WO2006116678A2 - Ciblage tissulaire de cellules souches - Google Patents

Ciblage tissulaire de cellules souches Download PDF

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Publication number
WO2006116678A2
WO2006116678A2 PCT/US2006/016235 US2006016235W WO2006116678A2 WO 2006116678 A2 WO2006116678 A2 WO 2006116678A2 US 2006016235 W US2006016235 W US 2006016235W WO 2006116678 A2 WO2006116678 A2 WO 2006116678A2
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cells
cell
hematopoietic
gene
stem
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PCT/US2006/016235
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WO2006116678A3 (fr
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Maria Grant
Sean Sullivan
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University Of Florida Research Foundation, Inc.
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Publication of WO2006116678A3 publication Critical patent/WO2006116678A3/fr

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    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0621—Eye cells, e.g. cornea, iris pigmented cells
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • Cellular replacement and tissue regeneration comprises isolated stem cells expressing tissue specific proteins.
  • Multicellular animals are derived from a clone of cells descended from a single original cell, the fertilized egg.
  • Embryogenesis involves the division and differentiation of multipotential cells, each cell having the ability to develop into multiple cellular lineages.
  • the cells of such lineages can vary substantially, such as blood cells, muscle cells and neural cells, being specialized.
  • a wide spectrum of diseases may be treated based upon both the possession of a population of cells having multi- lineage potential and an understanding of the mechanisms that regulate embryonic cell development.
  • the capacity to generate a new population of hematopoietic cells is the basis of bone marrow transplantation, which is currently used as a treatment for a growing number of diseases including anemia, leukemia and breast cancer.
  • transplantation of genetically altered multipotential cells has been considered as potential therapy for a variety of different diseases including AIDS.
  • Mammalian hematopoietic (blood) cells provide a diverse range of physiologic activities. Hematopoietic cells are divided into lymphoid, myeloid and erytliroid lineages.
  • the lymphoid lineage comprising B, T and natural killer (NK) cells, provides for the production of antibodies, regulation of the cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, and the like.
  • the myeloid lineage which includes monocytes, granulocytes, megakaryocytes, as well as cells, monitors for the presence of foreign bodies, provides protection against neoplastic cells, scavenges foreign materials, produces platelets, and the like.
  • the erythroid lineage provides the red blood cells, which act as oxygen carriers.
  • stem cells are capable of self-regeneration but may also divide into progenitor cells that are no longer pluripotent and have a limited self-regeneration. These progenitor cells divide repeatedly to form more mature cells which eventually become terminally differentiated to form the various mature hematopoietic cells. Thus the large number of mature hematopoietic cells is derived from a small reservoir of stem cells by a process of proliferation and differentiation.
  • progenitor cells mature into bipotential cells and then become lineage committed, that is, are incapable of maturing into more than one lineage.
  • progenitor or progenitor cells indicates cell populations which are no longer stem cells but which have not yet become terminally differentiated.
  • lymphoid, myeloid or erythroid in conjunction with progenitor indicates the potential cell populations into which the progenitor is capable of maturing.
  • Stem cells are homed to a specific tissue using specific tissue genes.
  • a particular advantage is using stem cells, which are assimilated by the target tissue or organ of interest and, due to the in vivo target micro environment, develop into the cell type of the desired target.
  • the therapeutic advantages are many and include the repair of tissues caused by a variety of factors, organs, cell linings, replacement of necrotic tissues and the like. The method is important especially in the areas of chemotherapy (creating drug resistant progenitor cells to repopulate after chemotherapy), cancers (mesenchymal stem cells have been shown to home to tumors.
  • an isolated stem cell comprises a vector expressing at least one homing gene.
  • the vector can be a non-integrating vector or can be an integrating vector.
  • the homing gene is any one of SCG 10; Na Channel II; glut-2, synapsin, epo, SCF , slili, wint, BMPs Ephrins, Pax-6, Emx-2, Mash -1, jagged 1 and 2, notch- and 2, ephrin B2 and ephrin B4, Bmi-1, and HOXB4.
  • the non-integrating vector transiently transfects the stem cells with at least one of homing gene.
  • the vector comprises plasmid DNA or messenger RNA.
  • expression of a homing gene comprises activating a stem cell endogenous gene.
  • the activating expression of the endogenous gene within a stem cell genome comprises a small molecule derived from a chemical library and screened for specific activation of the desired gene.
  • activating expression of the endogenous gene within a stem cell genome comprises administering a recombinant protein or peptide that transcriptionally activates the expression of the homing protein.
  • a transcriptional activator comprises a zinc finger-transcription activator fusion protein or a specific transcription activator protein that binds to the enhancer or promoter region controlling expression of the homing protein.
  • the vector is episomal or integrated into a stem cell genome.
  • Migration of stem cells to target tissues is enhanced by further genetic modification, e.g., introduction of an exogenous nucleic acid encoding a homing molecule into the cells.
  • homing molecules include chemoldne receptors, interleukin receptors, estrogen receptors, and integrin receptors.
  • the cells optionally contain an exogenous nucleic acid encoding a gene product, which increases endocrine action of the cell, e.g., a gene encoding a hormone, or a paracrine action of the cell.
  • stem cells are genetically modified to contain an exogenous nucleic acid encoding a bone morphogenetic factor and engrafted into bone, cartilage, or tooth tissue, e.g., to treat periodontitis.
  • the cells optionally also include nucleic acids encoding other biologically active or therapeutic proteins or polypeptides, e.g., angiogenic factors, extracellular matrix proteins, cytokines or growth factors.
  • cells to be engrafted into pancreatic tissue contain a nucleic acid(s) encoding insulin or insulin precursor molecules.
  • the cells also optionally include nucleic acids encoding gene products that decrease transplant rejection, e.g., CTLA4Ig CD40 ligand, or decrease development of transplant arteriosclerosis, e.g., inducible nitric oxide synthase (iNOS).
  • iNOS inducible nitric oxide synthase
  • Stem cells to be transplanted are obtained from bone marrow tissue of an adult subject, genetically modified ex vivo, and then engrafted into the same or different recipient.
  • the donor and recipient are of the same species; more preferably, the donor and recipient are genetically similar (or the same) at major histocompatibility loci.
  • the nucleic acid compositions are formulated in a vector.
  • Vectors include for example, an adeno-associated virus vector, a lentivirus vector and a retrovirus vector.
  • the vector is an adeno-associated virus vector.
  • the nucleic acid is operatively linked to a promoter such as a human cytomegalovirus immediate early promoter.
  • an expression control element such as a bovine growth hormone polyadenylation signal is operably linked to coding region the cell protective polypeptide.
  • the nucleic acid of the invention is flanked by the adeno-associated viral inverted terminal repeats encoding the required replication and packaging signals. Nucleic acid compositions are inserted into a stem cell through any suitable method known in the art.
  • Treatment is based, for example, the following sequence: harvest of stem cell- containing tissue, isolation and/or expansion of stem cells, transfection of stem cells with at least one terminal differentiation and/or homing gene, implantation of at least one recombinant stem cell into the patient, and in situ differentiation of the stem cells in the targeted tissue.
  • This approach differs from traditional tissue engineering in that undifferentiated stem cells are implanted and allowed to differentiate into their final form in the targeted tissue.
  • the stem cells can be administered in any part of the body, for example, by injection.
  • Biological, bioelectrical and/or biomechanical triggers from the host environment may be sufficient, or under certain circumstances, may be augmented as part of the therapeutic regimen to establish a fully integrated and functional tissue.
  • the stem cells are administered as a cell suspension in a pharmaceutically acceptable medium for injection.
  • Injection can be local, i.e. directly into the damaged portion of the retinal pigment epithelium, or systemic, i.e., injected into the peripheral circulatory system. Systemic administration is preferred.
  • RPE retinal pigment epithelium
  • homing elaboration of a composition from the injured tissue, e.g., injured retinal pigment epithelium (RPE), that recruits cells from the bone marrow or the circulation.
  • adhesion is meant binding of one cell to another or binding of a cell to an extracellular matrix. Adhesion encompasses movement of cells, e.g., rolling, in blood vessels.
  • Adhesion molecules are a diverse family of extracellular (e.g., laminin) and cell surface (e.g., NCAM) glycoproteins involved in cell-cell and cell-extracellular matrix adhesion, recognition, activation, and migration.
  • Cell engrafitment refers to the process by which cells, e.g., stem cells, become incorporated into a differentiated tissue and become part of that tissue.
  • stem cells bind to retinal tissue, differentiate into functional retinal pigment epithelial (RPE) cells, and become resident in the retinal epithelium.
  • RPE retinal pigment epithelial
  • the method is carried out by increasing the amount of a polypeptide on the surface of the cell such as a stem cell.
  • the method increases the number of stem cells in an area of injured tissue compared to the number of stem cells in the area in the absence of an exogenous stem cell-associated polypeptide or nucleic acid encoding such a polypeptide.
  • the cell is a stem cell such as a bone marrow-derived stem cell.
  • the amount of receptor on the surface of the cell is increase by contacting the cell with the protein or introducing into the cell a nucleic acid encoding said receptor under conditions that permit transcription and translation of the gene.
  • the gene product is expressed on the surface of the stem cell.
  • the stem cell receptor binds to a ligand that is expressed in injured tissue such as retinal pigment epithelium.
  • a method of enhancing migration, homing, adhesion, or engraftment of a cell such as a stem cell to an injured tissue is carried out by increasing the amount of an injury- associated polypeptide, e.g., a cytokine or adhesion protein, in the injured tissue.
  • T he method increases the number of stem cells in an area of injured tissue compared to the number of stem cells in the area in the absence of an exogenous injury-associated polypeptide or nucleic acid encoding such a polypeptide.
  • Identification of injury-associated polypeptides e.g., growth factors, activate endogenous mechanisms of repair in the tissues of interest such as proliferation and differentiation of progenitor cells.
  • the injured tissue is contacted with a nucleic acid encoding target protein or the protein itself, such as a cytokine or adhesion protein.
  • a nucleic acid encoding target protein or the protein itself such as a cytokine or adhesion protein.
  • the target protein or a nucleic acid encoding the protein or is directly injected into the retinal pigment epithelium.
  • cells such as fibroblast cells expressing exogenous nucleic acid molecules encoding the target proteins are introduced to the site of injury.
  • FIG. 1 is an electroretinogram showing restoration of retina function by engraftment of RPE65 infected endothelial stem cells.
  • the electroretinograms are the average retinal response following 5 flashes of dim light separated by 30 second intervals and measuring the response time.
  • the results show that the mice receiving the endothelial stem cells infected with the PRE65 lentivirus responded to the dim flash much better than the control treatment groups.
  • Stem cell compositions comprise a gene unique to the desired engrafted tissue which is introduced into the stem cell either ex vivo or in vivo. Expression of the unique gene results in the differentiation of the stem cell into the desired cell type.
  • a "pharmaceutically acceptable” component is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.
  • DNA construct and "vector” are used herein to mean a purified or isolated polynucleotide that has been artificially designed and which comprises at least two nucleotide sequences that are not found as contiguous nucleotide sequences in their natural environment.
  • plasmid refers to any nucleic acid encoding an expressible gene and includes linear or circular nucleic acids and double or single stranded nucleic acids.
  • the nucleic acid can be DNA or RNA and may comprise modified nucleotides or ribonucleotides, and may be chemically modified by such means as methylation or the inclusion of protecting groups or cap- or tail structures.
  • oligo-nucleotides include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form.
  • nucleotide as used herein as an adjective to describe molecules comprising RNA, DNA, or RNA/DNA hybrid sequences of any length in single-stranded or duplex form.
  • nucleotide is also used herein as a noun to refer to individual nucleotides or varieties of nucleotides, meaning a molecule, or individual unit in a larger nucleic acid molecule, comprising a purine or pyrimidine, a ribose or deoxyribose sugar moiety, and a phosphate group, or phosphodiester linkage in the case of nucleotides within an oligonucleotide or polynucleotide.
  • nucleotide is also used herein to encompass "modified nucleotides" which comprise at least one modifications (a) an alternative linking group, (b) an analogous form of purine, (c) an analogous form of pyrimidine, or (d) an analogous sugar, all as described herein.
  • modified nucleotides which comprise at least one modifications (a) an alternative linking group, (b) an analogous form of purine, (c) an analogous form of pyrimidine, or (d) an analogous sugar, all as described herein.
  • the phrase “having a length of N bases” or “having a length of N nucleotides” is used herein to describe lengths along a single nucleotide strand, of a nucleic acid molecule, consisting of N individual nucleotides.
  • binding refers to an interaction between the bases of an oligonucleotide which is mediated through base-base hydrogen bonding.
