EP0906440A1 - Procede de transduction de cellules dans des vaisseaux sanguins a l'aide de vecteurs de virus adeno-associes (vaa) recombinants - Google Patents

Procede de transduction de cellules dans des vaisseaux sanguins a l'aide de vecteurs de virus adeno-associes (vaa) recombinants

Info

Publication number
EP0906440A1
EP0906440A1 EP97907934A EP97907934A EP0906440A1 EP 0906440 A1 EP0906440 A1 EP 0906440A1 EP 97907934 A EP97907934 A EP 97907934A EP 97907934 A EP97907934 A EP 97907934A EP 0906440 A1 EP0906440 A1 EP 0906440A1
Authority
EP
European Patent Office
Prior art keywords
cell
cells
blood vessel
transducing
vector
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97907934A
Other languages
German (de)
English (en)
Inventor
Randolf L. Geary
Carmel M. Lynch
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wake Forest University
Ampliphi Biosciences Corp
Original Assignee
Wake Forest University
Targeted Genetics Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wake Forest University, Targeted Genetics Corp filed Critical Wake Forest University
Publication of EP0906440A1 publication Critical patent/EP0906440A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This invention relates to gene delivery, and more specifically to methods for transducing cells in blood vessels using recombinant aden ⁇ -associated virus (AAV) vectors.
  • AAV aden ⁇ -associated virus
  • Blood vessels a major component ofthe cardiovascular system, form a network that permits blood to flow from the heart to cells throughout the body and back to the heart.
  • blood vessels are composed of three layers, the intima, media and adventitia.
  • the intima in most undiseased arterial vessels generally comprises a luminal monolayer of endothelial cells.
  • the intima may contain smooth muscle cells; and, in diseases such as atherosclerosis, the intima may thicken with smooth muscle cells and inflammatory cells beneath the endothelial monolayer.
  • the intima is separated from the media by the internal elastic lamina.
  • the media generally comprises smooth muscle cells and their surrounding matrix material.
  • the media is composed of defined layers of smooth muscle cells separated by elastic fibers.
  • the adventitia forms the outermost layer of the artery wall and is separated from the media by the external elastic lamina.
  • the adventitia is generally composed of a loose matrix containing macrophages, fibroblasts, and other cell types, as well as the vasa vasorum (a rich network of adventitial micro vessels).
  • Microvessels may differ from the general structural model outlined above in that the three layers may not be well defined.
  • capillaries may comprise a monolayer of endothelial cells surrounded by a single layer of smooth muscle cells without any well-defined elastic layers.
  • Angiogenesis is the formation of new blood vessels. Angiogenesis occurs during fetal development ofthe vascular system, as well in a wide range of normal and postnatal pathological processes such as wound repair; neoplasia, and inflammation. [See, e.g., Diaz-Flores, 1994] Thus, a number of disease processes have abundant microvessels as key anatomical or pathological features.
  • microvessels may contribute directly or indirectly to the development of specific illnesses or may represent a benign morphological feature.
  • microvessels and angiogenesis are believed to play central roles in the pathogenesis of disease. These include, for example, the growth and metastasis of many cancers, diabetic retinopathy, retinitis, heart failure, arthritis, psoriasis, ischemia, wound healing, hemangiomas and other vascular malformations. [See, e.g., Diaz-Flores, 1994]
  • the cells comprising all tissues and organs require an extensive network of microvessels to support their normal function and viability. Since these microvessels may be functionally unique and distinct from larger blood vessels, microvessels provide unique targets for delivery of therapeutic polynucleotides to tissues.
  • the tissue or organ may be enhanced in a positive way.
  • retroviral vectors are advantageous because of their general potential for stable long-term gene expression, target cell replication is required for stable provirus integration.
  • most cells in the artery are generally quiescent.
  • Quiescent cells can sometimes be isolated and induced to divide in order to achieve significant and efficient gene transfer; but, this method often requires that the cells be induced and genetically modified ex vivo and thereafter transplanted back into the donor host.
  • Liposomal vector delivery systems have also been limited by inefficient uptake and transient episomal vector expression.
  • Adenoviral vectors that have been efficient at infecting endothelial cells have been limited by transient episomal vector expression and by antigenicity limiting the efficacy of repeated applications.
  • the present invention provides methods for transducing cells in blood vessels using recombinant AAV vectors.
  • Preferred embodiments ofthe present invention include the following: 1. A method of transducing a cell in a blood vessel of an individual, comprising introducing a recombinant adeno-associated viral (rAAV) vector to a blood vessel of said individual in vivo.
  • rAAV adeno-associated viral
  • a method of transducing a cell in a blood vessel according to embodiment 1 wherein said rAAV vector comprises a detectable marker gene.
  • said rAAV vector comprises a selectable marker gene.
  • a method of transducing a cell in a blood vessel according to embodiment 1 wherein said blood vessel is a microvessel selected from the group consisting of arteriole, capillary, venule, and adventitial microvessel.
  • a method of transducing a cell in a blood vessel according to embodiment 1 wherein said blood vessel is a microvessel and said cell is undergoing proliferation.
  • a method of transducing a cell in a blood vessel according to embodiment 1 wherein said cell is a proliferating cell.
  • a transduced microvascular cell produced by introducing a recombinant adeno-associated viral (rAAV) vector to said microvascular cell.
  • rAAV adeno-associated viral
  • a method for treating an individual for a disease condition comprising transducing a cell in a blood vessel of said individual according to the method of embodiment 4.
  • Polynucleotide refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or analogs thereof. This term refers only to the primary structure ofthe molecule. Thus, double- and single-stranded
  • DNA as well as double- and single-stranded RNA are included. It also includes modified polynucleotides such as methylated or capped polynucleotides.
  • Recombinant as applied to a polynucleotide, means that the polynucleotide is the product of various combinations of cloning, restriction and/or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature.
  • a “vector” refers to a recombinant plasmid or virus that comprises a polynucleotide to be delivered into a host cell, either in vitro or in vivo.
  • the polynucleotide to be delivered may comprise a coding sequence of interest in gene therapy.
  • a “recombinant AAV vector” refers to a vector comprising one or more polynucleotides of interest that are flanked by AAV inverted terminal repeat sequences (ITRs).
  • ITRs AAV inverted terminal repeat sequences
  • One possible method of replicating and packaging a rAAV vector into infectious viral particles may be to introduce the rAAV vector into a host cell expressing the AAV "rep” and “cap” genes and infected with a suitable helper virus.
  • a “gene” refers to a polynucleotide or portion of a polynucleotide comprising a sequence that encodes a protein. For most situations, it is desirable for the gene to also comprise a promoter operably linked to the coding sequence in order to effectively promote transcription. Enhancers, repressors and other regulatory sequences may also be included in order to modulate activity ofthe gene, as is well known in the art. (See, e.g., the references cited below).
