooth muscle cells, macrovascular endothelial cells, microvascular endothelial cells) A series of studies were performed to investigate the delivery of the rAAV vector. These preliminary Although the study was performed "in vitro", we used primary explant cultures. Use and emphasize that this should be distinguished from immortalized cell culture Is important. Primary explant cultures, which have a finite life span, are generally From an immortalized cell culture that has been transformed with an oncogene so that It is believed to be much more representative of cells in vivo. Furthermore, high level Transduction has been reported for many cell cultures, but AAV vectors are immortal Transduce primary cells with much lower efficiency than transformed cells [See, for example, Halbert, 1995]. Therefore, for in vivo gene therapy applications, The importance of using primary cells to evaluate AAV vectors You.   These primary explant culture studies have shown that rAAV vectors have been used in many different species (non-human). Various cell types typically found in blood vessels of origin (including primates and humans). It shows that a heterologous gene can be transferred into an ip and expressed.                                 Example 3 Uses ACAPSN introduced by intraluminal delivery without pretreatment In vivo transduction of microvascular cells in primates   In vivo, the methods of the invention have been performed herein using a non-human primate. Illustrated by research. In the following examples, the ACAPSN vector was Injection into the segment of the peripheral artery in cynomolgus monkeys with atherosclerosis Was. As described further below, transduction using the methods of the invention is treated. By varying the condition of the artery being used and / or the delivery method used, It was evaluated under the conditions. In this embodiment, the ACAPSN is unperturbed. It was delivered by intraluminal injection into the femoral artery.   Two atherosclerotic cynomolgus monkeys (Macaca fascicularis) (weight About 5 kg) from the Comparative Medicine Clinical Research Center of The Bow It was selected from the breeding colony of man Gray School of Medicine. Animal, Athero Consume a variable diet for a variable period of time, and consume this diet during the study period. I continued. Give aspirin 3 days before surgery for its antiplatelet effect (20 mg / kg, po). To label proliferating cells for further study (below Animals were vaccinated 1 hour and 16 hours before surgery with 5-bromo-2'- Deoxyuridine-5'-monophosphate (BrdU, 30 mg / kg.i.m., [3 0 mg / ml], Boehringer Mannheim Inc., Indianapolis, IN). Monkey with salt Sedated with ketamine acid (10-15 mg / kg i.m.), intubated, and halothane gas (4% Effective). Blood is collected from baseline serum, hematology, and chemistry I pulled it out.   The left and right femoral arteries were exposed in the femur using aseptic technique and It was controlled near the diameter and 4 cm distally. The animals were then treated with heparin (300 mg / kg i.v., E lkins-Sinn, Inc., Cherry Hill, NJ) with anticoagulation and a flexible cannula. A (20 gauge i.v. catheter) was inserted into each arterial branch. One of these arteries Occasional closures were made and flushed with 1 ml of sterile Ringer's solution. Example ACAPSN vector preparation (250-500 μl) generated according to procedure 1 was applied to the left artery Injected. Transduction was performed at 3 × 10⁹ACAP at the concentration of DNAse resistant vector particles Performed using SN preparation (DNAse resistance is characteristic of the encapsidated vector particles Is). Control (250-500 μl Ringer's lactate, “LRS”) Was injected. After 30 minutes, the ACAPSN vector preparation and LRS control were It was aspirated from the left and right arteries. The side branches are then ligated and blood flow reestablished Was.   Upon completion of the vector and control incubation, close the wound, The animals were then returned to single animal cages for recovery. Keep animals isolated for 48 hours And any signs of viral illness such as conjunctivitis, cough, and diarrhea Also closely observed. The animals were then anesthetized and treated left and right movements The pulse was surgically excised and fixed in 10% formalin for analysis. All of Animal care and procedures were performed according to state and United States laws. Animal proto Col has joined the Bowman Gray School of Medicine Aninlal Care and Use Committee. And approved by the National Institutes of Health in Publication No. 86-23, Guide for the Care and Use of Laboratory Animals Followed.   