WO1996033281A1 - Transduction ex vivo a productivite elevee de cellules souches hematopoietiques au moyen de preparations retrovirales xenotropes recombinees - Google Patents

Transduction ex vivo a productivite elevee de cellules souches hematopoietiques au moyen de preparations retrovirales xenotropes recombinees Download PDF

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WO1996033281A1
WO1996033281A1 PCT/US1996/005432 US9605432W WO9633281A1 WO 1996033281 A1 WO1996033281 A1 WO 1996033281A1 US 9605432 W US9605432 W US 9605432W WO 9633281 A1 WO9633281 A1 WO 9633281A1
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hematopoietic stem
stem cells
cells
population
recombinant
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PCT/US1996/005432
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Douglas J. Jolly
Joan M. Robbins
William G. Kerr
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Chiron Corporation
Systemix
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Priority to AU55571/96A priority Critical patent/AU5557196A/en
Priority to JP8531931A priority patent/JPH11514209A/ja
Priority to EP96912909A priority patent/EP0821740A1/fr
Publication of WO1996033281A1 publication Critical patent/WO1996033281A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2013—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/217—IFN-gamma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/37—Factors VIII
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/44—Vectors comprising a special translation-regulating system being a specific part of the splice mechanism, e.g. donor, acceptor

Definitions

  • the present invention relates generally to recombinant retroviruses and gene therapy, and more specifically, to recombinant retroviral particle preparations suitable for a variety of somatic cell gene therapy applications.
  • somatic gene therapy a process sometimes referred to as "somatic gene therapy"
  • viral vectors derived from retroviruses, adenoviruses, vaccinia viruses, herpes viruses, and adeno-associated viruses see Jolly, Cancer Gene Therapy 7(1):51-64, 1994
  • direct transfer techniques such as lipofection (Feigner et al. , Proc. Natl. Acad. Sci.
  • recombinant retroviral gene delivery methods have been most extensively utilized, in part due to: (1) the efficient entry of genetic material (the vector genome) into replicating cells; (2) an active, efficient process of entry into the target cell nucleus; (3) relatively high levels of gene expression; (4) the potential to target particular cellular subtypes through control of the vector-target cell binding and the tissue-specific control of gene expression; (5) a general lack of pre-existing host immunity; and
  • retroviruses are diploid positive-strand RNA viruses that replicate through an integrated DNA intermediate.
  • the retroviral genome is reverse-transcribed into DNA by a virally encoded reverse transcriptase that is carried as a protein in each retrovirus.
  • the viral DNA is then integrated pseudo-randomly into the host cell genome of the infecting cell, forming a "provirus" which is inherited by daughter cells.
  • Wild-type retroviral genomes (and their proviral copies) contain three genes (the gag, ol and env genes), which are preceded by a packaging signal ( ⁇ ), and two long terminal repeat (LTR) sequences which flank both ends.
  • the gag gene encodes the internal structural (nucleocapsid) proteins.
  • the pol gene codes for the RNA-dependent DNA polymerase which reverse transcribes the RNA genome, and the env gene encodes the retroviral envelope glycoproteins.
  • the 5' and 3' LTRs contain cis-acting elements necessary to promote transcription and polyadenylation of retroviral RNA.
  • Adjacent to the 5' LTR are sequences necessary for reverse transcription ofthe genome (the tRNA primer binding site) and for efficient encapsidation of retroviral RNA into particles (the psi sequence). Removal of the packaging signal prevents encapsidation (packaging of retroviral RNA into infectious virions) of genomic RNA, although the resulting mutant can still direct synthesis of all proteins encoded in the viral genome.
  • a foreign gene of interest may be incorporated into the retrovirus in place of a portion of the normal retroviral RNA.
  • the retrovirus injects its RNA into a cell, the foreign gene is also introduced into the cell, and may then be integrated into the host's cellular DNA as if it were the retrovirus itself.
  • retroviruses principally infect only replicating cells, thereby making efficient direct gene transfer difficult or impossible for cells characterized as largely non-replicating, such as hematopoietic stem cells. Indeed, some scientists have suggested that other, more efficient methods of gene transfer, such as direct administration of pure plasmid DNA, be utilized (Davis et al., Human Gene Therapy 4:133- 740, 1993) to introduce nucleic acid molecules into such cells.
  • the methods which have been suggested have principally been aimed at inducing the desired target cells to replicate, thereby allowing the retroviruses to infect the cells.
  • Such methods have included, for example chemical treatment with 10% carbon tetrachloride in mineral oil (Kaleko, et al. , Human Gene Therapy 2:21-32, 1991).
  • such techniques are not preferred for use in ex vivo techniques designed to introduce nucleic acid molecules encoding therapeutic gene products into mammalian hematopoietic cells.
  • Mammalian hematopoietic cells provide a diverse range of physiological activities. These cells are divided into lymphoid, myeloid and erythroid lineages.