  • One type of binding is "Watson-Crick-type” binding interactions in which adenine-thymine (or adenine-uracil) and guanine-cytosine base-pairs are formed through hydrogen bonding between the bases.
  • An example of this type of binding is the binding traditionally associated with the DNA double helix.
  • oligonucleotide refers to a polynucleotide fo ⁇ ned from naturally occurring bases and pentofuranosyl groups joined by native phosphodiester bonds. This term effectively refers to naturally occurring species or synthetic species formed from naturally occurring subunits or their close homologs.
  • oligonucleotide may also refer to moieties which function similarly to naturally occurring oligonucleotides but which have non-naturally occurring portions. Thus, oligonucleotides may have altered sugar moieties or intersugar linkages. Exemplary among these are the phosphorothioate and other sulfur-containing species which are known for use in the art.
  • the phosphodiester bonds of the oligonucleotide have been substituted with a structure which functions to enhance the ability of the compositions to penetrate into the region of cells where the RNA or DNA whose activity to be modulated is located. It is preferred that such substitutions comprise phosphorothioate bonds, methyl phosphonate bonds, or short chain alkyl or cycloalkyl structures.
  • the phosphodiester bonds are substituted with other structures which are, at once, substantially non-ionic and non-chiral, or with structures which are chiral and enantiomerically specific. Persons of ordinary skill in the art will be able to select other linkages for use in practice of the invention.
  • Oligonucleotides may also include species which include at least some modified base forms. Thus, purines and pyrimidines other than those normally found in nature may be so employed. Similarly, modifications on the pentofuranosyl portion of the nucleotide subunits may also be effected, as long as the essential tenets of this invention are adhered to. Examples of such modifications are 2'-O-alkyl- and 2'-halogen-substituted nucleotides.
  • T position of sugar moieties which are useful in the present invention are OH, SH, SCH 3 , F, OCH 3 , OCN, 0(CH 2 ) n NH 2 or 0(CH 2 ) n CH 3 where n is from 1 to about 10, and other substituents having similar properties.
  • administering a molecule to a cell refers to transducing, transfecting, microinjecting, electroporating, or shooting, the cell with the molecule, hi some aspects, molecules are introduced into a target cell by contacting the target cell with a delivery cell (e.g., by cell fusion or by lysing the delivery cell when it is in proximity to the target cell).
  • a delivery cell e.g., by cell fusion or by lysing the delivery cell when it is in proximity to the target cell.
  • Transforming DNA may or may not be integrated (covalently linked) with chromosomal DNA making up the genome of the cell.
  • the transforming DNA may be maintained on an episomal element, such as a plasmid.
  • a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA.
  • a "clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations (e.g., at least about 10).
  • safe and effective amount refers to the quantity of a component which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention.
  • therapeutically effective amount is meant an amount of a compound of the present invention effective to yield the desired therapeutic response.
  • the specific safe and effective amount or therapeutically effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal or animal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives.
  • a "pharmaceutical salt” include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids.
  • the salts are made using an organic or inorganic acid.
  • These preferred acid salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, and the like.
  • the most preferred salt is the hydrochloride salt.
  • Diagnostic or “diagnosed” means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity.
  • the "sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives”). Diseased individuals not detected by the assay are “false negatives.” Subjects who are not diseased and who test negative in the assay, are termed “true negatives.”
  • the "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive” rate is defined as the proportion of those without the disease who test positive.
  • patient or "individual” are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred.
  • the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates.
  • sample is used herein in its broadest sense.
  • a sample comprising polynucleotides, polypeptides, peptides, antibodies and the like may comprise a bodily fluid; a soluble fraction of a cell preparation, or media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA, polypeptides, or peptides in solution or bound to a substrate; a cell; a tissue; a tissue print; a fingerprint, skin or hair; and the like.
  • Treatment is an intervention performed with the intention of preventing the development or altering the pathology or symptoms of a disorder. Accordingly, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.
  • ameliorated refers to a symptom which is approaches a normalized value (for example a value obtained in a healthy patient or individual), e.g., is less than 50% different from a normalized value, preferably is less than about 25% different from a normalized value, more preferably, is less than 10% different from a normalized value, and still more preferably, is not significantly different from a normalized value as determined using routine statistical tests.
  • a normalized value for example a value obtained in a healthy patient or individual
  • allogeneic is used to refer to immune cells derived from non- self major histocompatibility complex donors. HLA haplotypes/allotypes vary from individual to individual and it is often helpful to determine the individual's HLA type. The HLA type may be determined via standard typing procedures.
  • host compatible or “autologous” cells means cells that are of the same or similar haplotype as that of the subject or "host” to which the cells are administered, such that no significant immune response against these cells occurs when they are transplanted into a host.
  • the term "pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives.
  • stabilizers and adjuvants see Martin Remington's Pharm. Sd., 15th Ed. (Mack Publ. Co., Easton (1975)).
  • the population of stem cells is purified.
  • a purified population of stem cells contains a significantly higher proportion of stem cells than the crude population of cells from which the stem cells are isolated.
  • the purification procedure should lead at least to a five fold increase, preferably at least a ten fold increase, more preferably at least a fifteen fold increase, most preferably at least a twenty fold increase, and optimally at least a twenty-five fold increase in stem cells with respect to the total population.
  • the purified population of stem cells should include at least 15%, preferably at least 20%, more preferably at least 25%, most preferably at least 35%, and optimally at least
  • Monoclonal antibodies are particularly useful for identifying markers associated with particular cell lineages and/or stages of differentiation.
  • a large proportion of terminally differentiated cells may be removed by initially using a "relatively crude” separation.
  • magnetic bead separations may be used initially to remove large numbers of lineage committed cells. Desirably, at least about 80%, usually at least 70% of the total hematopoietic cells will be removed.
  • Procedures for separation may include but are not limited to, magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic" agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, including but not limited to, complement and cytotoxins, and "panning" with antibody attached to a solid matrix, e.g., plate, elutriation or any other convenient technique.
  • Techniques providing accurate separation include but are not limited to, flow cytometry, which can have varying degrees of sophistication, e.g., a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc.
  • hematopoietic stem cells In adults, the large majority of pluripotent hematopoietic stem cells are found in the bone marrow. However, small but significant numbers of such cells can be found in the peripheral circulation, liver, spleen and cord blood. Human hematopoietic stem cells for use in the present invention may be derived from human bone marrow, human newborn cord blood, fetal liver or adult human peripheral blood, after appropriate mobilization. [0055] The frequency of hematopoietic stem cells can be dramatically increased by treatment of a subject with certain compounds including cytokines.
  • Such "mobilized" peripheral blood hematopoietic stem cells have become an important alternative to bone marrow-derived hematopoietic stem cells transplantation procedures primarily because engraftment is more rapid. (See, e.g., Tanaka, J, et al, bit JHematol 69(2):70-4, 1999.)
  • Such mobilization may be accomplished using for example, one or more of granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), thrombopoietin (Tpo), and a chemo therapeutic agent (i.e., cyclophosphamide), or a small molecule, such as AMD 3100, a CXCR4 agonist.
  • hematopoietic stem cell enrichment/isolation generally include obtaining bone marrow, newborn cord blood, fetal liver or adult human peripheral blood which contains hematopoietic stem cells. Once obtained, a hematopoietic stem cell population may be enriched by performing various separation techniques such as density gradient separation, immunoaffinity purification using positive and/or negative selection by panning, FACS or magnetic bead separation. Following such enrichment steps, the cell population is further characterized phenotypically and functionally.
  • Hematopoietic stem cells are initially characterized by immunophenotype, e.g., as lineage negative and either (1) CD34 + /Thyl + or (2) CD 34 +/ CD38 " cells that are also KDR + .
  • Human HSC may also be characterized by telomere length, where cells with high proliferative capacity have longer telomeres.
  • a population of cells is considered to be enriched for human HSC if greater than 0.1% of the CD 34 + cells have the immunophenotype, CD 34 + CD38 " KDR + or CD34+ Thyl.
  • Preferred cytokines for the culture of human hematopoietic stem cells include one or more of interleukin-3 (IL-3), interleuldn-6 (IL-6), interleukin- 11 (IL-I l), interleukin-12 • (IL-12) stem cell factor (SCF), fms-like tyrosine kinase-3 (flt-3), transforming growth factor- .beta. (TGF- .beta.), an early acting hematopoietic factor, described, for example in WO 91/05795, and thrombopoietin (Tpo).
  • IL-3 interleukin-3
  • IL-6 interleuldn-6
  • IL-I l interleukin-12 •
  • flt-3 stem cell factor
  • TGF- .beta. transforming growth factor- .beta.
  • Tpo thrombopoietin
  • Human adult hematopoietic stem cells are mostly quiescent or slow cycling. However, it has been demonstrated that when human stem cells are cultured under conditions which include exogenously provided cytokines, wherein TGF- ⁇ l is blocked; quiescent, hematopoietic multipotent progenitors grow in a short term culture assay in which the cells do not grow when TGF- ⁇ l is not blocked.
  • Molecules used for isolating populations of stem cells are advantageously conjugated with labels that expedite identification and separation.
  • Stem cell specific molecules include, but not limited to: CXCR4, CD 133, SCA-I 5 Tra-1-60, CD 44, CD 73, CD 90, CD 105 and Stro-1.
  • labels include magnetic beads; biotin, which may be identified or separated by means of its affinity to avidin or streptavidin; fluorocliromes, which may be identified or separated by means of a fluorescence-activated cell sorter (FACS, see below), and the like. Any technique may be used for isolation as long as the technique does not unduly harm the stem cells. Many such methods are known in the art.
  • the binding molecule is attached to a solid support.
  • suitable solid supports include nitrocellulose, agarose beads, polystyrene beads, hollow fiber membranes, magnetic beads, and plastic Petri dishes.
  • the binding molecule can be covalently linked to Pharmacia Sepharose 6 MB macro beads. The exact conditions and duration of incubation for the solid phase-linked binding molecules with the crude cell mixture will depend upon several factors specific to the system employed, as is well known in the art.
  • Cells that are bound to the binding molecule are removed from the cell suspension by physically separating the solid support from the remaining cell suspension.
  • the unbound cells may be eluted or washed away with physiologic buffer after allowing sufficient time for the solid support to bind the stem cells.
  • the bound cells are separated from the solid phase by any appropriate method, depending mainly upon the nature of the solid phase and the binding molecule.
  • bound cells can be eluted by enzymatically "nicking” or digesting an enzyme-sensitive "spacer" sequence between the solid phase and an antibody (e.g., antibodies directed to: CXCR4, CD 133, SCA-I 5 Tra-1-60, CD 44, CD 73, CD 90, CD 105 and Stro-1).
  • Suitable spacer sequences bound to agarose beads are commercially available from, for example, Pharmacia.
  • the eluted, enriched fraction of cells may then be washed with a buffer by centrifugation and preserved in a viable state at low temperatures for later use according to conventional technology.
  • the cells may also be used immediately, for example by being infused intravenously into a recipient.
  • unwanted cells in a starting cell population are labeled by an antibody, or by a cocktail of antibodies, to a cell surface protein characteristic of Lin + cells.
  • the unwanted antibody-labeled cells are removed by methods known in the art.
  • the labeled cells can be immobilized on a column that binds to the antibodies and captures the cells.
  • the antibody that binds the cell surface proteins can be linked to magnetic colloids for capture of unwanted cells on a column surrounded by a magnetic field. This system is currently available through StemCell Technologies Inc., Vancouver, British Columbia, Canada.
  • the remaining cells that flow through the column for collection are enriched in cells that do not express the cell surface proteins that the tetrameric antibodies were directed against.
  • the antibody cocktail that can be used to deplete unwanted Lin cells can be custom made to include antibodies against lineage specific markers, such as, for example, CD2, CD3, CD4, CD5, CD8, CDlO, CDl Ib, CD13, CD14, CD15, CD16, CD19, CD20, CD24, CD25, CD28, CD29, CD33, CD36, CD38, CD41, CD56, CD66b, CD66e, CD69, and glycophorin A.
  • the desired cells that lack these markers are not lineage committed, i.e. Lin " .
  • a labeled binding molecule is bound to the stem cells, and the labeled cells are separated by a mechanical cell sorter that detects the presence of the label.
  • the preferred mechanical cell sorter is a fluorescence activated cell sorter (FACS).
  • FACS machines are commercially available. Generally, the following FACS protocol is suitable for this procedure: a Coulter Epics Eliter sorter is sterilized by running 70% ethanol through the systems. The lines are flushed with sterile distilled water. Cells are incubated with a primary antibody diluted in Hank's balanced salt solution supplemented with 1% bovine serum albumin (HB) for 60 minutes on ice.