Formalin-fixed arterial segments were converted to human placental alkaline phosphatase as follows. The transduced cells expressing the ACAPSN vector were localized by staining for Asease: Wash tissue with three changes of phosphate buffered saline (PBS, 10 ml, 1 hour / wash) Clean and heat in a 65 ° C water bath for 30 minutes in PBS to remove any endogenous alkali. Phosphatase activity was also inactivated. The sample was then treated with 5-bromo-4-chloro- 3-indolyl phosphate (X-phos, 0.1 mg / ml, Boehringer Mannheim) and Nitro-blue tetrazolium (NBT, 1 mg / ml, Boehringer Mannheim, Buffer 3 [100mM Tris, 100mM NaCl, 50mM MgCl_Two-pH 9.5] in a solution at room temperature overnight in the dark Incubate to detect alkaline phosphatase activity (dark purple / black stain) . The samples are then washed in PBS and in paraffin for tissue sectioning. Embedded. Include a positive control in each AP assay, and (1) transduced with ACAPSN vector and 10% formalin Cytospin of cultured smooth muscle cells fixed with APN; or (2) ACAPSN vector , Pelleted, fixed in 10% formalin, embedded in paraffin, Cultured smooth muscle cells. Negative controls are included for each animal And it was one of the following: (1) Artery infused with Ringer's lactate Cut from an artery remote from the site of ACAPSN treatment Section.   Examination of the treated femoral artery tissue at regular intervals along the length of the treated vessel The sections were performed in a manner designed to systematically sample the sections. Generally, each The treatment section (about 1.5 inches long) was sectioned into six rings. Wrap every third ring in OCT Buried and frozen. Fix first, second, fourth and fifth rings in 10% formalin And stained for AP activity (as above), then sectioned (approximately 2 cell layer thickness). (2-3 sections per hoop) for embedding in paraffin.   However, the samples examined collectively produce very small images of the treated vascular tissue. It should be emphasized that only minutes (probably less than 1%) are represented. Therefore, the restriction Positive results observed in a given number of random examined sections were Although expected to reflect expression in the tissue, less frequent transduction events May have remained unobserved due to the limited number of samples tested .   In the case of the resting femoral artery, no expression events were observed in the examined sections Was. However, due to limited sampling, low levels of expression cannot be ruled out. No. These femoral arteries have scars from previous bleeding (multiple Fewer microvessels due to periarterial scarring) (due to the number of blood draws) Should be expected to exist. The next in vivo study is The carotid artery was used.                                 Example 4 Uses ACAPSN introduced by intraluminal delivery after pre-exposure treatment In vivo transduction of microvascular cells in primates   Endothelial cells act as barriers to rAAV gene delivery to deeper arterial vascular walls ACAPSN vector was used to determine whether or not Was delivered by intraluminal injection into the   Two animals received intraluminal delivery following acute balloon exposure pretreatment as follows. I did. ACAPSN vector preparation was generated according to the procedure of Example 1. Transduction 3 × 10 per ml⁹Performed using ACAPSN preparation at a concentration of DNAse resistant particles . Animals are generally prepared and selected as described in Example 3 with the following exceptions. Selected and maintained: (1) In this example, the carotid artery was studied; and And (2) intraluminal delivery of ACAPSN vector and control (left and right, respectively) Before carotid artery), exposure pretreatment was given as follows. Carotid endothelium, 3F Fogar ty balloon embolectomy catheter (V. Mueller Inc., McGaw Park, IL) Which was passed 3 cm through each carotid artery (three times), inflated and gently tensioned. It was put back under force and then removed.   Subsequent handling and preparation of animals and tissues is subject to the following three exceptions: The procedure outlined in Example 3 was followed: the treated artery was removed 60 hours after isolation And (2) the animals were then sedated with ketamine (15 mg / kg, i.m.) and Palin (300 units / kg, i.v.) treated and placed in a necropsy suite Transferred, where blood was drawn for hematological and chemical evaluation. Excess pent After sodium barbital (100 mg / kg, i.v.), Ringer's solution (about 75 Animals were bled while injecting 0 cc). Right and left carotid artery in 10% formalin Perfused-fixed and then removed for analysis. (3) Further treated neck movement Examination of the vascular tissue is made at regular intervals along the length of the treated vessel, distal to the injection site. Both proximal and proximal, in a manner designed to sample sections systematically. gave. Generally, each treatment section (about 2 inches long) was sectioned into nine rings and Each oval ring was embedded in paraffin. First, second, fourth, fifth, seventh and eighth rings Frozen and then 2-3 sections of each of these rings (approximately 2 cell layers thick) And stained for AP activity (as described in Example 3).   In the case of the exposed carotid artery, the expression was of the two carotid arteries exposed to the rAAV vector. One was observed in microvascular endothelium, but in any control artery Was not observed. Expression forms a clearly definable structure in the outer membrane Could be easily localized to the microvascular endothelium.                                 