  • polypeptide As used interchangeably to refer to polymers of amino acids of any length. These terms also include proteins that are post-translationally modified through reactions that include glycosylation, acetylation and phosphorylation.
  • AAV "rep” and “cap” genes encoding replication and encapsidatio ⁇ proteins, respectively, have been found in all AAV serotypes examined, as described in various references cited herein. Typically, the rep and cap genes are found adjacent to each other in the AAV genome, and they are generally conserved among AAV serotypes.
  • helper virus for AAV refers to a second virus that allows wild-type AAV, which is a defective parvovirus, to be replicated and packaged by a host cell.
  • helper viruses have been identified in the art, including adenoviruses, herpesviruses, and poxviruses such as vaccinia.
  • Packaging refers to a series of subcellular events that results in the assembly and encapsidation of a rAAV vector. Thus, when a suitable vector plasmid is introduced into a packaging cell line under appropriate conditions, it can be replicated and assembled into a viral particle.
  • Heterologous means derived from a genotypically distinct entity from that ofthe rest of the entity to which it is compared.
  • a polynucleotide derived from one cell type and introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide which, when expressed, can encode a heterologous polypeptide.
  • a promoter that is removed from its native coding sequence and operably linked to a different coding sequence is a heterologous promoter.
  • Promoter refers to a genomic region that controls the transcription of a gene or coding sequence to which it is operably linked.
  • “Operably linked” refers to a juxtaposition, wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a promoter is operably linked to a coding sequence if the promoter controls transcription of the coding sequence.
  • An operably linked promoter is usually in cis configuration with the coding sequence, but is not necessarily contiguous with it.
  • a “detectable marker gene” is a gene that allows cells carrying the gene to be specifically detected (e.g., distinguished from cells which do not carry the marker gene).
  • a large variety of such marker genes are known in the art. Preferred examples thereof include detectable marker genes which encode proteins appearing on cellular surfaces, thereby facilitating simplified and rapid detection and/or cellular sorting.
  • the inventors utilized an alkaline phosphatase ("AP") gene as a detectable marker, which allowed cells transduced with a vector carrying the AP gene to be detected based on expression of AP on the surface of transduced cells.
  • AP alkaline phosphatase
  • a “selectable marker gene” is a gene that allows cells carrying the gene to be specifically selected for or against, in the presence of a corresponding selective agent.
  • an antibiotic resistance gene can be used as a positive selectable marker gene that allows a host cell to be positively selected for in the presence ofthe corresponding antibiotic.
  • positive and negative selectable markers are knowii in the art, some of which are described below.
  • a “therapeutic polynucleotide” or “therapeutic gene” refers to a nucleotide sequence that is capable, when transferred to an individual, of eliciting a prophylactic, curative or other beneficial effect in the individual.
  • Cytokine refers to intercellular signaling molecules, the best known of which are involved in the regulation of mammalian somatic cells.
  • interleukins such as IL-l ⁇ , IL-l ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9 (P40), IL-10, IL-11, IL-12 and IL-13
  • CSF-type cytokines such as GM-CSF, G-CSF, M-CSF, LIF, EPO, TNF- ⁇ and TNF- ⁇
  • interferons such as IFN- ⁇ , IFN- ⁇ , IFN- ⁇
  • cytokines of the TGF- ⁇ family such as TGF- ⁇ 1 , TGF- ⁇ 2, TGF- ⁇ 3, inhibin A, inhibin B, activin A, activin B): chemotin
  • growth factors such as EGF, TGF- ⁇ , aFGF, bFGF, KGF, PDGF-A, PDGF-B, PD-ECGF
  • cytokines A number of other cytokines are also known to those of skill in the art. The sources, characteristics, targets and effector activities of these cytokines have been described and, for many of the cytokines, the DNA sequences encoding the molecules are also known; see, e.g., Callard & Gearing, The Cytokine Facts Book (Academic Press, 1994) and the particular publications reviewed and/or cited therein, which are hereby incorporated by reference in their entirety. As referenced in catalogs such as The Cytokine Facts Book, many ofthe DNA and/or protein sequences encoding such cytokines are also generally available from sequence databases such as GENBANK (DNA); and SWISSPROT (protein).
  • cytokines typically, cloned DNA encoding such cytokines will already be available as plasmids, although it is also possible to synthesize polynucleotides encoding the cytokines based upon the published sequence information. Polynucleotides encoding the cytokines can also be obtained using polymerase chain reaction (PCR) methodology, as described in the art.
  • PCR polymerase chain reaction
  • the detection, purification, and characterization of cytokines, including assays for identifying new cytokines effective upon a given cell type, have also been described in a number of publications as well as the references referred to herein. [See, e.g., Lymphokines and Interferons. (Clemens, J.J. et al. eds., IRL Press 1987); and DeMaeyer, 1988].
  • Transduction or “transducing” as used herein, are terms referring to the introduction of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion, which methods include well-known techniques such as transfection, lipofection, viral infection, transformation, and electroporation, as well as non-viral gene delivery techniques.
  • the introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
  • a “replicon” refers to a polynucleotide comprising an origin of replication, generally referred to as an on sequence, which allows for replication ofthe polynucleotide in an appropriate host cell.
  • examples include replicons of a target cell into which a desired nucleic acid might integrate, e.g., nuclear and mitochondrial chromosomes, and extrachromosomal replicons such as plasmids.
  • an “individual” as used herein refers to a mammal, preferably a human.
  • “Treatment” or “therapy” as used herein refers to administering cells, other agents, or combinations thereof, to an individual, that are capable of eliciting a prophylactic, curative or other beneficial effect in the individual.
  • Gene delivery refers to the introduction of an exogenous polynucleotide into a cell for gene transfer, and may encompass targeting, binding, uptake, transport, localization, replicon integration and expression.
  • Gene transfer refers to the introduction of an exogenous polynucleotide into a cell which may encompass targeting, binding, uptake, transport, localization and replicon integration, but is distinct from and does not imply subsequent expression ofthe gene.
  • Gene expression or “expression” refers to the process of gene transcription, translation, and posttranslational modification.
  • Vasculature or "vascular” are terms referring to the system of vessels carrying blood (as well as lymph fluids) throughout the mammalian body.
  • Blood vessel refers to any ofthe vessels ofthe mammalian vascular system, including arteries, arterioles, capillaries, venules, veins, sinuses, and vasa vasorum.
  • the wall of an artery consists typically of an outer layer (adventitia) separated by an external elastic lamina from a middle layer (media) which is separated by an internal elastic lamina from an inner layer (intima).
  • the adventitia is a layer of loose connective tissue which generally includes a network of microvessels (vasa vasorum), fibroblasts, and immune cells such as lymphocytes and macrophages.
  • the media comprises circular layers of smooth muscle cells and elastic fibers.
  • the intima is made up of a monolayer of endothelial cells overlying, in some instances, smooth muscle cells.