  • FACS fluorescence activated cell sorter
  • the cells are washed with HB and incubated with a secondary antibody labeled with fluorescein isothiocyanate (FITC) for 30 minutes on ice.
  • the secondary label binds to the primary antibody.
  • the sorting parameters such as baseline fluorescence, are determined with an irrelevant primary antibody.
  • the final cell concentration is usually set at one million cells per ml.
  • a sort matrix is determined using fluorescent beads as a means of aligning the instrument. Once the appropriate parameters are determined, the cells are sorted and collected in sterile tubes containing medium supplemented with fetal bovine serum and antibiotics, usually penicillin, streptomycin and/or gentamicin. After sorting, the cells are re-analyzed on the FACS to determine the purity of the sort.
  • a precursor cell population includes cells of a mesodermal derived cellular lineage, more particularly of hematopoietic lineage, endothelial lineage, muscle cell lineage, epithelial cell lineage and neural cell lineage.
  • a "precursor cell” can be any cell in a cell differentiation pathway that is capable of differentiating into a more mature cell. As such, the term “precursor cell population” refers to a group of cells capable of developing into a more mature cell.
  • a precursor cell population can comprise cells that are totipotent, cells that are pluripotent and cells that are stem cell lineage restricted (i.e. cells capable of developing into less than all hematopoietic lineages, or into, for example, only cells of erythroid lineage).
  • stem cell lineage restricted i.e. cells capable of developing into less than all hematopoietic lineages, or into, for example, only cells of erythroid lineage.
  • totipotent cell refers to a cell capable of developing into all lineages of cells.
  • totipotent population of cells refers to a composition of cells capable of developing into all lineages of cells.
  • pluripotent cell refers to a cell capable of developing into a variety ⁇ albeit not all) lineages and are at least able to develop into all hematopoietic lineages (e.g., lymphoid, erythroid, and tlirombocytic lineages).
  • hematopoietic lineages e.g., lymphoid, erythroid, and tlirombocytic lineages.
  • hematopoietic lineages e.g., lymphoid, erythroid, and tlirombocytic lineages.
  • a pluripotent cell can differ from a totipotent cell by having the ability to develop into all cell lineages except endothelial cells.
  • a "pluripotent population of cells” refers to a composition of cells capable of developing into less than all lineages of cells but at least into all hematopoietic lineages.
  • a totipotent cell or composition of cells is less developed than a pluripotent cell or compositions of cells.
  • the terms “develop”, “differentiate” and “mature” all refer to the progression of a cell from the stage of having the potential to differentiate into at least two different cellular lineages to becoming a specialized cell. Such terms can be used interchangeably for the purposes of the present application.
  • the term “population” refers to cells having the same or different identifying characteristics.
  • the term “lineage” refers to all of the stages of the development of a cell type, from the earliest precursor cell to a completely mature cell (i.e. a specialized cell).
  • a stem cell population of the present invention is capable of developing into cells of mesodermal cell lineage, of ectodermal cell lineage or of endodermal cell lineage.
  • mesodermal cells include cells of connective tissue, bone, cartilage, muscle, blood and blood vessel, lymphatic and lymphoid organ, notochord, pleura, pericardium, peritoneum, kidney and gonad.
  • Ectodermal cells include epidermal tissue cells, such as those of nail, hair, glands of the skin, the nervous system, the external sense organs (e.g., eyes and ears) and mucous membranes (such as those of the mouth and anus).
  • Endodermal cells include cells of the epithelium such as those of the pharynx, respiratory tract (except the nose), digestive tract, bladder and urethra cells.
  • Preferred cells within a stem cell population of the present invention include cells of at least one of the following cellular lineages: hematopoietic cell lineage, endothelial cell lineage, epithelial cell lineage, muscle cell lineage and neural cell lineage.
  • Other preferred cells within a stem cell population of the present invention include cells of erythroid lineage, endothelial lineage, leukocyte lineage, thrombocyte lineage, erythroid lineage (including primitive and definitive erythroid lineages), macrophage lineage, neutrophil lineage, mast cell lineage, megakaryocyte lineage, natural killer cell lineage, eosinophil lineage, T cell lineage, endothelial cell lineage and B cell lineage.
  • stem cells expressing a tissue specific protein home to tissue expressing the protein.
  • a gene encoding a protein unique to the pigmented epithelium, RPE65 was introduced into syngeneic mouse endothelial stem cells using a lentivirus vector. After 7 days some migration of the stem cells expressing the RPE 65 into the depleted retinal pigmented epithelial layer was observed. This population significantly increased one month after administration. The results have far reaching implications for developing stem cell therapies.
  • compositions and methods of the invention includes tissue replacement using adult or embryonic stem cells.
  • the immediate application is for treatment of loss of vision due to the loss of retinal detachment.
  • eye diseases that can be treated by replacing the loss of critical cell types necessary for vision.
  • the far reaching application is ' for tissue replacement of other tissue types such as hepatocytes, islet cells, neurons, etc.
  • stem cells are transiently transfected with a homing gene to a specific tissue for regeneration of cells.
  • integration of a vector into the stem cell genome is avoided. Lack of integration into the stem cell genome is accomplished by using plasmid DNA or messenger RNA.
  • the expression of the endogenous gene within the stem cell genome is activated using either small molecules derived from a chemical library and screened for specific activation of the desired gene and/or adding a recombinant protein or peptide that transcriptionally activates the expression of the homing protein.
  • the transcriptional activation can use any transcriptional activator, for example, a zinc finger-transcription activator (additional DNA binding protein motifs include helix-turn-helix and leucine zipper) fusion protein or a specific transcription activator protein that binds to the enhancer or promoter region responsible for controlling expression of the homing protein.
  • a zinc finger-transcription activator additional DNA binding protein motifs include helix-turn-helix and leucine zipper
  • fusion protein or a specific transcription activator protein that binds to the enhancer or promoter region responsible for controlling expression of the homing protein.
  • Bmi-1 is expressed in stem cells and is essential for their maintenance.
  • a gene delivery vehicle comprises use of a non- integrating vector.
  • gene delivery vehicle is meant a carrier which can deliver at least one nucleic acid to a host cell.
  • the nucleic acid that is delivered to a host cell may comprise a nucleic acid sequence encoding an amino acid sequence, such as for example, SCG 10; Na Channel II; glut-2, synapsin, epo, SCF , shh, wint, BMPs Ephrins, Pax-6, Emx-2, Mash -1, jagged 1 and 2, notch- and 2, ephrin B2 and ephrin B4, Bmi-1, HOXB4.
  • the nucleic acid may further comprise at least one promoter, and/or enhancer, and/or terminator. It may also comprise transcription initiation sites, and the like.
  • a nucleic acid By delivering a nucleic acid to a host cell, the nucleic acid is moved from the outside to the inside of the host cell. Transient expression of the trans gene is sufficient to trigger cells to differentiate into the desired mature tissue cells. Therefore, a preferred non-integrating vector is adenovirus (adenovirus has other affects on the cells, such as immunostimulatory properties that may results in elimination of the infected cell.) or non-replicating, n ⁇ n-integrating plasmids.
  • a non-replicating, non-integrating plasmid is a nucleic acid which when transfected into a host cell does not replicate and does not specifically integrate into the host cell's genome (i.e. does not integrate at high frequencies and does not integrate at specific sites). In other preferred embodiments, the plasmid is non-integrating but does replicate and can be used in those instances where larger numbers of transduced cells are required.
  • Replicating plasmids can be identified using standard assays including the standard replication assay of Ustav et al., EMBO J, 10, 449-457, 1991.
  • a potential stabilized episome plasmid is derived from the Epstein bar virus gene, EBNA-I, that causes the plasmid to exist as an episome but is limited to B cell lineages.
  • a non-replicating, non-integrating plasmid is used.
  • This type of plasmid cannot be stably maintained in cells, independently of genomic DNA replication, and which does not persist in progeny cells for three or more cell divisions without a significant loss in copy number of the plasmid in the cells, i.e., with a loss of greater than an average of about 50% of the plasmid molecules in progeny cells between a given cell division.
  • the self-replicating function is provided by using a viral origin of replication and providing one or more viral replication factors that are required for replication mediated by that particular viral origin.
  • transiently transfecting, non-integrating plasmid herein means the same as the term "non- replicating, non-integrating plasmid" as defined above.
  • the plasmid is a naked nucleic acid.
  • naked refers to a nucleic acid molecule that is free of direct physical associations with proteins, lipids, carbohydrates or proteoglycans, whether covalently or through hydrogen bonding.
  • the term does not refer to the presence or absence of modified nucleotides or ribonucleotides, or chemical modification of the all or a portion of a nucleic acid molecule by such means as methylation or the inclusion of protecting groups or cap- or tail structures.
  • a non-integrating vector comprises a nucleic acid encoding for any one of SCG 10; Na Channel II; glut-2, synapsin, epo, SCF , shli, wint, BMPs Ephrins, Pax-6, Emx-2, Mash -1, jagged 1 and 2, notch- and 2, ephrin B2 and ephrin B4, Bmi-1, HOXB4.
  • the vector comprises nucleic acids expressing targeting agents.
  • targeting agents may include, but are not limited to, EGF, FGF, SDF-I, transferrin, and endothelial specific peptides and bone specific ligands or antibodies to cell surface markers, such as CD34.
  • a targeting agent may comprise an antibody, cytokine, growth factor, hormone, lymphokine, receptor protein, such as, for example CD4 (T-helper cell surface marker), CD8 (cytotoxic lymphocyte cell surface marker) or soluble fragments thereof, a nucleic acid which bind corresponding nucleic acids through base pair complementarity, or a combination thereof (U.S. Pat. No. 6,071,533, incorporated herein by reference).
  • the targeting ligand may comprise a cellular receptor- targeting ligand, a fusogenic ligand, a nucleus targeting ligand, or a combination thereof (U.S. Pat. No. 5,908,777, incorporated herein by reference).
  • the targeting ligand may comprise an integrin receptor ligand, described in U.S. Pat. No. 6,083,741, incorporated herein by reference.
  • the vector integrates into the stem cell genome.
  • the vector expresses SCG 10; Na Channel II; glut-2, synapsin, epo, SCF , shh, wint, BMPs Ephrins, Pax-6, Emx-2, Mash -1, jagged 1 and 2, notch- and 2, ephrin B2 and ephrin B4, Bmi-1, HOXB4 and the integration is site-specific.
  • Vectors include chemical conjugates such as described in WO 93/04701, which has a targeting moiety (e.g. a ligand to a cellular surface receptor), and a nucleic acid binding moiety (e.g. polylysine), viral vector (e.g. a DNA or RNA viral vector), fusion proteins such as described in PCT/US95/02140 (WO 95/22618) which is a fusion protein containing a target moiety (e.g. an antibody specific for a target cell) and a nucleic acid binding moiety (e.g. a protamine), plasmids, phage etc.
  • a targeting moiety e.g. a ligand to a cellular surface receptor
  • nucleic acid binding moiety e.g. polylysine
  • viral vector e.g. a DNA or RNA viral vector
  • fusion proteins such as described in PCT/US95/02140 (WO 95/22618) which is a fusion protein containing a
  • the genetic construct(s) When taken up by a cell, the genetic construct(s) remain present in the cell as a functioning extrachromosomal molecule.
  • DNA may be introduced into cells where it remains as separate genetic material in the form of a plasmid or plasmids.
  • RNA may be administered to the cell. It is also contemplated to provide the genetic construct as a linear minichromosome including a centromere, telomeres and an origin of replication.
  • Vectors can include regulatory elements necessary for gene expression of a nucleic acid molecule. The elements include: a promoter, an initiation codon, a stop codon, and a polyadenylation signal.
  • enhancers may be required for gene expression of the sequence of choice, variants or fragments thereof. It is necessary that these elements be operably linked to the sequence that encodes the desired proteins and that the regulatory elements are operable in the individual to whom they are administered. [0087] Initiation codons and stop codons are generally considered to be part of a nucleotide sequence that encodes the desired tissue specific protein. However, it is necessary that these elements are functional in the individual to whom the gene construct is administered. The initiation and termination codons must be in frame with the coding ⁇ sequence.
  • Promoters and polyadenylation signals used must be functional within the cells of the individual.
  • promoters useful to practice the present invention include but are not limited to promoters from Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HFV) such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV, Cytomegalovirus (CMV) such as the CMV immediate early promoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters from human genes such as human Actin, human Myosin, human Hemoglobin, human muscle creatine and human metallothionein.