Example 5 Introduced by intraluminal delivery after exposure and hyperfocal dilation pretreatment ACAPSN Vivo transduction of microvessel cells in primates using DNA   Effectiveness of enhancing access to the outer adventitia layer of the blood vessel wall (where microvessels are primarily located) To further evaluate the results, the ACAPSN vector was gently exposed and focused. It was delivered by intraluminal injection into the taut carotid artery.   Two animals had mild endothelial cell exposure and focal balloon overload as follows. Following the dilation pretreatment, it received intraluminal delivery. ACAPSN vector preparation was prepared as in Example 1. Generated according to the procedure described above. Transduction was performed at 1 × 10^TenDNAse resistant particle concentration Performed using the ACAPSN preparation. Animals are generally designated with the following exceptions: Prepared, selected, and maintained as described in Example 3: (1) This example , The carotid artery was studied; and (2) ACAPSN vector and control Before endoluminal delivery of the carotid artery (left and right carotid artery respectively), gently gently carotid endothelium Exposed and subjected to hyperfocal extension. This pretreatment was performed as follows: 3F  Fogarty balloon embolectomy catheter (V. Mueller Inc., McGaw Park, IL) Pass the carotid artery 4 cm (once), inflate, and use very little tension The endothelium was removed by pulling toward the catheter insertion site. Then the catheter The arterial segment immediately adjacent to the insertion site is over-focused by over-inflation of the balloon The balloon was removed (in a 0.5 cm long carotid artery) and the balloon was then removed.   Subsequent animal and tissue handling and preparation should be performed at 48 The procedure outlined in Example 4 was followed except that it was removed shortly thereafter.   In the case of a mildly exposed and hyperfocal carotid artery, the expression was In the endothelium of a small number of microvessels in the adventitia of both carotid arteries exposed to But was not observed in any of the control arteries. rAAV vector Adventitia cells of both carotid arteries exposed to, which were clearly identified as endothelial cells There was also a somewhat lower level of expression observed in Was not observed in any of the control arteries.                                 Example 6 Use ACAPSN introduced by adventitia injection delivery after prior balloon injury pre-treatment In vivo transduction of proliferating microvascular cells in primates   To further study the effect of enhancing access of the vessel wall to the adventitia, ACAP Direct injection into the adventitia of the carotid artery, which was also stimulated several days before delivery with the SN vector Delivered by Specifically, the arteries were swallowed 5 days prior to delivery of ACAPSN. Stimulation was achieved by repeated intraluminal passage of a catheter. This is before delivery of the vector In addition, it is expected that the rate of cell proliferation in the vessel wall will be increased. Previous time-course study From that maximum proliferation of cells in the arterial wall is induced within 4-7 days of balloon injury [See, eg, Geary, 1996]. Thus, pre-balloon injured blood vessels grow Physiological characteristics of blood vessels (during angioplasty and at arterial angioplasty or other forms of blood vessels (As would occur after reconstruction).   One animal received adventitia injection following pre-balloon injury pretreatment as follows. Received. ACAPSN vector preparation was generated according to the procedure of Example 1. Trait Introduce 3 × 10 per ml⁹Of DNAse resistant particles using ACAPSN preparation gave. Animals were generally prepared as described in Example 3 with the following exceptions: Selected and maintained: (1) In this example, the carotid artery was studied (2) both carotids received the ACAPSN vector; and (3) delivery was direct Produced by membrane injection. Adventitia injection was performed using the lumen described in Example 3 with the following exceptions: Performed by the procedure used for intradermal delivery: an alternative to placing a cannula in the lumen Instead, add 250 μl of ACAPSN vector preparation per side directly into the Injections were made using a syringe and a 27 gauge needle. (4) Left and right neck movement Five days prior to delivery of the vector to the pulse, the right carotid artery was inflated under mild tension. 3F Fogarty balloon catheter (V. Mueller Inc., McGaw Park, IL) The balloon was injured by passing through the lumen. The left carotid artery was not previously manipulated.   