  • Microvessel “microvascular” or “microvasculature,” as used herein, are terms referring to the arterioles, capillaries, venules, and adventitial microvessels. Microvessels generally comprise endothelial cells surrounded by one or a few layers of smooth muscle cells. Arteriole refers to a minute arterial branch, especially one just proximal to a.capillary. Capillary refers to any one ofthe minute vessels that connect the arterioles and venules, forming a network in virtually all organs and tissues. Venules refer to any ofthe small vessels that collect blood from the capillary plexuses and join to form veins.
  • Advanced microvessel refers to microvessels that supply blood to the adventitia of larger blood vessels such as arteries.
  • the network of these adventitial microvessels is commonly referred to as the vasa vasorum.
  • adventitial microvessels are believed to be supplied with blood from the lumen ofthe parent vessel (e.g., the artery) via small microvessels traversing the vessel intima and media.
  • Microvascular cell refers to cells that make up the structure of microvessels.
  • Endothelium refers to the layer of cells (i.e., “endothelial cells”) that generally lines the cavities ofthe heart and blood vessels, as well as vessels ofthe lymphatic system.
  • vessels such as arteries can contain both endothelial cells and smooth muscle cells which are distinguishable in terms of origin, functionality, and attributes such as cell surface markers.
  • endothelial cells derive from embryonic ectoderm, form the layer of cells that make up the endothelium, provide a non-thrombogenic surface, and can be readily distinguished using a number of well-known cell surface markers, including, by way of illustration, vWF, as exemplified below.
  • smooth muscle cells derive from embryonic mesoderm, provide structure and contractile function for blood vessel walls, do not provide a non-thrombogenic surface, and can be readily distinguished using a number of well-known surface markers, including, by way of illustration, alpha-actin, as exemplified below.
  • "Proliferating” or “proliferation” are terms referring to growth by cell multiplication.
  • Angiogenesis is the formation of new blood vessels which occurs during fetal development of the vascular system, as well in a wide range of normal and postnatal pathological processes such as wound repair, neoplasia, and inflammation.
  • Additional references describing delivery and logistics of surgery which may be used in the methods ofthe present invention include the following: The Textbook of Interventional Cardiology. 2nd Ed. (E. Topol ed., W.B. Saunders Co. 1994); Rutherford, R.B., Vascular Surgery. 3rd Ed. (W.B. Saunders Co. 1989); Interventional Radiology. 2nd Edition (W. Castaneda-Zuniga & S. Tadavarthy eds., Williams & Wilkins 1992); Textbook of Respiratory Medicine. 2nd Ed. (J. Murray & J. Nadel eds., W.B. Saunders Co. 1994); and Textbook of Sureerv. 14th Ed. (D. Sabiston, Jr., W.B. Saunders Co. 1991).
  • Additional references describing cell types found in the blood vessels, and the structure ofthe vasculature which may be used in the methods ofthe present invention include the following: W. Bloom & D. Fawcett, A Textbook of Histology. 10th Ed.. (W.B. Saunders Co. 1975). Additional references describing AAV vectors which may be used in the methods ofthe present invention include the following: Carter, B., Handbook of Parvoviruses. vol. I, pp. 169-228 (1990); Berns, Virology, pp. 1743-1764 (Raven Press 1990); Muzyczka. N.. Current Topics in Microbiology and Immunology. 158: 92-129 (1992); and Kotin, R., Human Gene Therapy. 5: 793-801 (1994).
  • Carter, B. Handbook of Parvoviruses. vol. I, pp. 169-228 (1990). Carter, B., Current Opinions in Biotechnology. 3: 533-539 (1992).
  • the invention described involves methods for transducing cells in blood vessels using recombinant AAV vectors.
  • the transduction of cells in blood vessels in vivo presents problems not encountered in the transduction of other cells in vivo or in vitro.
  • AAV vectors are among a small number of recombinant virus vector systems which have been shown to have utility as in vivo gene transfer agents and thus are potentially of great importance for human gene therapy.
  • AAV vectors are capable of high-frequency gene transfer and expression in a variety of cell lines ex vivo. [Carter, 1992; Egan, 1992; Flotte, 1992; Flotte (1 ), 1993; Flotte (2), 1993: Kaplitt, 1994; Kotin, 1994; Muzyczka, 1992; and Walsh, 1992]
  • AAV is a DNA parvovirus often found in association with adenovirus infections of humans.
  • AAV has not been shown to cause disease in man and is not a transforming or oncogenic virus.
  • most ofthe other proposed viral systems such as retroviruses, adeno viruses, herpesviruses, or poxviruses are disease-causing viruses. Indeed, greater than 85% of adults are believed to be seropositive for one of four AAV serotypes.
  • AAV is also replication-defective, and can replicate only within the nucleus of cells simultaneously infected with a helper virus such as adenovirus, herpes virus, or in some cases poxviruses such as vaccinia.
  • AAV is believed to be capable of infecting and replicating (albeit at different efficiencies) in virtually any cell line of human, simian or rodent origin if an appropriate helper is present.
  • AAV has a clear advantage over retroviruses, especially in tissues such as the human airway epithelium where most cells are terminally differentiated and non-dividing, because AAV does not require active cell division for gene transfer.
  • retroviruses are capable of transducing quiescent cells in situ and have recently been shown to be effective at transducing monkey pulmonary epithelium in vivo with the CFTR gene for cystic fibrosis. [See, e.g., Flotte, 1994] These studies have recently been carried into human trials approved by the NIH Recombinant DNA Advisory Committee.
  • AAV can be modified to create a vector for the delivery of heterologous genes.
  • such AAV vectors will have no wild-type coding sequences and will be incapable of replication, even in the presence of helper virus.
  • the process of modification involves deleting all wild-type AAV coding sequences (rep and cap) so that only the inverted terminal repeat sequences (which are required in cis for vector replication) remain.
  • a gene of interest can be inserted between the viral inverted terminal repeat sequences and then packaged.
  • AAV vectors can be constructed in AAV recombinant plasmids by substituting portions of the AAV coding sequence with heterologous DNA to generate a vector plasmid.
  • the terminal (ITR) portions ofthe AAV sequence play an important role in cis for several functions including excision from the plasmid after transfection, replication of the vector genome and integration and rescue from a host cell genome.
  • the vector can then be packaged into an AAV particle to generate an AAV transducing virus by transfection ofthe vector plasmid into cells that are: (1) infected by an appropriate helper virus such as adenovirus or herpesvirus, and (2) capable of providing AAV replication and encapsidation functions in trans (since these functions were deleted in construction ofthe vector plasmid).
  • helper virus such as adenovirus or herpesvirus
  • AAV replication and encapsidation functions in trans since these functions were deleted in construction ofthe vector plasmid.
  • recombinant AAV vector preparations can comprise AAV ITR regions and a transcription promoter operably linked to any gene of interest that is to be transduced to the recipient cell, including for example, detectable genes, selectable genes, and/or therapeutic genes.
  • detectable genes include detectable genes, selectable genes, and/or therapeutic genes.
  • the inventors used human placental alkaline phosphatase (AP) as a detectable gene.
  • AP placental alkaline phosphatase
  • other recombinant AAV vectors containing one or more detectable, selectable, and/or therapeutic genes can be readily employed.