  • SV40 Simian Virus 40
  • MMTV Mouse Mammary Tumor Virus
  • HBV Human Immunodeficiency Virus
  • LTR HIV Long Terminal Repeat
  • ALV a virus
  • CMV Cytomegalovirus
  • EBV Epstein Barr Virus
  • polyadenylation signals useful to practice the present invention, especially in the production of a genetic vaccine for humans include but are not limited to SV40 polyadenylation signals and LTR polyadenylation signals, human growth hormone poly A signal.
  • the SV40 polyadenylation signal which is in pCEP4 plasmid (Invitrogen, San Diego Calif), referred to as the SV40 polyadenylation signal can be used.
  • other elements may also be included in the DNA molecule. Such additional elements include, zinc fingers, enhancers.
  • the enhancer may be selected from the group including but not limited to: human Actin, human Myosin, human Hemoglobin, human muscle creatine and viral enhancers such as those from CMV, RSV and EBV.
  • Genetic constructs can be provided with mammalian origin of replication in order to maintain the construct extrachromosomally and produce multiple copies of the construct in the cell.
  • plasmids pCEP4 and pREP4 from Invitrogen contain the Epstein Barr virus origin of replication and nuclear antigen EBNA-I coding region which produces high copy episomal replication without integration.
  • regulatory sequences may be selected which are well suited for gene expression in the cells the construct is administered into.
  • codons may be selected which are most efficiently transcribed in the cell.
  • One having ordinary skill in the art can produce DNA constructs which are functional in the cells.
  • the nucleic acid molecule is delivered to the cells in conjunction with administration of a facilitating agent.
  • Facilitating agents are also referred to as polynucleotide function enhancers or genetic vaccine facilitator agents.
  • Facilitating agents are described in e.g. International Application No. PCT/US94/00899 filed Jan. 26, 1994 and International Application No. PCT7US 95/04071 filed Mar. 30, 1995, both incorporated herein by reference.
  • Facilitating agents which are administered in conjunction with nucleic acid molecules may be administered as a mixture with the nucleic acid molecule or administered separately simultaneously, before or after administration of nucleic acid molecules.
  • the genetic constructs of the invention are formulated with or administered in conjunction with a facilitator selected from the group consisting of, for example, benzoic acid esters, anilides, amidines, urethans and the hydrochloride salts thereof such as those of the family of local anesthetics.
  • a facilitator selected from the group consisting of, for example, benzoic acid esters, anilides, amidines, urethans and the hydrochloride salts thereof such as those of the family of local anesthetics.
  • the facilitating agent is administered prior to, simultaneously with or subsequent to the genetic construct.
  • the facilitating agent and the genetic construct may be formulated in the same composition.
  • the genetic constructs are administered free of facilitating agents, that is in formulations free from facilitating agents using administration protocols in which the genetic constructions are not administered in conjunction with the administration of facilitating agents, (cationic lipid/helper lipid formulations, electroporation, ultrasound, cationic polymers (PEI, poly lysine, poly-L-ornithine), non-interacting polymers, such as Poloxamer or polyvinylpyrolidone).
  • Nucleic acid molecules which are delivered to cells according to the invention may serve as genetic templates for proteins that function as tissue specific maturation agents.
  • the nucleic acid molecules comprise the necessary regulatory sequences for transcription and translation of the coding region in the cells of the animal.
  • nucleic acid sequences of choice can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification.
  • Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson et al, 1978, J. Biol. Chem. 253: 6551; Zoller and Smith, 1984, DNA 3:479-488; Oliphant et al., 1986, Gene 44: 177; Hutchinson et al., 1986, Proc. Natl.
  • PCR techniques are preferred for site directed mutagenesis (see Higuchi, 1989, "Using PCR to Engineer DNA", in PCR Technology: Principles and Applications for DNA Amplification, H. Erlich, ed., Stockton Press, Chapter 6, pp. 61-70).
  • Various methods known to those skilled in the art can be used to express nucleic acid sequences in the non-immortalized cells.
  • the identified and isolated gene can be inserted into an appropriate cloning vector.
  • a large number of vector-host systems known in the art may be used.
  • Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used.
  • examples of vectors include, but are not limited to, E. coli, bacteriophages such as lambda derivatives, or plasmids such as pBR322 derivatives or pUC plasmid derivatives, e.g., pGEX vectors, pmal-c, pFLAG, etc.
  • the insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector that has complementary cohesive termini.
  • the ends of the DNA molecules may be enzymatically modified.
  • any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences.
  • Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated.
  • the cloned gene is contained on a shuttle vector plasmid, which provides for expansion in a cloning cell, e.g., E.
  • a shuttle vector which is a vector that can replicate in more than one type of organism, can be prepared for replication in both E. coli and Saccharomyces cerevisiae by linking sequences from an E. coli plasmid with sequences from the yeast 2 ⁇ plasmid.
  • Another method comprises ligating the differentiation protein into an artificial replicating chromosome and microinject it into the stem cell.
  • the percent of transformed cells may not be important because the disease state selects for the transformed stem cell. Larger numbers of stem cells may be transduced if a critical mass is needed to seed the tissue replacement or tissue correction site.
  • the desired gene may be identified and isolated after insertion into a suitable cloning vector in a "shot gun" approach.
  • Enrichment for the desired gene for example, by size fractionation, removal of highly-repetitive sequences, subtractive or otherwise selective hybridization, and other methods as may be known in the art, can be done before insertion into the cloning vector.
  • the nucleotide sequence can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. Such elements are termed herein a "promoter.”
  • a promoter a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence.
  • the nucleic acid encoding a desired protein, functional fragments, derivatives or analogs thereof is operationally associated with a promoter in an expression vector of the invention. Both cDNA and genomic sequences can be cloned and expressed under control of such regulatory sequences.
  • An expression vector also preferably includes a replication origin. The necessary transcriptional and translational signals can be provided on a recombinant expression vector.
  • Potential host-vector systems include but are not limited to mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA.
  • virus e.g., vaccinia virus, adenovirus, etc.
  • insect cell systems infected with virus e.g., baculovirus
  • microorganisms such as yeast containing yeast vectors
  • bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA e.g., bacteriophage, DNA, plasmid DNA, or cosmid DNA.
  • the expression elements of vectors vary in their strengths and specificities. Depending on the host- vector system utilized, any one of a number of suitable transcription and translation elements may be used.
  • a recombinant protein may be expressed chromosomally, after integration of the coding sequence by recombination.
  • any of a number of amplification systems maybe used to achieve high levels of stable gene expression (See Sambrook et al., 1989, supra).
  • nucleic acid condensing agents include the use of nucleic acid condensing agents, electroporation, complexation with asbestos, polybrene, DEAE cellulose, Dextran, liposomes, cationic liposomes, lipopolyamines, polyomithine, particle bombardment and direct microinjection (reviewed by Kucherlapati and Skoultchi, Crit. Rev. Biochem. 16:349- 379 (1984); Keown et al., Methods Enzymol. 185:527 (1990)).
  • a vector of the invention may be delivered to a host cell via a viral or non- viral means of delivery.
  • Preferred delivery methods of viral origin include viral particle packaging cell lines as transfection recipients for the vector of the present invention into which viral packaging signals have been engineered, such as those of adenovirus, herpes viruses and papovaviruses.
  • Preferred non-viral based gene delivery means and methods may also be used in the invention and include direct naked nucleic acid injection, nucleic acid condensing peptides and non-peptides, cationic liposomes and encapsulation in liposomes.
  • the viral vectors are replication defective, that is, they are unable to replicate autonomously in the target cell.
  • the genome of the replication defective viral vectors which are used within the scope of the present invention lack at least one region which is necessary for the replication of the virus in the infected cell. These regions can either be eliminated (in whole or in part), be rendered non- functional by any technique known to a person skilled in the art. These techniques include the total removal, substitution (by other sequences, in particular by the inserted nucleic acid), partial deletion or addition of one or more bases to an essential (for replication) region. Such techniques may be performed in vitro (on the isolated DNA) or in situ, using the techniques of genetic manipulation or by treatment with mutagenic agents.
  • the replication defective virus retains the sequences of its genome which are necessary for encapsulating the viral particles.
  • DNA viral vectors include an attenuated or defective DNA virus, such as but not limited to adenovirus, adeno-associated virus (AAV), herpes simplex virus (HSV), papillomavirus, Epstein-Barr virus (EBV), and the like.
  • Defective viruses which entirely or almost entirely lack viral genes, are preferred. Defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HSVl) vector [Kaplitt et al, Molec. Cell. Neurosci.
  • HSVl herpes virus 1
  • Adenovirus vectors in one preferred embodiment, the vector is an adenovirus vector.
  • Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a nucleic acid of the invention to a variety of cell types.
  • Various serotypes of adenovirus exist.
  • adenoviruses of animal origin preference is given, within the scope of the present invention, to using type 2 or type 5 human adenoviruses (Ad 2 or Ad 5) or adenoviruses of animal origin (see WO94/26914).
  • Those adenoviruses of animal origin which can be used within the scope of the present invention include adenoviruses of canine, bovine, murine (example: Mavl, Beard 75 (1990) 81), ovine, porcine, avian, and simian (example: SAV) origin.
  • the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus (e.g., Manhattan or A26/61 strain (ATCC VR-800), for example).
  • Adenovirus vectors, adeno- associated virus vectors, parvovirus vectors and herpes simplex virus vectors are preferred for introducing the nucleic acid e.g.
  • the adenovirus vector results in a shorter term expression (about 2 months) than adeno-associated virus (about 4 months), which in turn is shorter than HSV vectors.
  • the vectors can be introduced by standard techniques, e.g. infection, transfection, transduction or transformation.
  • the replication defective adenoviral vectors of the invention comprise the ITRs, an encapsidation sequence and the nucleic acid of interest.
  • at least the El region of the adenoviral vector is non-functional. The deletion in the El region preferably extends from nucleotides 455 to 3329 in the sequence of the Ad5 adenovirus (PvuII-Bglll fragment) or 382 to 3446 (HinfII-Sau3A fragment).
  • the adenoviral vector has a deletion in the El region (Ad 1.0). Examples of El-deleted adenoviruses are disclosed in EP 185,573, the contents of which are incorporated herein by reference. In another preferred embodiment, the adenoviral vector has a deletion in the El and E4 regions (Ad 3.0).
  • the adenoviral vector has a deletion in the El region into which the E4 region and the nucleic acid sequence are inserted.
  • the replication defective recombinant adenoviruses according to the invention can be prepared by any technique known to the person skilled in the art (Levrero et al, Gene 101 (1991) 195, EP 185 573; Graham, EMBOJ. 3 (1984) 2917). In particular, they can be prepared by homologous recombination between an adenovirus or modified adenovirus genome and a plasmid which carries, inter alia, the DNA sequence of interest. The homologous recombination is effected following cotransfection of the said adenovirus and plasmid into an appropriate cell line.
  • the cell line which is employed should preferably (i) be transformable by the said elements, and (ii) contain the sequences which are able to complement the part of the genome of the replication defective adenovirus, preferably in integrated form in order to avoid the risks of recombination.
  • Examples of cell lines which may be used are the human embryonic kidney cell line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which contains the left-hand portion of the genome of an Ad5 adenovirus (12%) integrated into its genome, and cell lines which are able to complement the El and E4 functions, as described in applications WO94/26914 and WO95/02697.
  • Recombinant adenoviruses are recovered and purified using standard molecular biological techniques, which are well known to one of ordinary skill in the art.
  • Adeno-associated viruses hi a preferred embodiment, the vector is an adeno- associated viruses.
  • the adeno-associated viruses are DNA viruses of relatively small size which can integrate, in a stable and site-specific manner, into the genome of the cells which they infect. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies.
  • the AAV genome has been cloned, sequenced and characterized. It encompasses approximately 4700 bases and contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as an origin of replication for the virus.
  • ITR inverted terminal repeat
  • the remainder of the genome is divided into two essential regions which carry the encapsidation functions: the left-hand part of the genome, which contains the rep gene involved in viral replication and expression of the viral genes; and the right-hand part of the genome, which contains the cap gene encoding the capsid proteins of the virus.
  • the use of vectors derived from the AAVs for transferring genes in vitro and in vivo has been described (see WO 91/18088; WO 93/09239; U.S. Pat. No. 4,797,368, U.S. Pat. No. 5,139,941, EP 488 528).
  • the replication defective recombinant AAVs according to the invention can be prepared by cotransfecting a plasmid containing the nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions, and a plasmid carrying the AAV encapsidation genes (rep and cap genes), into a cell line which is infected with a human helper virus (for example an adenovirus).