Subsequent animal and tissue handling and preparation was performed with the following exceptions: The procedure outlined in Example 4 was followed: (1) The treated artery was removed 72 hours after isolation And (2) immunocytochemical analysis was taken from the right carotid artery as follows Conducted on the organization.   To positively identify specific cell types that express ACAPSN in the outer membrane First, a frozen formalin-fixed section from the right carotid artery was cut, and smooth muscle cells (α- Actin, Boehringer Mannheim), endothelial cells (vWF, DAKO), macrophages ( Immunostaining with antibodies specific for CD-68, DAKO) and lymphocytes (CD-3, DAKO). Replace the primary antibody with an appropriate biotinylated secondary antibody (Vector Laboratories Inc.) and a tertiary Localized by avidin-biotin-peroxidase staining (Vector Laboratories Inc.) It has become. Sections were counterstained with hematoxylin and stained with a standard light microscope. Inspected. For the right carotid artery, much of the AP activity is Co-localized with cell staining for any of the muscle cell markers Was. Few AP activities are co-localized with cell staining for macrophage markers Was. Many AP positive cells are positive with these cell type-specific antibodies Could not be identified.   Very high frequency of delivery by adventitia injection to the carotid artery, which was previously stimulated Expression was observed in cells histologically identified within the outer membrane microvessels. This For arteries, expression is also significant in the adventitia of the area of injection As described above, these were positive with cell type-specific antibodies. Not identified, and perhaps, for example, fibroblasts, lymphocytes, other Could be leukocytes, and nerve cells. In control arteries that were not manipulated , A small number of AP positive cells were observed, and these few cells And was histologically consistent with structure and dominance. These results show that The method of the invention may also be used in other situations where vesicle growth is present or enhanced. To the expectation that frequent transduction can be achieved.                                 Example 7 Uses ACAPSN introduced by intraluminal delivery after pre-balloon injury preparation In vivo transduction of proliferating microvascular cells in primates   As described above, the adventitia microvessels pass through small microvessels that traverse the vascular medium. It is believed that blood is supplied from the lumen of the parent blood vessel (eg, an artery). This study The introduction of the rAAV vector into the outer membrane has also been pre-treated by pre-balloon injury. Could be achieved indirectly by direct injection into the lumen of the affected artery. Designed for As in Example 6, the arteries were removed 5 days prior to the delivery of ACAPSN. Stimulation was achieved by repeated intraluminal passage of a large inflated balloon catheter. this Is expected to increase the rate of cell proliferation in the vascular wall prior to delivery of the vector You.   One animal received intraluminal delivery following pre-balloon injury pretreatment as follows: Received. ACAPSN vector preparation was generated according to the procedure of Example 1. Transduction 1 x 10 per ml^TenOf DNAse resistant particles using ACAPSN preparation gave. Animals were generally prepared as described in Example 6 with the following exceptions. Selected, and maintained: As described in Example 3, delivery was to intraluminal delivery. Generated. Therefore, in this example, (1) both carotid arteries received ACAPSN And (2) 5 days before delivery of the vector to the left and right carotid arteries, Was inflated under gentle tension with a 3F Fogarty balloon catheter (V. Mueller).  Inc., McGaw Park, IL) for balloon injuries by three intraluminal passes. left The carotid artery was not manipulated beforehand.   Subsequent handling and preparation of the animals and tissues will allow the treated arteries to be isolated The procedure outlined in Example 4 was followed, with the exception that it was removed after an hour.   For intraluminal delivery to the pre-stimulated carotid artery, expression occurs in the adventitia microvessels. Were observed in histologically identified cells (but not in control arteries). Was not observed in The frequency of AP positive cells is determined by direct injection into the outer membrane Although not as high as the frequency observed for Clearly demonstrate that introduction can be achieved by injection into the lumen of a blood vessel .Summary of in vivo studies   These in vivo studies show that, for the first time, rAAV vectors Demonstrated that it could be used successfully to transduce cells in it.   Significant levels of transduction occur in arteries that have been treated with prior balloon injury. Was observed. As discussed above, such arteries are responsible for the physiology of the growing blood vessels. It is expected to mimic architectural features (as occurs during angiogenesis). these The results show that the method of the present invention can also be used in other situations where vascular growth is enhanced. To achieve high frequency transduction.   