  • the methods ofthe present invention can employ rAAV vectors comprising (in place of or in addition to a detectable and/or selectable gene) a therapeutic gene that is used to alter the activity ofthe transduced recipient cell so that the recipient cell and/or its progeny have a beneficial effect on an individual receiving such cells.
  • a typical example would be the transduction of cells in a blood vessel with a therapeutic gene that enhances the level of a beneficial protein or other agent in the cell and or its progeny, or that reduces the level of a deleterious protein or other agent in the cell and/or its progeny, or that provides resistance to a cytotoxic or other harmful agent.
  • the present invention can be used to transduce cells in a blood vessel with a gene or genes that encode secreted proteins or that encode proteins involved in the secretion of other agents from the cell and/or its progeny, which secreted proteins or other agents have a beneficial effect on the recipient individual.
  • the present invention can be used to transduce cells in a blood vessel with a polynucleotide, gene or genes that affect the interaction between a cell and/or its progeny and other cells in the recipient individual.
  • the therapeutic gene might render the transduced cells and/or their progeny more or less susceptible to activation by other cells, more or less resistant to a chemotherapeutic agent, or more or less resistant to an infectious agent such as a virus or a toxic agent such as a chemotherapeutic drug, to name just a few examples.
  • the present invention thus can be used to "deliver" any of a wide variety of genes to cells within the vasculature of a mammal, preferably a human.
  • a few examples of specific therapeutic strategies taking advantage ofthe invention and its ability to transduce cells in blood vessels, especially microvessels, for recombinant gene delivery are outlined below. These additional examples are provided for purposes of further illustrating exemplary applications ofthe present invention. Numerous other genes can be delivered using the methods ofthe present invention, as will be clear to those of skill in the art.
  • a number of genes can be expressed in the vasculature, especially microvessels, to increase cell growth of microvascular cells (e.g., promote angiogenesis) and/or to increase cell growth in a target tissue or organ supplied by the transduced microvessels.
  • genes include, for example, human growth factors, platelet-derived growth factor (PDGF), vascular endothelial growth factors (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor-beta (TGF- ⁇ ), and epidermal growth factor (EGF).
  • PDGF platelet-derived growth factor
  • VEGF vascular endothelial growth factors
  • bFGF basic fibroblast growth factor
  • TGF- ⁇ transforming growth factor-beta
  • EGF epidermal growth factor
  • a number of genes can be expressed in the vasculature, especially microvessels, to inhibit growth of microvascular cells and/or to decrease cell growth in a target tissue or organ supplied by transduced microvessels.
  • growth factors have specific cell-surface receptors to which the respective growth factors bind to initiate their growth-promoting effects.
  • the corresponding receptors have also been identified. [See, e.g., Callard, R. & Gearing, A., The Cytokine Facts Book.
  • mutant receptors that, for example, bind growth factor without initiating a growth-promoting signal. This could also be achieved using soluble (e.g., secreted) forms ofthe mutant receptors. Thus, such mutant receptors could serve as antagonists for their respective growth factors; expression of such mutant receptors in cells in the vasculature, especially microvessels, which supply organs or tissues can inhibit growth of these cells and/or cells ofthe organ and tissue itself.
  • inhibitors of cell growth that could be delivered to the vasculature, especially microvessels, using the methods ofthe invention include, for example, the nonphosphorylated form ofthe retinoblastoma (Rb) gene product, which inhibits cell cycle progression by binding to specific cellular transcription factors.
  • Rb retinoblastoma
  • large artery cell proliferation could be inhibited by over-expressing genes such as endothelial cell nitric oxide synthase (which synthesizes nitric oxide, an inhibitor of vascular smooth muscle cell proliferation) in the vasa vasorum of a parent vessel.
  • the methods ofthe invention could be used to reduce atherosclerosis and restenosis following arterial reconstructions.
  • numerous other genes can be expressed in the vasculature, especially microvessels, to promote cell death.
  • expression of such "suicide" genes in transduced microvascular cells can result in the death of the transduced cells and death of adjacent cells in the target organ or tissue via a "bystander" effect.
  • Such genes include herpes simplex virus thymidine kinase which encodes a protein rendering cells susceptibile to antiviral pro-drug ganciclovir.
  • programmed cell death in cells in blood vessels, especially microvascular cells, can be promoted by over-expressing, for example, p53 tumor suppressor gene or other genes in the apoptosis pathways.
  • p53 tumor suppressor gene or other genes in the apoptosis pathways.
  • the rAAV vectors may also contain one or more detectable markers.
  • detectable markers include, by way of illustration, the bacterial beta-galactosidase (lacZ) gene; the human placental alkaline phosphatase (“AP”) gene and genes encoding various cellular surface markers which have been used as reporter molecules both in vitro and in vivo.
  • the rAAV vectors may also contain one or more selectable markers.
  • the rAAV vector may also comprise a "suicide" gene that allows recipient cells in a blood vessel to be selectively eliminated at will.
  • a suicide gene is a type of negative selectable marker gene that causes host cells to be inhibited or eliminated in the presence ofthe corresponding selective agent. Such suicide genes can thereby be used to selectively eliminate the host cells should that become necessary or desirable.
  • Recombinant AAV vectors can also comprise polynucleotides that do not encode proteins, including, e.g., polynucleotides encoding for antisense mRNA (the complement of mRNA) which can be used to block the translation of normal mRNA by forming a duplex with it, and polynucleotides that encode ribozymes (RNA catalysts).
  • polynucleotides that do not encode proteins including, e.g., polynucleotides encoding for antisense mRNA (the complement of mRNA) which can be used to block the translation of normal mRNA by forming a duplex with it, and polynucleotides that encode ribozymes (RNA catalysts).
  • rAAV vectors by the methods ofthe present invention may involve use of any number of delivery techniques (both surgical and non- surgical) which are available and well known in the art.
  • delivery techniques include vascular catheterization, cannulization, injection, inhalation, inunction, topical, oral, percutaneous, intra-arterial, intravenous, and/or intraperitoneal administrations.
  • Vectors can also be introduced by way of bioprostheses, including, by way of illustration, vascular grafts (PTFE and dacron), heart valves, intravascular stents, intravascular paving as well as other non-vascular prostheses.
  • PTFE and dacron vascular grafts
  • Heart valves intravascular stents
  • intravascular paving intravascular paving as well as other non-vascular prostheses.
  • the methods of the present invention can be quite beneficial in a number of disease conditions in which blood vessels and/or angiogenesis play a role.
  • the ability to transduce cells in blood vessels, especially microvessels, of a specific tissue or organ would allow one to locally modify gene expression and to locally enhance the tissue or organ in a positive way.
  • angiogenesis There are many clinical situations associated with angiogenesis where the ability to limit or eliminate angiogenic potential using the methods ofthe present invention can be very beneficial.