  • ITR AAV inverted terminal repeat
  • the AAV recombinants which are produced are then purified by standard techniques.
  • the invention also relates, therefore, to an AAV-derived recombinant virus whose genome encompasses a sequence encoding a nucleic acid encoding a tissue maturation factor, (e.g. SCG 10; Na Channel II; glut-2, synapsin, epo, SCF , shh, wint, BMPs Ephrins, Pax-6, Emx-2, Mash -1, jagged 1 and 2, notch- and 2, ephrin B2 and ephrin B4, Bmi-1, different homeobox genes, such as, HOXB4), flanked by the AAV ITRs.
  • a tissue maturation factor e.g. SCG 10; Na Channel II; glut-2, synapsin, epo, SCF , shh, wint
  • the invention also relates to a plasmid encompassing a sequence encoding a nucleic acid encoding a desired gene flanked by two ITRs from an AAV.
  • a plasmid can be used as it is for transferring the nucleic acid sequence, with the plasmid, where appropriate, being incorporated into a liposomal vector (pseudo-virus).
  • Retrovirus vectors in another embodiment the gene can be introduced in a retroviral vector, e.g., as described in Anderson et al, U.S. Pat. No. 5,399,346; Mann et al., 1983, Cell 33:153; Temin et al, U.S. Pat. No.
  • the retroviruses are integrating viruses which infect dividing cells.
  • the retrovirus genome includes two LTRs, an encapsidation sequence and three coding regions (gag, pol and env).
  • the gag, pol and env genes are generally deleted, in whole or in part, and replaced with a heterologous nucleic acid sequence of interest.
  • retrovirus can be constructed from different types of retrovirus, such as, HIV, MoMuLV ("murine Moloney leukaemia virus” MSV ("murine Moloney sarcoma virus”), HaSV ("Harvey sarcoma virus”); SNV ("spleen necrosis virus”); RSV ("Rous sarcoma virus”) and Friend virus.
  • Defective retroviral vectors are disclosed in WO95/02697.
  • a plasmid is constructed which contains the LTRs, the encapsidation sequence and the coding sequence.
  • This construct is used to transfect a packaging cell line, which cell line is able to supply in trans the retroviral functions which are deficient in the plasmid.
  • the packaging cell lines are thus able to express the gag, pol and env genes.
  • Such packaging cell lines have been described in the prior art, in particular the cell line PA317 (U.S. Pat. No. 4,861,719); the PsiCRIP cell line (WO90/02806) and the GP + envAm-12 cell line (WO89/07150).
  • the recombinant retroviral vectors can contain modifications within the LTRs for suppressing transcriptional activity as well as extensive encapsidation sequences which may include a part of the gag gene (Bender et ah, J. Virol. 61 (1987) 1639).
  • Recombinant retroviral vectors are purified by standard techniques known to those having ordinary skill in the art.
  • Retroviral vectors can be constructed to function as infectious particles or to undergo a single round of transfection. hi the former case, the virus is modified to retain all of its genes except for those responsible for oncogenic transformation properties, and to express the heterologous gene. Non-infectious viral vectors are prepared to destroy the viral packaging signal, but retain the structural genes required to package the co-introduced virus engineered to contain the heterologous gene and the packaging signals. Thus, the viral particles that are produced are not capable of producing additional virus. Targeted gene delivery is described in International Patent Publication WO 95/28494, published October 1995.
  • Lentiviral Vectors include members of the bovine lentivirus group, equine lentivirus group, feline lentivirus group, ovinecaprine lentivirus group and primate lentivirus group.
  • the development of lentiviral vectors for gene therapy has been reviewed in Klimatcheva et al, 1999, Frontiers in Bioscience 4: 481-496.
  • the design and use of lentiviral vectors suitable for gene therapy is described, for example, in U.S. Pat. No. 6,207,455, issued Mar. 27, 2001, and U.S. Pat. No. 6,165,782, issued Dec. 26, 2000.
  • lentiviruses examples include, but are not limited to, HIV-I, HIV-2, HIV-l/HIV-2 pseudotype, HIV-1/SIV, FIV, caprine arthritis encephalitis virus (CAEV), equine infectious anemia virus and bovine immunodeficiency virus. HIV-I is preferred.
  • Autonomous parvoviruses are small DNA viruses that replicate autonomously in rapidly dividing cells. The genomes of autonomous parvoviruses do not integrate, at least not at a detectable level. Autonomous parvovirus genomes are single-stranded DNA molecules about 5 kilobases (kb) in size.
  • the genomes are organized such that the NS gene encoding the nonstructural polypeptides NSl and NS2 is located on the left side of the genome and the VP gene encoding the structural polypeptides required for capsid formation are on the right side of the genome.
  • Expression of the nonstructural polypeptides is controlled by a transcription control sequence called P4 in most parvoviruses, which is located at about map unit position 4 of the genome (assuming the entire genome is 100 map units and numbering is from left to right).
  • Expression of the structural polypeptides is controlled by a transcription control sequence called P38, P39 or P40 in most parvoviruses, which is located at about map unit position 38 to about 40, depending on the autonomous parvovirus.
  • NSl serves as a trans-activator of the latter transcription control sequence.
  • NSl is also essential for virus replication and appears to be the primary mediator of parvovirus cytotoxicity, particularly against tumor cells.
  • Autonomous parvovirus genomes also have inverted repeat sequences (i.e., palindromes) at each end which contain essential signals for replication and encapsidation of the virus.
  • inverted repeat sequences i.e., palindromes
  • Suitable autonomous parvovirus nucleic acid sequences include, but are not limited to, LuIII parvovirus (LuIII), minute virus of mice (MVM; e.g., MVMi and MVMp), hamster parvovirus (e.g., Hl), feline panleukopenia virus, canine parvovirus, porcine parvovirus, latent rat virus, mink enteritis virus, human parvovirus (e.g., B19), bovine parvovirus, and Aleutian mink disease parvovirus nucleic acid sequences.
  • LuIII parvovirus is a parvovirus of unknown origin that was isolated as a contaminant of a substrain of human permanent cell line LuI 06.
  • Non-viral Vectors alternatively, the vector can be introduced in vivo as nucleic acid free of transfecting excipients, or with transfection facilitating agents, e.g., lipofection.
  • transfection facilitating agents e.g., lipofection.
  • liposomes for encapsulation and transfection of nucleic acids in vitro.
  • Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker [Feigner, et. al, Proc. Natl. Acad. Sci. U.S.A.
  • cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes [Feigner and Ringold, Science 337:387-388 (1989)].
  • Particularly useful lipid compounds and compositions for transfer of nucleic acids are described in International Patent Publications WO95/18863 and WO96/17823, and in U.S. Pat. No. 5,459,127.
  • a nucleic acid in vivo such as a cationic oligopeptide (e.g., International Patent Publication WO95/2193 1), peptides derived from DNA binding proteins (e.g., International Patent Publication WO96/25508), or a cationic polymer (e.g., International Patent Publication WO95/21931).
  • a cationic oligopeptide e.g., International Patent Publication WO95/21931
  • peptides derived from DNA binding proteins e.g., International Patent Publication WO96/25508
  • a cationic polymer e.g., International Patent Publication WO95/21931
  • Naked DNA vectors can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter [see, e.g., Wu et al, J. Biol. Chem. 267:963-967 (1992); Wu and Wu, J. Biol. Chem. 263:14621-14624 (1988); Williams et al, Proc. Natl. Acad. ScL USA 88:2726-2730 (1991)].
  • Receptor-mediated DNA delivery approaches can also be used [Curiel et al, Hum. Gene Ther.
  • Regulatory Regions expression of, for example, SCG 10; Na Channel II; glut-2, synapsin, epo, SCF , shh, wint, BMPs Ephrins, Pax-6, Emx-2, Mash -1, jagged 1 and 2, notch- and 2, ephrin B2 and ephrin B4, Bmi-1, different homeobox genes, such as, HOXB4, from a vector of the invention may be controlled by any regulatory region, i.e., promoter/enhancer element known in the art.
  • the regulatory regions may comprise a promoter region for functional transcription in the tissue of interest, as well as a region situated in 3' of the gene of interest, and which specifies a signal for termination of transcription and a polyadenylation site. All these elements constitute an expression cassette.
  • Promoters that may be used in the present invention include both constitutive promoters and regulated (inducible) promoters.
  • the promoter may be naturally responsible for the expression of the nucleic acid. It may also be from a heterologous source.
  • it may be promoter sequences of eukaryotic or viral genes.
  • it may be promoter sequences derived from the genome of the cell which it is desired to infect.
  • it may be promoter sequences derived from the genome of a virus, including the adenovirus used.
  • the promoters of the ElA, MLP, CMV and RSV genes and the like may be mentioned, for example, the promoters of the ElA, MLP, CMV and RSV genes and the like.
  • the promoter may be modified by addition of activating or regulatory sequences or sequences allowing a tissue-specific or predominant expression (enolase and GFAP promoters and the like).
  • the nucleic acid may be inserted, such as into the virus genome downstream of such a sequence.
  • Some promoters useful for practice of this invention are heat shock protein promoters (hsp), ubiquitous promoters (e.g., HPRT, vimentin, actin, tubulin), intermediate filament promoters (e.g., GFAP, desmin, neurofilaments, keratin), therapeutic gene promoters (e.g., MDR type, CFTR, factor VIII), tissue-specific promoters (e.g., actin promoter in smooth muscle cells), promoters which are preferentially activated in dividing cells, promoters which respond to a stimulus (e.g., steroid hormone receptor, retinoic acid receptor), tetracycline-regulated transcriptional modulators, cytomegalovirus immediate-early, retroviral LTR, metallothionein, SV-40, ElA, and MLP promoters.
  • hsp heat shock protein promoters
  • ubiquitous promoters e.g., HPRT, vimentin, actin, tubulin
  • the promoters which may be used to control gene expression include, but are not limited to, GFAP, HSP promoters, the cytomegalovirus (CMV) promoter, the SV40 early promoter region (Benoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc.
  • CMV cytomegalovirus
  • promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter.
  • nucleic acid condensing peptides which are particularly useful for condensing the vector and delivering the vector to a cell, are described in WO 96/41606.
  • Functional groups may be bound to peptides useful for delivery of a vector according to the invention, as described in WO 96/41606. These functional groups may include a ligand that targets a specific cell-type such as a monoclonal antibody, insulin, transferrin, asialoglycoprotein, or a sugar. The ligand thus may target cells in a nonspecific manner or in a specific manner that is restricted with respect to cell type.
  • Nucleic acid condensing agents useful in the invention include spermine, spermine derivatives, histones, cationic peptides, cationic non-peptides such as polyethyleneimine (PEI) and polylysine.
  • spermine derivatives refers to analogues and derivatives of spermine and include compounds as set forth in International Patent Application. WO 93/18759 (published Sep. 30, 1993).
  • Delivery vehicles for delivery of DNA constructs to cells are known in the art and include DNA/poly-cation complexes which are specific for a cell surface receptor, as described in, for example, Wu and Wu 3 J. Biol. Chem. 263:14621 (1988); Wilson et al, J. Biol. Chem. 267:963(1992); and U.S. Pat. No. 5,166,320).
  • the delivery vehicle comprises a functional group.
  • the functional groups also may comprise a lipid, such as palmitoyl, oleyl, or stearoyl; a neutral hydrophilic polymer such as polyethylene glycol (PEG), or polyvinylpyrrolidine (PVP); a fusogenic peptide such as the HA peptide of influenza virus; or a recombinase or an integrase.
  • the functional group also may comprise an intracellular trafficking protein such as a nuclear localisation sequence (NLS) and endosome escape signal or a signal directing a protein directly to the cytoplasm.
  • NLS nuclear localisation sequence
  • endosome escape signal or a signal directing a protein directly to the cytoplasm.
  • the stem cells are transformed with nucleic acids which encode for desired tissue specific targeting markers, such as for example, endothelial markers - VEGFR, Tie-1, EC-I, EnPo 1.
  • tissue specific targeting markers such as for example, endothelial markers - VEGFR, Tie-1, EC-I, EnPo 1.
  • the invention provides methods for the targeting and tracking of stem cells to specific locations within an animal's body.
  • the methods used herein are also useful in the therapeutic applications of repairing or colonizing specifically targeted areas within an animal, with stem cells, which then differentiate into mature cells of the specific cell type of the targeted area.
  • the method of the invention optionally comprises vectors expressing antibodies specific to antigens in a desired target area.
  • a second antibody can be used for the in vivo tracking of the stem cell from any area in the animal's body to the desired target area.
  • stem cells from a patient are harvested, sorted, purified and identified.