In addition, these in vivo studies demonstrate that atherosclerotic arteries found in humans Atherosclerosis, the most representative disease artery model of sclerosis disease state Demonstration of transduction of cells in primate arteries. Arteries in these macaques Atherosclerotic state of the disease was not severe, but Physical access is due to plaques, fat deposits, and / or thickened smooth muscle cells in blood vessels Due to the presence of layers, it can be achieved even in mild atherosclerotic vessels. Is expected to be more difficult. Thus, atheromatous using the method of the invention The ability to successfully transduce cells in blood vessels in the atherosclerosis macaque model That atherosclerotic vascular transduction can be achieved and that higher levels Even transduction of bells is reached in non-atherosclerotic vessels where access conditions are less stringent Indicates that it can be done.                                 Example 8 Assessing post-injury cell proliferation using antibody staining   One hour and six hours before euthanasia, all animals were injected with BrdU as described above. Enables measurement of cell proliferation in histological sections showing positive ACAPSN staining I made it. Transduction is most efficient for the most actively growing cells It is considered.   Selected unstained deparaffinized sections (5 microns thick) from the treated arteries were Exposure to monoclonal antibody to dU (Boehringer Mannheim) Localized by tinylated secondary antibody and avidin-biotin-peroxidase tertiary antibody staining Become Proliferating cells are identified as nuclei with brown antibody staining. BrdU data Indicates that rAAV uptake and expression are proliferating cells (eg, those associated with angiogenesis). Cells can be provided. In addition, ocular microvascular cells Alternatively, an angiogenesis model, such as the yolk sac model, can be added to primary tissue in primary cells. Can be used to quantify similar results.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE, LS, MW, S D, SZ, UG), EA (AM, AZ, BY, KG, KZ , MD, RU, TJ, TM), AL, AM, AT, AU , AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, G B, GE, GH, HU, IL, IS, JP, KE, KG , KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, N O, NZ, PL, PT, RO, RU, SD, SE, SG , SI, SK, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU (72) Inventor Gary, Randol Fel.             United States North Carolina             27104, Winston-Salem, Rich             Field Road 620 (72) Inventor Lynch, Carmel M.             United States Washington 98034             Kland, N. E. 14 Tea H               Place 10031

Claims (1)

[Claims] 1. A method of transducing cells in a blood vessel of an individual, said method comprising transducing the blood vessel of said individual in vivo. Introducing a recombinant adeno-associated virus (rAAV) vector into the cell. 2. 2. The blood of claim 1, wherein said rAAV vector comprises a detectable marker gene. A method of transducing cells in a tube. 3. The blood of claim 1, wherein the rAAV vector comprises a selectable marker gene. A method of transducing cells in a tube. 4. 2. The cell of claim 1, wherein said rAVV vector comprises a therapeutic gene. How to introduce quality. 5. The group of arterioles, capillaries, venules, and adventitia microvessels; Transducing cells in a blood vessel according to claim 1, which is a microvessel selected from the group consisting of: Method. 6. 6. Transducing cells in blood vessels according to claim 5, wherein said blood vessels are adventitia microvessels. How to enter. 7. 2. The method of claim 1, wherein the blood vessels are microvessels and the cells are proliferating. A method for transducing cells in a blood vessel. 8. 2. Transduce cells in blood vessels according to claim 1, wherein said cells are primate cells. Way. 9. 9. Transduce cells in blood vessels according to claim 8, wherein said cells are human cells. Method. 10. 2. Transducing cells in a blood vessel according to claim 1, wherein said cells are proliferating cells. How to enter. 11. The cells of claim 10, wherein said cells are proliferating microvascular cells. A method for transducing vesicles. 12. 2. Transducing cells in blood vessels according to claim 1, wherein said cells are microvascular cells. How to enter. 13. 13. The cell in a blood vessel according to claim 12, wherein the cell is a microvascular endothelial cell. A method for transducing. 14． 2. The rAAV vector is introduced into the adventitia of an artery of the individual. A method for transducing cells in blood vessels. 15. Introduction of recombinant adeno-associated virus (rAAV) vector into microvascular cells And the transduced microvascular cells produced by the method. 16. A method for treating an individual for a disease state, comprising the steps of claim 4. Transducing cells in the blood vessels of said individual according to the method.