  • bom benign and malignant neoplasms typically require extensive blood supply to support growth and metastasis, so that inhibition of angiogenesis can be used to inhibit or eliminate these neoplasms.
  • the malignant potential of many tumors can be directly correlated to the extent of microvascular content.
  • the methods ofthe invention can be used to target the blood supply ofthe tumor itself and, in a minimally invasive fashion, promote its elimination without systemic or permanent side effects.
  • cardiac ischemia is characterized by insufficient blood and oxygen flow to the heart and is an example of a system that would be benefited by application of the present invention.
  • rAAV vectors e.g., comprising polynucleotides that can transduce and modify growth of microvascular cells in thai area
  • the ability to transduce cells in blood vessels, especially microvessels, of an organ or tissue allows for gene expression that can be used to directly influence the tissue or organ supplied without necessarily affecting the microvessel itself.
  • tissue such as coronary arteries blocked with atherosclerosis
  • scarring frequently results in a recurrent blockage (e.g., restenosis) at the site of reconstruction.
  • a recurrent blockage e.g., restenosis
  • 30-60% of treated arteries develop restenosis.
  • the methods ofthe invention to transduce cells in the vasa vasorum of an affected artery with, for example, an inhibitor of vascular smooth muscle cell proliferation the atherosclerosis, scarring and subsequent restenosis following such arterial reconstructions can be reduced.
  • Example 1 Generation of rAAV Preparations For purposes of illustrating the present invention, the inventors prepared several rAAV vectors. For a number ofthe primary explant culture and in vivo studies described below, the inventors employed an adeno-associated virus-based vector called "ACAPSN" which comprises AAV ITR sequences, the human cytomegalovirus (CMV) promoter operably linked to the human placental alkaline phosphatase (AP) cDNA, the simian virus 40 promoter operably linked to the E. coli transposon Tn5 neomycin phosphotransferase (neo) gene, and a synthetic polyadenylation site.
  • ACAPSN adeno-associated virus-based vector
  • ACAPSN adeno-associated virus-based vector
  • CMV human cytomegalovirus
  • AP placental alkaline phosphatase
  • simian virus 40 promoter operably linked to the E. coli transposon Tn5
  • ACAPSN vector was as follows: the ITR sequences and plasmid backbone were derived from AAV-CFTR (which contains AAV2 nucleotides 1-145 comprising the left-hand ITR, the cystic fibrosis transmembrane regulator (CFTR) cDNA nucleotides 133 to 4573, a synthetic polyadenylation signal based on murine ⁇ -globin [See, e.g., Flotte (1), 1993] and AAV2 nucleotides 4490 to 4680 containing the right-hand ITR inserted in a plasmid backbone of pBR322 nucleotides 2295 (Ndel) to 4284 (Aat2)).
  • AAV-CFTR which contains AAV2 nucleotides 1-145 comprising the left-hand ITR, the cystic fibrosis transmembrane regulator (CFTR) cDNA nucleotides 133 to 4573, a synthetic polyadenylation signal based on murine ⁇ -globin [
  • the AAV-CFTR vector was digested with Xho 1 and SnaB 1 and the ITRs and plasmid backbone were gel isolated.
  • An Xhol to SnaBl fragment containing a portion of the CMV promoter (nucleotides -671 to -464) [See, e.g., Boshart, 1985] was gel isolated and ligated to the ITR plasmid backbone fragment derived from AAV-CFTR to generate "pAAV- CMV (SnaBl)."
  • an Spel to SnaBl fragment containing the synthetic polyadenylation signal was inserted into Spel/SnaBl digested pAAV-CMV (SnaBl) to generate "pAAV-CMV (Spel)-spA.”
  • the pAAV-CMV (Spel)-spA vector contains nucleotides -671 to -584 of the CMV promoter.
  • AAV-CMV-AP vector Construction of AAV-CMV-AP vector The inventors also employed a second adeno-associated virus-based vector
  • AAV-CMV-AP in their primary explant culture studies.
  • the construction of vector AAV-CMV-AP was as follows: the AAV vector pTRF46 [See, e.g., Flotte (1), 1993] was restriction endonuclease digested with Hind3 and Asp718 and the fragment containing the ITR sequences, synthetic polyadenylation site (and including the pBR322 derived plasmid backbone) was gel isolated.
  • neomycin phosphotransferase gene which had been engineered with a Kozak consensus eukaryotic translation initiation sequence was PCR amplified using primers that gave a Hind3 site at the 5 ' end and an Asp718 site at the 3 ' end ofthe neo sequences.
  • the Hind3/Asp718 digested neo PCR product was ligated to the pTRF46 derived fragment to generate "pAAVneoBR.”
  • the AAV and neo sequences were excised from the pBR322 derived plasmid backbone at the Bgl2 sites flanking the ITRs and subcloned into a Bluescript vector in which the multiple cloning site was substituted with a Bgl2 linker to create "pAAVneo.”
  • a Sail fragment containing the human placental alkaline phosphatase cDNA sequence was isolated from the retroviral vector DAP.
  • a 2 base pair fill-in was performed and the fragment inserted into pAAVneo which had been linearized with BamHl and partially filled-in to accept the insert.
  • the resulting intermediate construct "pAAVneoAP” was digested with Hind3 to remove the neo sequences.
  • the construct was religated to create "pAAVAP.”
  • a 2 base pair fill-in was performed on an Spel to Nhel fragment containing the CMV immediate early promoter sequences [See, e.g., Boshart, 1985] (from nucleotide -583 to +71) and then was inserted into pAAVAP which had been linearized with Hind3 and also partially filled-in.
  • the resulting construct was vector "AAV-CMV-AP.”
  • rAAV vectors In order to investigate the delivery of rAAV vectors to a variety of cell types typically found in blood vessels, the inventors examined the transduction of smooth muscle cells in human primary explant cultures using the AAV-CMV-AP vector.
  • Human smooth muscle cells were derived from a biopsy sample by enzymatic digestion using procedures as described in the art. Primary explant cultures were characterized and confirmed to contain predominantly smooth muscle cells using immunocytochemical staining for alpha-actin, as described in Example 6 below. Cells were seeded at a density of 1x10 cells/ 12 wells such that the cultures were semi-confluent (50% coverage ofthe dish) 24 hours later at the time of exposure to AAV-CMV-AP vector.
  • AAV-CMV-AP vector Cells were exposed to lO ⁇ l of AAV-CMV-AP vector in a total volume of 1 ml complete medium (containing a final concentration of 10% fetal bovine serum) for 24 hours. Smooth muscle cells were transduced with three independent preparations of AAV-CMV-AP vector. The vector preparations were estimated by slot-blot analysis of DNA genomes to contain on the order of 10 particles per ml. Transductions were thus performed with approximately 100 particles per cell. Virus was removed from the cells at the end of the 24 hour exposure period and the cells were cultured in fresh medium for an additional 24 hours.
  • the cells were harvested using trypsin and 2.5 x 10 cells were transferred onto glass slides using a cytospin. The slides were allowed to air dry, and the cells were then fixed in 0.5% glutaraldehyde in PBS. The cells were washed in three changes of PBS and heat treated for 30 minutes at 65 °C to inactivate any endogenous alkaline phosphatase activity.
  • the cells were incubated in 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, in Buffer 3 [100 mM Tris, 100 mM NaCl, 50 mM MgCl2 - pH 9.5]) for 3 hours at room temperature in the dark, which resulted in dark purple/black staining of cells expressing alkaline phosphatase.
  • the slides were then rinsed in PBS and coverslipped using aqueous mounting medium.
  • the percent of cells expressing alkaline phosphatase was determined by counting cells under light microscopy in at least two fields using a eye piece grid. The mean transduction frequency from the three independent infections was approximately 5.37%.