  • the stem cells are then transduced with an expression vector comprising nucleic acid sequence encoding an antibody which will target the stem cell to the targeted location.
  • this procedure can be used for different antigen specificities of stem cells.
  • Different isotypes of the arming antibody e.g. IgGl, etc.
  • the secondary antibody can come from different sources, e.g. rat, sheep, goat etc; the important property being that it is targeted against the species of origin of the primary antibody.
  • secondary antibodies conjugated with different fluorochromes can be used, e.g. PE, FITC, APC, etc.
  • antibodies can be conjugated to a stem cells surface using linkers, chemical conjugates and the like.
  • the stem cells may be transformed with DNA which codes for different tissue specific markers and or antibodies for targeting of the stem cells to a specific tissue.
  • the stem cells differentiate into mature cells representative of the target area. In other cases it is desirable to target the stem cells to areas whereby diseases such as cancer have destroyed certain target areas such as for example, colon cancer. Stem cells can be targeted to areas which have been removed by surgery or have been affected by chemotherapy and allowed to repopulate the area. In other cases, such as in hemophiliacs, it is desirable to target stem cells to the blood vessel lining thereby repairing the blood vessels and prevent further bleeding.
  • the methods of the invention have many advantages over gene therapy or organ transplantation, skin grafts and-the like.
  • the stem cells are immature and are able to repopulate without an immune response being mounted.
  • exogenous DNA segments for example, SCG 10; Na Channel II; glut-2, synapsin, epo, SCF , shh, wint, BMPs Ephrins, Pax-6, Emx-2, Mash -1, jagged 1 and 2, notch- and 2, ephrin B2 and ephrin B4, Bmi-1, different homeobox genes HOXB4, typically include an expression control DNA sequence operably linked to a tissue- specific promoter. Examples of tissue specific promoters are shown in Table 1.
  • Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.
  • NCAM Neural Cell Adhesion Molecule
  • SAA Human Serum Amyloid A
  • TPA Collagenase Phorbol Ester
  • tissue-specific promoters or elements as well as assays to characterize their activity, is well known to those of skill in the art.
  • regions include the human LIMK2 gene (Nomoto et al. Gene, 236(2):259-271, 1999), the somatostatin receptor 2 gene (Kraus et al., FEBS Lett., 428(3): 165-70, 1998), murine epididymal retinoic acid-binding gene (Lareyre et al., J. Bio. Chem., 274(12):8282-90, 1999), human CD4 (Zhao-Emonet et al., Biochim. Biophys.
  • mice alpha 2 (XI) collagen Tsumaki, et al., 1998), DlA dopamine receptor gene (Lee, et al., DNA Cell Biol, 16(11):1267-75. 1997), insulin-like growth factor II (Wu et al., Biochem. Biophys. Res. Commun., 233(l):221-6, 1997; Wu et al., J. Med. Virol., 52:83-85. 1997), human platelet endothelial cell adhesion molecule- 1 (Almendro et al., J Immunol., 157(12):5411-21, 1996).
  • the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting stem host cells, but control sequences for prokaryotic hosts may also be used.
  • Vectors can be constructed which also comprise a detectable/selectable marker gene.
  • these marker genes are fluorescent proteins such as green fluorescent protein (GFP), cyan- (CFP), yellow- (YFG), blue- (BFP), red- (RFP) fluorescent proteins; enhanced green fluorescent protein (EGFP), EYFP, EBFP, Nile Red, dsRed, mutated, modified, or enhanced forms thereof, and the like.
  • the "green-fluorescence protein” is a gene construct which in transfected or infected cells, respectively, shines green under ultraviolet light and thus enables the detection of a cell transfected or infected, respectively, with GFP in a simple manner.
  • Uses of green fluorescent protein for the study of gene expression and protein localization are well known.
  • the compact structure makes GFP very stable under diverse and/or harsh conditions such as protease treatment, making GFP an extremely useful reporter in general.
  • Green fluorescent protein such as a "humanized” GFP DNA, the protein product of which has increased synthesis in mammalian cells.
  • One such humanized protein is “enhanced green fluorescent protein” (EGFP).
  • EGFP enhanced green fluorescent protein
  • Other mutations to green fluorescent protein have resulted in blue-, cyan- and yellow-green light emitting versions.
  • Endogenously fluorescent proteins have been isolated and cloned from a number of marine species including the sea pansies Renilla reniformris, R. kollikeri and R. mullerei and from the sea pens Ptilosarcus, Stylatula and Acanthoptilum, as well as from the Pacific Northwest jellyfish, A equorea victoria; Szent-Gyorgyi et al. (SPIE conference 1999), D. C. Prasher et al., Gene, 111:229-233 (1992) and several species of coral (Matz et al. Nature Biotechnology, 17 969-973 (1999). These proteins are capable of forming a highly fluorescent, intrinsic chromophore through the cyclization and oxidation of internal amino acids within the protein that can be spectrally resolved from weakly fluorescent amino acids such as tryptophan and tyrosine.
  • stem cells comprise vectors expressing desired chemokines.
  • Chemokines and cytokines play a powerful role in the development of an immune response. The role of chemokines in leukocyte trafficking is reviewed by Baggiolini (1998) Nature 392:565-8, in which it is suggested that migration responses in the complicated trafficking of lymphocytes of different types and degrees of activation will be mediated by chemokines. The use of small molecules to block chemokines is reviewed by Baggiolini and Moser (1997) J. Exp. Med. 186:1189-1191.
  • SLC secondary lymphoid- tissue chemokine
  • HEV high endothelial venule
  • These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombination (genetic recombination). Expression of proteins, may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression.
  • Expression vectors containing a nucleic acid encoding a desired polypeptide can be detected or identified by four general approaches: (a) PCR amplification of the desired plasmid DNA or specific mRNA, (b) nucleic acid hybridization, (c) presence or absence of selection marker gene functions, and (d) expression of inserted sequences.
  • the nucleic acids can be amplified by PCR to provide for detection of the amplified product.
  • the presence of a foreign gene inserted in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted marker gene.
  • the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "selection marker" gene functions (e.g., ⁇ -galactosidase activity, thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of foreign genes in the vector.
  • selection marker e.g., ⁇ -galactosidase activity, thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.
  • Gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods.
  • regulatory sequences may comprise promoters, enhancers, Scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences.
  • sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting, including polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules).
  • the targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence (for example, inserting a new promoter or enhancer or both upstream of a gene).
  • the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.
  • the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally-occurring elements.
  • the naturally occurring sequences are deleted and new sequences are added.
  • the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the host cell genome.
  • the identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.
  • Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine- guanine phosphoribosyltransferase (gpt) gene.
  • cell maturity and differentiation it is desirable to determine cell maturity and differentiation.
  • Several different ways, to assess maturity and cell differentiation, are available. For example, one such method is by measuring cell phenotypes. The phenotypic changes can be evaluated by flow cytometry after irnmunofiuorescent staining using monoclonal antibodies that will bind membrane proteins characteristic of various cell types.
  • the amount of stem cells administered to the patient will also vary depending on the condition of the patient and should be determined via consideration of all appropriate factors by the practitioner. Preferably, however, about IxIO 6 to about IxIO 12 , more preferably about IxIO 8 to about IxIO 11 , more preferably, about lxl0 9 to about lxl ⁇ lo stem cells are utilized for adult humans. These amounts will vary depending on the age, weight, size, condition, sex of the patient, the type of tumor to be treated, the route of administration, whether the treatment is regional or systemic, and other factors. Those skilled in the art should be readily able to derive appropriate dosages and schedules of administration to suit the specific circumstance and needs of the patient.
  • Methods of re-introducing cellular components are known in the art and include procedures such as those exemplified in U.S. Pat. No. 4,844,893 to Honsik, et al. and U.S. Pat. No. 4,690,915 to Rosenberg.
  • administration of activated CD8 + cells via intravenous infusion is appropriate.
  • the cell preparations and compositions of the invention can be used in a variety of methods (e.g. transplantation) and they have numerous uses in the field of medicine. They may be used for the replacement of body tissues, organs, components or structures which are missing or damaged due to trauma, age, metabolic or toxic injury, disease, idiopathic loss, or any other cause.
  • the stem cell compositions of the invention do not need to be transplanted. Transplantation is one other option that can be carried out. Transplantation as used herein, also means administering the stem cell compositions to a patient, such as by, for example, injection; i.v. i.m., etc.
  • Transplantation or grafting can include the steps of isolating a cell preparation according to the invention and transferring cells in the preparation into a mammal or a patient.
  • Transplantation can involve transferring the cells into a mammal or a patient by injection of a cell suspension into the mammal or patient, surgical implantation of a cell mass into a tissue or organ of the mammal or patient, or perfusion of a tissue or organ with a cell suspension.
  • the route of transferring the cells may be determined by the requirement for the cells to reside in a particular tissue or organ and by the ability of the cells to find and be retained by the desired target tissue or organ.
  • the transplanted cells are to reside in a particular location, they can be surgically placed into a tissue or organ or simply injected into the bloodstream and migrate to the desired target organ. See, the examples which follow.
  • the invention may be used for autografting (cells from an individual are used in the same individual), allografting cells (cells from one individual are used in another individual) and xenografting (transplantation from one species to another).
  • the cells, cell preparations and cellular compositions of the invention maybe used in autologous or allogeneic transplantation procedures to improve a non-hematopoietic cell or hematopoietic cell deficit or to repair tissue.
  • the newly created cellular compositions comprising cells with potential or increased potential to form non-hematopoietic cells, or non- hematopoietic cells differentiated therefrom, can be used in both cell therapies and gene therapies aimed at alleviating disorders and diseases involving the non-hematopoietic cells.
  • the invention obviates the need for human tissue to be used in various medical and research applications.
  • the cell therapy approach involves the use of transplantation of the newly created cellular compositions comprising cells with the potential or increased potential to form non- hematopoietic cells, or non-hematopoietic cells differentiated therefrom, as a treatment for injuries and diseases.
  • the steps in this application include: (a) producing a cellular composition comprising cells with the potential or increased potential to form non- hematopoietic cells, or non-hematopoietic cells differentiated therefrom, as described herein; and (b) allowing the cells to -form functional connections either before or after a step involving transplantation of the cells.
  • the gene therapy approach also involves cellular compositions comprising cells with the potential or increased potential to form non- hematopoietic cells, however, following the culturing step in proliferation conditions, the newly created cells are transfected with an appropriate vector containing a cDNA for a desired protein, followed by a step where the modified cells are transplanted.
  • cells with potential or increased potential to form non-hematopoietic cells and hematopoietic cells in cellular compositions of the present invention, or cells or tissues differentiated from the cells can be transplanted in, or grafted to, a patient in need.
  • the cells with potential to form non- hematopoietic cells or differentiated cells therefrom can be used to replace non-hematopoietic cells in a patient in a cell therapy approach, useful in the treatment of tissue injury, and diseases.
  • These cells can be also used as vehicles for the delivery of specific gene products to a patient.
  • One example of how these newly created cells or cell differentiated therefrom can be used in a gene therapy method is in treating the effects of Parkinson's disease.
  • tyrosine hydrolase a key enzyme in dopamine synthesis
  • a cell preparation of the invention comprising cells that are capable of differentiating into neuronal cells, or transplantation of neuronal cells differentiated from the cells, which have been transfected with a vector suitable for the expression of tyrosine hydrolase.
  • the invention also provides a method of treating a patient with a condition involving a non-hematopoietic cell comprising transferring a cellular composition comprising cells with the potential or increased potential to form non-hematopoietic cells into the patient, wherein the cell differentiates into the non-hematopoietic cells.
  • the invention provides a method for obtaining non-hematopoietic cells for autologous transplantation from a patient's own hematopoietic cells comprising (a) obtaining a sample comprising hematopoietic cells from the patient, preferably from fresh or cryopreserved umbilical cord blood; (b) separating out an enriched cell preparation comprising hematopoietic stem cells and hematopoietic progenitor cells,; and (b) culturing the cells under proliferation conditions to produce a cellular composition comprising cells with the potential or increased potential to form non-hematopoietic cells.
  • the cellular composition obtained from (b) can be cultured with a differentiating factor, or cells of the composition can be transferred to the patient.
  • the invention also contemplates a pharmaceutical composition
  • a pharmaceutical composition comprising cells, a cell preparation, or cellular composition of the invention, and a pharmaceutically acceptable carrier, excipient, or diluent.
  • the pharmaceutical compositions herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective amount of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985).
  • compositions include, albeit not exclusively, solutions of the cells, cell preparations, or cellular compositions in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
  • kits for producing cellular compositions of the invention comprising cells that have potential or increased potential to form cells capable of differentiating into cells of multiple tissue types both in vitro and in vivo.