  • AAV-CMV-AP Vector In order to further investigate the delivery of rAAV vectors to a variety of species, the inventors also examined the transduction of smooth muscle cells in rat primary explant cultures using the AAV-CMV-AP vector.
  • Rat smooth muscle cell cultures were derived from Fischer 344 rat aorta by enzymatic digestion and confirmed to contain predominantly smooth muscle cells using immunocytochemical staining for alpha-actin, as described above. [See, e.g. ,
  • Rabbit smooth muscle cells were derived from rabbit aorta by enzymatic digestion, characterized and confirmed to contain predominantly smooth muscle cells using immunocytochemical staining for alpha-actin, as described above. Cells were transduced with a single preparation of AAV-CMV-AP vector at 100 particles per cell, as described in Example 2 (a). Human and rat smooth muscle cells were also transduced for comparison purposes. Rabbit smooth muscle cells were transduced at a frequency of approximately 10.26%, or twice the frequency of human smooth muscle cell (approximately 5.41%) in the same study. Rat smooth muscle cells were again transduced at a frequency two orders of magnitude lower (approximately 0.016%).
  • the inventors also examined the transduction of smooth muscle cells in monkey primary explant cultures using the ACAPSN vector.
  • Smooth muscle cells were derived from a Macaca fascicularis biopsy sample by enzymatic digestion, characterized and confirmed to contain predominantly smooth muscle cells by immunocytochemical staining for alpha-actin, as described above.
  • Cells were transduced with ACAPSN vector as described for human smooth muscle cell transductions in Example 2 (a), except that approximately 1000 particles per cell were used. The mean frequency of the transduction was approximately 13.19%.
  • Example 2 Transduction of Umbilical Vein Endothelial Cells in Human Primary Explant Cultures Using the ACAPSN Vector.
  • Human umbilical vein endothelial cells were derived from a biopsy sample following standard protocols for preparation of primary umbilical vein endothelial cell explant cultures. [See, e.g., Jaffe, 1973] Primary explant cultures were characterized and confirmed to contain predominantly endothelial cells by immunocytochemical staining for von Willebrand's Factor (vWF), as described in Example 6 below. Cells were transduced with the ACAPSN vector as described for human smooth muscle cell transductions in Example 2 (a), except that approximately 1000 particles per cell were used. The mean frequency ofthe transduction was approximately 4.56% .
  • vWF von Willebrand's Factor
  • Human microvascular endothelial cells were derived from omentum (fatty tissue) using enzymatic digestion and sequential plating on fibronectin coated plastic cultureware to remove contaminating fibroblasts as described in the art. Primary explant cultures were characterized and confirmed to contain predominantly endothelial cells by immunocytochemical staining for vWF, as described above.
  • Example 2 fgl Transduction of Microvascular Endothelial Cells in Macaque Primary Explan Cultures Using the ACAPSN Vector
  • the inventors also examined the transduction of microvascular endothelial cells in monkey primary explant cultures using the ACAPSN vector. Macaque microvascular endothelial cells were derived from omentum and characterized and confirmed to contain predominantly endothelial cells using immunocytochemical staining for vWF as described above. Cells were transduced with the ACAPSN vector as described for human smooth muscle cell transductions in Example 2 (a), except that approximately 1000 particles per cell were used. The mean frequency of the transduction was approximately 4.93%.
  • the inventors conducted a series of investigations to explore the delivery of rAAV vectors to cells typically found in blood vessels (e.g., smooth muscle cells, large vessel endothelial cells, microvascular endothelial cells) from a variety of different species. It is important to emphasize that while these preliminary studies were conducted "in vitro," the inventors utilized primary explant cultures, which should be distinguished from immortalized cell cultures. Primary explant cultures, which have a finite lifespan, are believed to be far more representative of cells in vivo than immortalized cell culture lines which have generally been transformed with an oncogene so that they may grow indefinitely.
  • blood vessels e.g., smooth muscle cells, large vessel endothelial cells, microvascular endothelial cells
  • rAAV vectors can transfer and express a heterologous gene in a variety of the cell types typically found in blood vessels from a number of different species, including non-human primates and humans.
  • Example 3 In Vivo Transduction of Microvascular Cells in Primates Using ACAPSN Introduced by Intraluminal Delivery Without Pre-Treatment The methods ofthe present invention are illustrated herein by in vivo studies conducted with non-human primates. In the following examples, the ACAPSN vector was infused into a segment of a peripheral artery in cynomolgus monkeys with established atherosclerosis.
  • transductions using the methods ofthe present invention were evaluated under a variety of conditions by varying the state of the artery being treated and/or the delivery method employed.
  • ACAPSN was delivered by intraluminal infusion to unperturbed femoral arteries.
  • the animals received 5-bromo- 2'-deoxyuridine-5'-monophosphate (BrdU, 30 mg/kg, i.m., in saline [30 mg/ml], Boehringer Mannheim Inc., Indianapolis, IN) one and sixteen hours before surgery, to label proliferating cells for further studies (described in Example 8 below).
  • the monkeys were sedated with ketamine hydrochloride (10-15 mg/kg i.m.), intubated and anesthetized with halothane gas (4% to effect). Blood was drawn for baseline serum, hematology and chemistries.
  • the transduction was conducted using an ACAPSN preparation with a concentration of 3 x 10° DNAse-resistant vector particles per ml (DNAse- esistance beinu characteristic of encapsidated vector particles).
  • a control 250-500 ⁇ l of Lactate Ringer's solution, "LRS" was infused into the right artery. After thirty minutes, ACAPSN vector preparation and LRS control were aspirated from the left and right arteries, respectively. The side branches were then ligated, and blood flow was re- established.
  • the formalin-fixed arterial segments were stained for human placental alkaline phosphatase to localize transduced cells expressing the ACAPSN vector as follows: tissues were washed in three changes of phosphate buffered saline (PBS, 10 ml, 1 hr/wash) and heated in PBS for thirty minutes in a 65°C water bath to inactivate any endogenous alkaline phosphatase activity.
  • PBS phosphate buffered saline
  • Positive controls were included with each AP assay and were either: (1) cytospins of cultured smooth muscle cells transduced with the ACAPSN vector and fixed with 10% formalin; or (2) cultured smooth muscle cells transduced with the ACAPSN vector, pelleted, fixed with 10% formalin, embedded in paraffin and sectioned.
  • Negative controls were included for each animal and were either: (1) sections cut from arteries infused with Lactate Ringer's Solution; or (2) sections cut from arteries remote from the site of ACAPSN treatment.
  • Example 4 In Vivo Transduction of Microvascular Cells in Primates Using ACAPSN Introduced hv Intraluminal Delivery Following Denudation Pre-Treatment In order to determine whether endothelial cells function as a barrier to rAAV gene delivery deeper into the artery vessel wall, the ACAPSN vector was delivered by intraluminal infusion to carotid arteries which had been denuded of endothelium.
  • ACAPSN preparation with a concentration of 3 x 10 9 DNAse-resistant particles per ml.
  • the animals were generally prepped, selected and maintained as described in Example 3, with the following exceptions: (1) the carotid arteries were studied in this Example; and (2) prior to intraluminal delivery ofthe ACAPSN vector and control (to the left and right carotids, respectively), a denudation pre-treatment was given as follows.
  • the carotid arteries were denuded of endothelium using a 3F
  • Fogarty balloon embolectomy catheter V. Mueller Inc., McGaw Park, IL which was passed (3 times) 3 cm into each carotid artery, inflated, retrieved under gentle tension and then removed.
  • Example 3 The subsequent handling and preparation of animals and tissue followed the procedures outlined in Example 3, with the following three exceptions: the treated arteries were removed after 60 hours of quarantine; and (2) animals were then sedated with ketamine (15 mg/kg, i.m.), heparinized (300 units/kg, i.v.), and transported to the necropsy suite where blood was drawn for hematologic and chemistry assessments. After an overdose of sodium pentobarbital (100 mg/kg, i.v.) the animals were exsanguinated while infusing Ringer's solution (approximately 750 cc) at 100 mm Hg. The right and left carotid arteries were perfusion- fixed in 10% formalin and then removed for analysis.
  • ketamine 15 mg/kg, i.m.
  • heparinized 300 units/kg, i.v.
  • the ACAPSN vector was delivered by intraluminal infusion to carotid arteries which had been gently denuded and focally distended.
  • ACAPSN vector preparation was generated according to the procedures of Example 1. The transduction was conducted using an ACAPSN preparation with a concentration of 1 x 10 10 DNAse-resistant particles per ml. The animals were generally prepped, selected and maintained as described in Example 3, with the following exceptions: (1) the carotid arteries were studied in this Example; and (2) prior to intraluminal delivery ofthe ACAPSN vector and control (to the left and right carotids, respectively), the carotid arteries were gently denuded of endothelium and subjected to focal over-distention. This pre-treatment was performed as follows: a 3F Fogarty balloon embolectomy catheter (V.
  • J J adventitial cells that were not clearly identified as endothelial cells of both carotid arteries exposed to rAAV vector, but was not observed in either control artery.
  • the ACAPSN vector was also delivered by injection directly into the adventitia of a carotid artery that had been stimulated several days prior to delivery.
  • the artery was stimulated by repeated intraluminal passage of an inflated balloon catheter five days prior to delivery of ACAPSN, which would be expected to increase the rate of cellular proliferation ofthe vessel wall prior to delivery ofthe vector.
  • the maximum proliferation of cells throughout the artery wall is induced within 4-7 days of balloon injury.
  • An ACAPSN vector preparation was generated according to the procedures of Example 1. The transduction was conducted using an ACAPSN preparation with a concentration of 3 x 10 9 DNAse- resistant particles per ml. The animal was generally prepped, selected, and maintained as described in Example 3, with the following four exceptions: ( 1 ) the carotid arteries were studied in this Example; (2) both carotids received the
  • ACAPSN vector preparation was injected per side, directly into the subadventitial plane, using a 1 cc syringe and 27 gauge needle.
  • Example 4 The subsequent handling and preparation of animals and tissue followed the procedures outlined in Example 4, with the following exceptions: ( 1 ) the treated arteries were removed after 72 hours of quarantine; and (2) immunocytochemical analysis was performed on tissue taken from the right carotid as follows.
  • endothelial cell vWF, DAKO
  • macrophage CD-68, DAKO
  • lymphocyte CD-3, DAKO
  • Primary antibodies were localized with appropriate biotinylated secondary antibodies (Vector Laboratories Inc.) and tertiary avidin-biotin-peroxidase staining (Vector Laboratories Inc.). Sections were counter- stained with hematoxylin and examined by standard light-microscopy. For the right carotid, much ofthe AP activity co-localized with cells staining for either endothelial cell or smooth muscle cell markers. A minority of the AP activity co-localized with cells staining for a macrophage marker.
  • a number of AP positive cells could not be positively identified with these cell type-specific antibodies.
  • a very high frequency of expression was observed in cells identified histologically within adventitial microvessels For this artery, expression was also observed in a significant number of other cells in the adventitia at the region of injection; as described above, these were not positively identified with cell-type specific antibodies and could possibly be, for example, fibroblasts, lymphocytes, other leukocytes, and nerve cells
  • a small number of AP positive cells was observed, and these few cells were predominantly associated with structures histologically consistent with microvessels
  • adventitial microvessels are believed to be supplied with blood from the lumen of the parent vessel (e.g., the artery) via small microvessels traversing the vessel media.
  • This study was designed to demonstrate that introduction of an rAAV vector into the adventitia can also be achieved indirectly, by injection directly into the lumen of an artery that had been pre-treated by prior balloon injury.
  • the artery was stimulated by repeated intraluminal passage of a large inflated balloon catheter five days prior to delivery of ACAPSN, which would be expected to increase the rate of cellular proliferation of the vessel wall prior to delivery of the vector.
  • An ACAPSN vector preparation was generated according to the procedures of Example 1. The transduction was conducted using an ACAPSN preparation with a concentration of 1 x 10 10 DNAse-resistant particles per ml. The animal was generally prepped, selected and maintained as described in Example 6, with the following exception: delivery occurred by intraluminal delivery as described in Example 3. Thus, in this Example, (1) both carotids received
  • the right carotid artery was subjected to balloon injury by three intraluminal passages of an inflated 3F Fogarty balloon catheter (V. Mueller Inc., McGaw Park, IL) under moderate tension.
  • the left carotid was previously unmanipulated..
  • the subsequent handling and preparation of animals and tissue followed the procedures outlined in Example 4, with the exception that the treated arteries were removed after 72 hours of quarantine.
  • the ability to successfully transduce cells in blood vessels in an atherosclerotic macaque model using the methods ofthe present invention indicates that transduction of atherosclerotic vessels can be achieved and that even higher levels of transduction may be achieved in non-atherosclerotic vessels in which access conditions are less stringent.
  • the BrdU data may provide further confirmation that rAAV uptake and expression is associated with cells that are proliferating, such as those associated with angiogenesis.
  • angiogenesis models such as ocular microvascular cells or egg-yolk sac models can be used to quantify similar results in primary tissue in primary cells.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Les techniques actuelles visant à exprimer les gènes recombinants dans les cellules de vaisseaux sanguins après le transfert direct de gènes in vivo, se heurtent à des problèmes ou limitations connexes. En outre, aucun procédé efficace n'a été trouvé qui permette d'effectuer la transduction de cellules microvasculaires et/ou de cellules jouant un rôle dans la formation de nouveaux vaisseaux sanguins (angiogenèse). Cette invention concerne des procédés permettant d'effectuer la transduction de cellules dans des vaisseaux sanguins à l'aide de vecteurs de virus adéno-associés (VAA) recombinants.
EP97907934A 1996-03-04 1997-02-28 Procede de transduction de cellules dans des vaisseaux sanguins a l'aide de vecteurs de virus adeno-associes (vaa) recombinants Withdrawn EP0906440A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US61066096A 1996-03-04 1996-03-04
US610660 1996-03-04
PCT/US1997/003134 WO1997032990A1 (fr) 1996-03-04 1997-02-28 Procede de transduction de cellules dans des vaisseaux sanguins a l'aide de vecteurs de virus adeno-associes (vaa) recombinants