  • the kit includes the reagents for a method of the present invention for producing a cellular composition.
  • This kit preferably would include at least one homing gene, e.g.of SCG 10; Na Channel II; glut-2, synapsin, epo, SCF , shh, wint, BMPs Ephrins, Pax-6, Emx-2, Mash -1, jagged 1 and 2, notch- and 2, ephrin B2 and ephrin B4, Bmi-1, and HOXB4. Positive growth factors can also be included, and instructions for use.
  • cells, cell preparations, and cellular compositions disclosed herein can be used for toxicity testing for drug development testing.
  • Toxicity testing may be conducted by culturing cells, cell preparations, and cellular compositions or cells differentiated therefrom in a suitable medium and introducing a substance, such as a pharmaceutical or chemical, to the culture. The cells or differentiated cells are examined to determine if the substance has had an adverse effect on the culture.
  • Drug development testing may be done by developing derivative cell lines which may be used to test the efficacy of new drags. Affinity assays for new drags may also be developed from the cells, differentiated cells, or cell lines.
  • the cellular compositions of the invention may be used to screen for potential therapeutics that modulate development or activity of cells with the potential to form non- hematopoietic cells or cells differentiated there from.
  • the cells of a cellular composition of the invention may be subjected to a test substance, and the effect of the test substance may be compared to a control (e.g. in the absence of the substance) to determine if the test substance modulates development or activity of the cells with the potential to form non-hematopoietic cells or cells differentiated therefrom.
  • a method for using cells with the potential or increased potential to form non-hematopoietic cells or cells differentiated therefrom to assay the activity of a test substance comprising the steps of a) culturing cells in an enriched hematopoietic cell preparation comprising hematopoietic stem cells and progenitor cells under proliferation conditions to obtain a cellular composition comprising cells which have potential or increased potential to form non-hematopoietic cells; b) optionally culturing the cells which have potential or increased potential to form non- hematopoietic cells under differentiation conditions in vitro; c) exposing the cultured cells in step (a) or (b) to a test substance; and d) detecting the presence or absence of an effect of the test substance on the survival of the cells or on a morphological functional, or physiological characteristic and/or molecular biological property of the cells, whereby an effect altering cell survival, a morphological, functional, or physiological characteristic and/or
  • a method for using cells with the potential or increased potential to form non-hematopoietic cells or cells differentiated therefrom to screen a potential new drag to treat a disorder involving the non-hematopoietic cells comprising the steps of: (a) obtaining hematopoietic cells from a sample from a patient with a disorder involving non-hematopoietic cells; (b) preparing from the hematopoietic cells an enriched hematopoietic cell preparation comprising hematopoietic stem cells and progenitor cells; (c) culturing the enriched hematopoietic cell preparation under proliferation conditions to obtain cells with potential or increased potential to form the non-hematopoietic cells; (d) optionally culturing the cells with potential or increased potential to form the non-hematopoietic cells under differentiation conditions in vitro; (e) exposing the cultured cells in (c) or (d) to a potential new drug; and (f
  • the invention also relates to the use of cells, cell preparations, and cellular compositions in drug discovery.
  • the invention provides methods for drag development using the cells, cell preparations, and cellular compositions of the invention.
  • Cells, cell preparations, and cellular compositions of the invention may comprise cells that secrete novel or known biological molecules or components.
  • culturing in the absence of serum may provide cells that, have minimal interference from serum molecules and thus, may be more physiologically and topologically-accurate. Therefore, proteins secreted by cells described herein maybe used as targets for drug development.
  • drugs can be made to target specific proteins on cells that have the potential or increased potential to form non-hematopoietic cells.
  • Binding of the drug may promote differentiation of cells into specific non-hematopoietic cells.
  • drags specific for regulatory proteins of non-hematopoietic cells may be used to arrest growth of a particular type of cell. Any of the proteins can be used as targets to develop antibody, protein, antisense, aptamer, ribozymes, or small molecule drags.
  • Agents, test substances, or drugs identified in accordance with a method of the invention or used in a method of the invention include but are not limited to proteins, peptides such as soluble peptides including Ig-tailed fusion peptides, members of random peptide libraries and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids, phosphopeptides (including members of random or partially degenerate, directed phosphopeptide libraries), antibodies [e.g. polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, single chain antibodies, fragments, (e.g.
  • An agent, substance or drag may be an endogenous physiological compound or it may be a natural or synthetic compound.
  • the cells, cell preparations, and cellular compositions disclosed herein can be used in various bioassays.
  • the cells are used to determine which biological factors are required for proliferation or differentiation.
  • the cells are used to determine which biological factors are required for proliferation or differentiation.
  • different biological compounds such as hormones, specific growth factors, etc.
  • one or more specific biological compounds can be found to induce differentiation to non-hematopoietic cells.
  • Other uses in a bioassay for the cells are differential display (i.e. mRNA differential display) and protein-protein interactions using secreted proteins from the cells. Protein-protein interactions can be determined with techniques such as a yeast two-hybrid system.
  • Proteins from cells, cell preparations and cellular compositions of the invention can be used to identify other unknown proteins or other cell types that interact with the cells. These unknown proteins may be one or more of the following: growth factors, hormones, enzymes, transcription factors, translational factors, and tumor suppressors. Bioassays involving cells, cell preparations, and cellular compositions of the invention, and the protein-protein interactions these cells form and the effects of protein- protein or cell-cell contact may be used to determine how surrounding tissue contributes to proliferation or differentiation of non-hematopoietic and hematopoietic cells.
  • cells with potential or increased potential for forming non-hematopoietic cells obtained after culturing a preparation from cord blood stem cells maybe used to repair cell or tissue injury. They may also be used in the treatment of genetic defects that result in nonfunctional cells.
  • the cord blood stem cells grown in proliferation medium may be transplanted directly to the site of defective cells in order to rescue the defect or delivered via the blood stream by injecting the cells into the vein.
  • gene therapy vectors may be integrated into the cord blood stem cells followed by engraftment of these engineered cells to their target tissues. The introduction of gene therapy vectors requires cell proliferation. The successful long term engraftment of the cells to the target tissue requires they maintain a stem cell characteristic. High proliferation rates of cord stem cells has been achieved without differentiation, which should lead to successful gene therapy.
  • hepatocytes obtained from differentiating cells of a cellular composition of the invention may be used to restore a degree of liver function to a subject needing such therapy, perhaps due to an acute, chronic, or inherited impairment of liver function.
  • they may be used to treat liver disease or repair liver damage.
  • hepatocytes obtained in accordance with the present invention may be used to treat a number of degenerative liver diseases. Non- functional liver cells where there is no apparent physical damage may be treated through partial hepatectomy, followed by therapy using hepatocytes obtained using the present invention.
  • the hepatocytes may be encapsulated, or part of a bioartificial liver device.
  • endothelial cells obtained from differentiating cells of a cellular composition of the invention may be used for vascular repair and they can be used in cardiopulmonary bypass surgery. Endothelial cells may be transfected with genes which produce angiogenic factors and used in gene therapy to stimulate angiogenesis in patients with vascular or cardiac insufficiency.
  • muscle cells obtained from differentiating cells of a cellular composition of the invention may be employed to repair muscle, in particular striated or cardiac muscle.
  • the present invention may be used to treat degenerative muscle disease.
  • the cells may be used in treating muscular dystrophy, cardiomyopathy, congestive heart failure; and myocardial infarction, for example.
  • Genetic muscle disorders and cardiac muscle disorders may be treated using precursor muscle cells obtained using methods of the present invention. If muscle loss is due to the lack of neuronal connection (neuro-muscular disease), both the neural and muscle tissues can be replaced using cells obtained using the present invention.
  • neural cells obtained from differentiating cells of a cellular composition of the invention preferably derived from umbilical cord blood, or precursor cells thereof maybe used for treating neurodegenerative disorders, a brain or spinal cord injury, or neurological deficit.
  • Neurodegenerative disorders which can be treated include for example, Parkinson's disease, Huntington's disease, multiple sclerosis, Alzheimer's disease, Tay Sach's disease, lysosomal storage disease, brain and/or spinal cord injury due to ischemia, stroke, head injury, cerebral palsy, spinal cord and brain damage/injury, depression, epilepsy, schizophrenia, and ataxia and alcoholism.
  • Neural cells generated in accordance with a method of the invention may be transfected with a vector that can express growth factors, growth factor receptors, and peptide neurotransmitters, or express enzymes involved in the synthesis of neurotransmitters. These transfected cells maybe transplanted into regions of neurodegeneration.
  • bone or cartilage cells obtained from differentiating cells of a cellular composition of the invention preferably derived from umbilical cord blood, or precursor cells thereof, may be used to repair bone, and in reconstructive surgery or degenerative diseases. Artificial substrates or matrices can be used in combination with the cells to reconstitute tissues, implanted into the joints of patients to replace or repair damaged or deficient cartilage.
  • the cartilage cells may be useful in the treatment of diseases of the joint, for example, osteoarthritis, inflammatory arthropathies, septic arthritis, and crystalline arthropathies, and they can be used to enhance healing of bone fractures when inserted into the site of a fracture.
  • the cells can also be used in the study and treatment of chondrodysplasias, and to test angiogenic factors.
  • Retinal cells or precursor cells thereof generated in accordance with a method of the invention may be used to restore vision lost when retinal cells are damaged, (see, the examples which follow) and they can be used as in vivo targets for stimulation by growth factors in order to produce healthy tissue.
  • the cells may be used to treat conditions such as glaucoma, macular degeneration, diabetic retinopathies, inherited retinal degeneration such as retinitis pigmentosa, retinal detachment or injury and retinopathies (whether inherited, induced by surgery, trauma, a toxic compound or agent, or, photically; in particular, diabetic retinopathy).
  • the stem cells express the products of homing genes, such as:SCG 10; Na Channel II; glut-2, synapsin, epo, SCF , shh, wint, BMPs Ephrins, Pax-6, Emx-2, Mash -1, jagged 1 and 2, notch- and 2, ephrin B2 and ephrin B4, Bmi-1, and HOXB4
  • homing genes such as:SCG 10; Na Channel II; glut-2, synapsin, epo, SCF , shh, wint, BMPs Ephrins, Pax-6, Emx-2, Mash -1, jagged 1 and 2, notch- and 2, ephrin B2 and ephrin B4, Bmi-1, and HOXB4
  • Connective tissue cells or precursor cells thereof, generated in accordance with a method of the invention may be seeded onto matrices or substrates and used to repair or regenerate damaged tissue (e.g. tendons).
  • tissue e.g. tendons
  • the invention contemplates a method for de novo formation of connective tissue in vivo by introducing connective tissue cells produced by a method of the invention into a site for de novo connective tissue formation in a patient in need thereof.
  • Renal cells or precursors thereof, generated in accordance with a method of the invention may be used to treat kidney disorders or damage, or renal cancer.
  • the cells, or tissue or a functioning kidney regenerated therefrom, may be administered to a patient to treat acute or chronic decline in renal function.
  • Functional renal cells or regenerated kidney can be implanted into the donor of the hematopoietic cells from which the renal cells are derived or into another patient.
  • Renal cells or precursors there of may be used to construct an artificial kidney system (e.g. a system based on a hollow fiber filtration system.
  • Corneal cells or precursors thereof, generated in accordance with a method of the invention may be used to treat a variety of corneal and/or conjunctival epithelial cell injuries, degenerations and/or abnormalities, including subjects having ocular surface diseases such as Stevens- Johnson's Syndrome, chemical and thermal burns, ocular surface tumors, immunological conditions, radiation injury, inherited syndromes such as aniridia, ocular pemphigoid, macular degeneration, and the like.
  • the corneal cells or precursors thereof may be particularly useful in treating patients where the normal stem cell population of the corneal limbus is depleted, non-functional or otherwise inadequate to promote healing of the corneal damage.
  • the cells, cell preparations, and cellular compositions of the invention may be used as immunogens that are administered to a heterologous recipient.
  • Administration of non-hematopoietic and hematopoietic cells obtained in accordance with the invention may be accomplished by various methods.
  • Methods of administering cells as immunogens to a heterologous recipient include without limitation immunization, administration to a membrane by direct contact (e.g. by swabbing or scratch apparatus), administration to mucous membranes (e.g. by aerosol), and oral administration.
  • Immunization may be passive or active and may occur via different routes including intraperitoneal injection, intradermal injection, and local injection.
  • the route and schedule of immunization are in accordance with generally established conventional methods for antibody stimulation and production.
  • Mammalian subjects, particularly mice, and antibody producing cells therefrom may be manipulated to serve as the basis for production of mammalian hybridoma cell lines.