Publications (1)

Publication Number Publication Date
EP0906440A1 true EP0906440A1 (fr) 1999-04-07

Family

ID=24445931

Family Applications (1)

Application Number Title Priority Date Filing Date
EP97907934A Withdrawn EP0906440A1 (fr) 1996-03-04 1997-02-28 Procede de transduction de cellules dans des vaisseaux sanguins a l'aide de vecteurs de virus adeno-associes (vaa) recombinants

Country Status (5)

Country Link
EP (1) EP0906440A1 (fr)
JP (1) JP2002514899A (fr)
AU (1) AU1980697A (fr)
CA (1) CA2247099A1 (fr)
WO (1) WO1997032990A1 (fr)

Families Citing this family (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6924128B2 (en) 1994-12-06 2005-08-02 Targeted Genetics Corporation Packaging cell lines for generation of high titers of recombinant AAV vectors
US5652224A (en) 1995-02-24 1997-07-29 The Trustees Of The University Of Pennsylvania Methods and compositions for gene therapy for the treatment of defects in lipoprotein metabolism
EP0871512A1 (fr) * 1995-12-22 1998-10-21 Localmed, Inc. Administration intravasculaire localisee de facteurs de croissance pour la stimulation de l'angiogenese
EP0888086B1 (fr) 1996-02-15 2005-07-27 Biosense Webster, Inc. Sonde d'excavation
US6989264B2 (en) 1997-09-05 2006-01-24 Targeted Genetics Corporation Methods for generating high titer helper-free preparations of released recombinant AAV vectors
US6566118B1 (en) 1997-09-05 2003-05-20 Targeted Genetics Corporation Methods for generating high titer helper-free preparations of released recombinant AAV vectors
US6642051B1 (en) 1997-10-21 2003-11-04 Targeted Genetics Corporation Amplifiable adeno-associated virus(AAV) packaging cassettes for the production of recombinant AAV vectors
JP2001520051A (ja) * 1997-10-21 2001-10-30 ターゲテッド ジェネティックス コーポレイション 組換えａａｖベクターの産生のための増幅可能アデノ随伴ウイルス（ａａｖ）パッケージングカセット
US6346415B1 (en) 1997-10-21 2002-02-12 Targeted Genetics Corporation Transcriptionally-activated AAV inverted terminal repeats (ITRS) for use with recombinant AAV vectors
EP1563866B1 (fr) 1998-02-05 2007-10-03 Biosense Webster, Inc. Appareil pour l'administration de medicament intracardiaque
US7078387B1 (en) 1998-12-28 2006-07-18 Arch Development Corp. Efficient and stable in vivo gene transfer to cardiomyocytes using recombinant adeno-associated virus vectors
US6893865B1 (en) 1999-04-28 2005-05-17 Targeted Genetics Corporation Methods, compositions, and cells for encapsidating recombinant vectors in AAV particles
CA2374597A1 (fr) 1999-05-28 2000-12-07 Targeted Genetics Corporation Techniques et compositions permettant d'abaisser le niveau de facteur de necrose tumorale (tnf) dans les troubles associes au tnf
AU6893100A (en) * 1999-08-02 2001-02-19 Avigen, Inc. Use of recombinant adeno-associated virus vector (raav) for the prevention of smooth muscle cell proliferation in vascular graft
JP4827353B2 (ja) 1999-08-09 2011-11-30 ターゲティッド ジェネティクス コーポレイション 鎖内塩基対を形成するような配列の設計による、組換えウイルスベクターからの一本鎖の異種ヌクレオチド配列の発現の増大
EP1916258B1 (fr) 1999-08-09 2014-04-23 Targeted Genetics Corporation Améliorations de l'expression d'une séquence de nucléotides hétérologues à brin unique à partir de vecteurs viraux recombinants par la désignation de la séquence de manière à ce qu'elle forme des paires de bases intrabrins
EP1870473A1 (fr) * 2000-08-17 2007-12-26 Keiya Ozawa Libération de facteurs angiogéniques à médiation virale et adéno associée
AU2001294096A1 (en) * 2000-08-17 2002-02-25 Uichi Ikeda Adeno-associated virus-mediated delivery of angiogenic factors
HUE057606T2 (hu) 2009-05-02 2022-05-28 Genzyme Corp Génterápia neurodegeneratív rendellenességekre
US20140155469A1 (en) 2011-04-19 2014-06-05 The Research Foundation Of State University Of New York Adeno-associated-virus rep sequences, vectors and viruses

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5658565A (en) * 1994-06-24 1997-08-19 University Of Pittsburgh Of The Commonwealth System Of Higher Education Inducible nitric oxide synthase gene for treatment of disease
CA2176117C (fr) * 1993-11-09 2006-01-03 Terence R. Flotte Production de titres eleves de vecteurs d'aav recombinants
EP0750503A4 (fr) * 1994-03-07 1999-07-21 Immusol Inc Therapie a base de ribozymes pour traiter la restenose
US6162796A (en) * 1995-09-27 2000-12-19 The Rockefeller University Method for transferring genes to the heart using AAV vectors

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9732990A1 *

Also Published As

Publication number Publication date
JP2002514899A (ja) 2002-05-21
WO1997032990A1 (fr) 1997-09-12
AU1980697A (en) 1997-09-22
CA2247099A1 (fr) 1997-09-12

Similar Documents

Publication Publication Date Title
EP0906440A1 (fr) Procede de transduction de cellules dans des vaisseaux sanguins a l'aide de vecteurs de virus adeno-associes (vaa) recombinants
US5693531A (en) Vector systems for the generation of adeno-associated virus particles
US7078387B1 (en) Efficient and stable in vivo gene transfer to cardiomyocytes using recombinant adeno-associated virus vectors
Ponnazhagan et al. Adeno-associated virus type 2-mediated transduction of murine hematopoietic cells with long-term repopulating ability and sustained expression of a human globin gene in vivo
US5780447A (en) Recombinant adeno-associated viral vectors
US20060292117A1 (en) Improved rAAv vectors
EP0844887B1 (fr) Transduction de myoblastes par vecteurs de virus adeno-associes
Raper et al. Selective gene transfer into the liver of non-human primates with E1-deleted, E2A-defective, or E1-E4 deleted recombinant adenoviruses
US20020001581A1 (en) Methods for transducing cells in blood vessels using recombinant AAV vectors
Eslami et al. Gene delivery to in situ veins: differential effects of adenovirus and adeno-associated viral vectors
CA2274146C (fr) Modification genetique de cellules souches de repopulation hematopoietiques de primates
Conte et al. Genetic interventions for vein bypass graft disease: a review
US20020159978A1 (en) Muscle-directed gene transfer by use of recombinant AAV-1 and AAV-6 virions
CN102712920A (zh) 缺氧调节的条件沉默性aav表达血管生成诱导因子
EP1027451A1 (fr) Systeme de vecteurs recombinants bases sur des parvovirus chimeres et specifiquement diriges sur le lignage erythrocitaire
AU763049B2 (en) Efficient and stable (in vivo) gene transfer to cardiomyocytes using recombinantadeno-associated virus vectors
US20100028312A1 (en) Stably transformed bone marrow-derived cells and uses thereof
AU2646901A (en) Methods for transducing cells in blood vessels using recombinant AAV vectors
WO1999064569A1 (fr) Procedes et compositions servant a generer des vecteurs de virus adeno-associes de recombinaison
WO2000056368A1 (fr) Therapeutique genique
CN110760542A (zh) 一种共表达znf580和vegf165双基因的质粒及应用
Raper et al. Ex vivo hepatocyte-directed gene therapy
WO2003024384A2 (fr) Procedes ameliores de traitement par vecteurs viraux
Lin et al. Disease-Inducible Transgene Expression
Miyake et al. 900. Development of models for RPS19 deficient diamond-blackfan anemia using regulatable expression of siRNA against RPS19

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19980831

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

17Q First examination report despatched

Effective date: 20030902

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20040113