  • the cellular compositions of the invention may be used to prepare model systems of disease.
  • the cellular compositions of the invention can also be used to produce growth factors, hormones, etc.
  • the invention provides a culture system from which genes, proteins, and other metabolites involved in proliferation or differentiation of hematopoietic or non- hematopoietic cells can be identified and isolated.
  • the cells in a culture system of the invention may be compared with other cells (e.g. differentiated cells) to determine the mechanisms and compounds that stimulate production of non-hematopoietic and hematopoietic cells.
  • the cellular compositions of the invention can be used to screen for genes expressed in or essential for differentiation of non-hematopoietic cells. Screening methods that can be used include Representational Difference Analysis (RDA) or gene trapping with for example SA-lacZ (25). Gene trapping can be used to induce dominant mutations (e.g. by deleting particular domains of the gene product) that affect differentiation or activity of non- hematopoietic cells and allow the identification of genes expressed in or essential for differentiation of these cells.
  • RDA Representational Difference Analysis
  • SA-lacZ 25
  • Gene trapping can be used to induce dominant mutations (e.g. by deleting particular domains of the gene product) that affect differentiation or activity of non- hematopoietic cells and allow the identification of genes expressed in or essential for differentiation of these cells.
  • the expanded cell preparations of the invention comprising increased numbers of hematopoietic stem cells and progenitor cells may be used for enhancing the immune system of a patient. The cell preparations will facilitate enhancement or recon
  • the cellular compositions of the invention are used in the treatment of leukemia (e.g. acute myelogenous leukemia, chronic myelogenous leukemia), lymphomas (e.g. non-Hodgkii ⁇ s lymphoma), neuroblastoma, testicular cancer, multiple myeloma, melanomas, breast cancer, solid tumors that have a stem cell etiology, or other cancers in which therapy results in the depletion of hematopoietic cells.
  • leukemia e.g. acute myelogenous leukemia, chronic myelogenous leukemia
  • lymphomas e.g. non-Hodgkii ⁇ s lymphoma
  • neuroblastoma e.g. non-Hodgkii ⁇ s lymphoma
  • testicular cancer multiple myeloma
  • melanomas melanomas
  • breast cancer solid tumors that have a stem cell etiology, or other cancers in which
  • a cellular composition of the invention with or without genetic modification to provide resistance to HIV, is used to treat subjects infected with HIV-I that have undergone severe depletion of their hematopoietic cell compartment resulting in a state of immune deficiency.
  • the hematopoietic stem cells and progenitor cells in the expanded cell preparation may also be transfected with a desired gene that can be used for treatment of genetic diseases.
  • Hematopoietic cell-related genetic diseases can be treated by grafting the expanded cell preparation with cells transfected with a gene that can make up for the deficiency or the abnormality, of the gene causing the diseases.
  • a normal wild type gene that causes a disease such as .beta.
  • -thalassemia (Mediterranean anemia), sickle cell anemia, ADA deficiency, recombinase deficiency, recombinase regulatory gene deficiency and the like, can be transferred into the hematopoietic stem cells or progenitor cells by homologous or random recombination and the cells can be grafted into a patient. Further, a preparation comprising normal hematopoietic stem cells and progenitor cells free from abnormalities of genes (from a suitable donor) can be used for treatment.
  • Another application of gene therapy permits the use of a drug in a high concentration, which is normally considered to be dangerous, by providing drug resistance to normal hematopoietic stem cells by transferring a drag resistant gene into the cells.
  • a drug in a high concentration, which is normally considered to be dangerous
  • it is possible to carry out the treatment using an anticancer drag in high concentration by transferring a gene having drag resistance against the anticancer drug, e.g., a multiple drag resistant gene into an expanded cell preparation comprising hematopoietic stem cells and progenitor cells.
  • a deficient protein can be induced and expressed by transferring a gene encoding a target protein into the hematopoietic stem cells or progenitor cells under the control of a suitable promoter. The expression of the protein can be controlled to obtain the same activity as that obtained by the natural expression in vivo.
  • a gene encoding a ribozyme, an antisense nucleic acid or the like or another suitable gene into the hematopoietic stem cells or progenitor cells to control expression of a specific gene product in the cells or to inhibit susceptibility to diseases.
  • the hematopoietic stem cells and progenitor cells can be subjected to gene modification to express an antisense nucleic acid or a ribozyme, which can prevent growth of hematic pathogens such as HIV, HTLV-I, HTLV-II and the like in hematopoietic stem cells or cells differentiated from hematopoietic stem cells.
  • the cell preparations comprising hematopoietic stem cells and progenitor cells can be introduced in a vertebrate, which is a recipient of cell grafting, by, for example, conventional intravenous administration.
  • the invention also relates to a method for conducting a regenerative medicine business, comprising: (a) a service for accepting and logging in samples from a client comprising hematopoietic cells capable of forming cells that have the potential to form hematopoietic and non-hematopoietic cells; (b) a system for culturing cells dissociated from the samples, which system provides conditions for producing cells that have the potential to form hematopoietic and non-hematopoietic cells; (c) a cell preservation system for preserving cells generated by the system in (b) for later retrieval on behalf of the client or a third party.
  • the method may further comprise a billing system for billing the client or a medical insurance provider thereof.
  • the invention features a method for conducting a stem cell business comprising identifying agents which influence the proliferation, differentiation, or survival of cells that have the potential to form hematopoietic and non-hematopoietic cells.
  • agents are small molecules, antibodies, and extracellular proteins.
  • Identified agents can be profiled and assessed for safety and efficacy in animals.
  • the invention contemplates methods for influencing the proliferation, differentiation, or survival of cells that have the potential to form hematopoietic and non-hematopoietic cells by contacting the cells with an agent or agents identified by the foregoing method.
  • the identified agents can be formulated as a pharmaceutical preparation, and manufactured, marketed, and distributed for sale.
  • the invention provides a method for conducting a stem cell business comprising (a) identifying one or more agents which affect the proliferation, differentiation, function, or survival of cells that have the potential to fo ⁇ n hematopoietic and non-hematopoietic cells of the invention; (b) conducting therapeutic profiling of agents identified in (a); or analogs thereof for efficacy and toxicity in animals; and (c) formulating a pharmaceutical composition including one or more agents identified in (b) as having an acceptable therapeutic profile.
  • the method may further comprise the step of establishing a distribution system for distributing the pharmaceutical preparation for sale.
  • the method may also comprise establishing a sales group for marketing the pharmaceutical preparation.
  • the invention also contemplates a method for conducting a drug discovery business comprising identifying factors that influence the proliferation, differentiation, function, or survival of cells that have the potential to form hematopoietic and non-hematopoietic cells of the invention, and licensing the rights for further development.
  • Non-Integrating Vectors can be avoided by: (a) transiently transfecting the stem cells with the homing gene, such as SCG 10; Na Channel II; glut-2, synapsin, epo, SCF , shh, wint, BMPs Ephrins, Pax-6, Emx-2, Mash -1, jagged 1 and 2, notch- and 2, ephrin B2 and eplirin B4, Bmi-1, HOXB4.
  • the homing gene such as SCG 10; Na Channel II; glut-2, synapsin, epo, SCF , shh, wint, BMPs Ephrins, Pax-6, Emx-2, Mash -1, jagged 1 and 2, notch- and 2, ephrin B2 and eplirin B4, Bmi-1, HOXB4.
  • plasmid DNA or messenger RNA activating the expression of the endogenous gene with in the stem cell genome using either small molecules derived from a chemical library and screened for specific activation of the desired gene, or adding a recombinant protein or peptide that transcriptionally activates the expression of the homing protein.
  • This can either be a zinc finger-transcription activator fusion protein or a specific transcription activator protein that binds to the enhancer or promoter region responsible for controlling expression of the homing protein.
  • Example 1 Homing of stem cells to sites of retinal pigment epithelium (RPE) cell injury and maturation into RPE when transduced to express an RPE-specific protein.
  • RPE retinal pigment epithelium
  • HSC Hematopoietic stem cells
  • progeny, endothelial precursor cells have marked plasticity and are involved in tissue repair.
  • Embryonic stem cells can be forced to differentiate into specific cell types by infecting the cells with viral vectors expressing terminal differentiation markers.
  • RPE65 is apparently sufficient to drive the differentiation of HSC to RPE.
  • No gfp+ endothelial cells also expressed RPE65.
  • FIG. 1 shows electroretinograms from three different treatment groups of five mice.
  • the electroretinograms analyzes the function of the retinal pigmented epithelial cells. Strong, medium and dim light is flashed at each eye. There are five flashes 30 seconds apart. The depolarization of the membrane is measured over time (microseconds).
  • the null treatment group are mice that received sodium iodate but no endothelial stem cells.
  • the lac-Z treatment group received endothelial stem cells infected with a LacZ lenti virus as a control for lenti viral infected cells.
  • the RPE65 group are mice that received endothelial stem cells infected with RPE65 lentivirus.
  • the electroretinograms are from mice receiving the dim flash. The response is the average of the five flashes for each treatment group.

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Abstract

La présente invention concerne des compositions de cellules souches comprenant un gène particulier au tissu greffé désiré, qui est introduit dans la cellule souche soit ex vivo soit in vivo. En conséquence de l'expression du gène particulier, la cellule souche se niche et se différencie en le type de cellule souhaité en un emplacement spécifique in vivo.
PCT/US2006/016235 2005-04-28 2006-04-28 Ciblage tissulaire de cellules souches WO2006116678A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007090647A1 (fr) * 2006-02-08 2007-08-16 Universität Ulm TRANSFECTION D'ARNm DE CELLULES PROGÉNITRICES ADULTES POUR UNE RÉGÉNÉRATION TISSULAIRE SPÉCIFIQUE
US7816140B2 (en) * 2005-06-14 2010-10-19 The United States Of America As Represented By The Department Of Veterans Affairs Composition and methods for osteogenic gene therapy
JP2013535184A (ja) * 2010-07-07 2013-09-12 ドイチェス クレブスフォルシュンクスツェントルム がん幹細胞（ｃｓｃ）を除去するためのパルボウイルスの使用
US9603899B2 (en) 2010-10-01 2017-03-28 The Trustees Of Columbia University In The City Of New York PDGF induced cell homing

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US6153383A (en) * 1997-12-09 2000-11-28 Verdine; Gregory L. Synthetic transcriptional modulators and uses thereof
US20020086383A1 (en) * 2000-02-23 2002-07-04 Guy Sauvageau Hematopoietic stem cell expansion enhancing factor and method of use
US20020099196A1 (en) * 1996-08-27 2002-07-25 Kyowa Hakko Kogyo Co., Ltd. And Atsunobu Hiraoka Hematopoietic stem cell growth factor (scgf)
US20050063961A1 (en) * 2002-07-25 2005-03-24 The Scripps Research Institute Hematopoietic stem cells and methods of treatment of neovascular eye diseases therewith

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US20020099196A1 (en) * 1996-08-27 2002-07-25 Kyowa Hakko Kogyo Co., Ltd. And Atsunobu Hiraoka Hematopoietic stem cell growth factor (scgf)
US6153383A (en) * 1997-12-09 2000-11-28 Verdine; Gregory L. Synthetic transcriptional modulators and uses thereof
US20020086383A1 (en) * 2000-02-23 2002-07-04 Guy Sauvageau Hematopoietic stem cell expansion enhancing factor and method of use
US20050063961A1 (en) * 2002-07-25 2005-03-24 The Scripps Research Institute Hematopoietic stem cells and methods of treatment of neovascular eye diseases therewith

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7816140B2 (en) * 2005-06-14 2010-10-19 The United States Of America As Represented By The Department Of Veterans Affairs Composition and methods for osteogenic gene therapy
US8772571B2 (en) 2005-06-14 2014-07-08 The United States Of America As Represented By The Department Of Veterans Affairs Compositions and methods for osteogenic gene therapy
US9458215B2 (en) 2005-06-14 2016-10-04 Loma Linda University Compositions and methods for osteogenic gene therapy
WO2007090647A1 (fr) * 2006-02-08 2007-08-16 Universität Ulm TRANSFECTION D'ARNm DE CELLULES PROGÉNITRICES ADULTES POUR UNE RÉGÉNÉRATION TISSULAIRE SPÉCIFIQUE
JP2013535184A (ja) * 2010-07-07 2013-09-12 ドイチェス クレブスフォルシュンクスツェントルム がん幹細胞（ｃｓｃ）を除去するためのパルボウイルスの使用
US9603899B2 (en) 2010-10-01 2017-03-28 The Trustees Of Columbia University In The City Of New York PDGF induced cell homing

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