  • the lymphoid lineages comprising B-cells, T-cells, and NK-cells, provides for the production of antibodies, regulation of the cellular immune system, detection of foreign agents in the blo od, detection of cells foreign to the host, and the like.
  • the myeloid lineage which includes monocytes, granulocytes, megakaryocytes as well as other 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 hematopoietic progenitor cells
  • stem cells are capable of self-regeneration and may become lineage restricted progenitors, which further differentiate and expand into specific lineages.
  • stem cells or “hematopoietic stem cells” refers to hematopoietic cells and not stem cells of other cell types. Further, unless indicated otherwise, “stem cells” refers to human hematopoietic stem cells.
  • Hematopoietic cells constitute only a small percentage of the total number of hematopoietic cells. Hematopoietic cells are identifiable by the presence of a variety of cell surface
  • markers Such markers may be either specific to a particular lineage or progenitor cell or be present on more than one cell type.
  • CD34 is a marker found on stem cells and a significant number of more differentiated progenitor cells.
  • U.S. Patent No. 4,714,680 describes a population of cells expressing the CD34 marker.
  • Table 1 summarizes probable phenotypes of stem cells in fetal, adult, and mobilized peripheral blood.
  • myelomonocytic stands for myelomonocytic associated markers
  • NK stands for natural killer cells
  • FBM and ABM refer to fetal and adult bone marrow, respectively
  • AMPB stands for adult mobilized peripheral blood.
  • the negative sign or, uppercase negative sign, (-) means that the level of the specified marker is undetectable above Ig isotype controls by FACS analysis, and includes cells with very low expression of the specified marker.
  • stem cells The ability of stem cells to undergo substantial self-renewal as well as the ability to proliferate and differentiate into all of the hematopoietic lineages makes stem cells the target of choice for a number of gene therapy applications.
  • Successful gene transfer into stem cells should provide long-term repopulation of an individual with the modified cells and their progeny, which will express the desired gene product.
  • gene transfer into more mature hematopoietic cells, such as T-cells provides only transient therapeutic benefit.
  • retroviral vectors have been the primary vehicle due to the generally high rate of gene transfer obtained in experiments with cell lines, and the ability to obtain stable integration of the genetic material, which ensures that the progeny of the modified cell will contain the transferred genetic material.
  • retroviral vectors have been the primary vehicle due to the generally high rate of gene transfer obtained in experiments with cell lines, and the ability to obtain stable integration of the genetic material, which ensures that the progeny of the modified cell will contain the transferred genetic material.
  • stem cells typically are quiescent in culture; retroviral vectors require target cells to be cycling for stable integration of the retroviral DNA.
  • Cytokines have been used to cause stem cells to cycle, which improves gene transfer efficiency, but the effect of various cytokines in driving stem cells to differentiation remains in question.
  • the range of host cells that may be infected by a retrovirus or retroviral vector is determined by the viral envelope protein. Therefore, a lack or deficiency of the receptor for the given envelope protein would limit transduction efficiency. In addition, a lack ofthe requisite cellular factors involved in viral binding, penetration, uncoating of the retroviral vectors, viral replication or integration would limit transduction efficiency.
  • the transduced stem cells may then be re- administered to the patient by standard techniques, e.g., intravenous infusion to achieve a desired therapeutic benefit.
  • the present invention provides compositions and methods for transducing hematopoetic stem cells
  • a method for the production of transduced hematopoetic stem cells comprising obtaining a population of hematopoetic stem cells from a patient and transducing the population of hematopoetic stem cells with a recombinant retroviral particles substantially free from contamination with replication competent retrovirus, wherein the recombinant retroviral particles carry a vector construct encoding a gene of interest.
  • the vector construct encodes a molecule selected from the group consisting of a protein, an active portion of a protein and a RNA molecule with intrinsic biological activity.
  • the protein or active portion of a protein is selected from the group consisting of a cytokine, a colony stimulating factor, a clotting factor, and a hormone.
  • methods are provided for treating a genetic disease, cancer, infectious disease, degenerative disease, inflammatory disease, cardiovascular disease, and autoimmune disease by administering to a patient a composition or reintroduction of a therapeutically effective amount of the population of transduced hematopoetic stem cells.
  • the transduced population of hematopoetic stem cells are characterized as CD34 + Thy-1 + Lin".
  • the recombinant reroviral vectors used to the transduce the hematopoitic stem cells are xenotropic retroviral vectors.
  • the rerotroviral vector preparations used to transduce the hematopoietic stem cells are high titer preparations.
  • the hematopoetic stem cells are expanded in vitro prior to reintroduction of the cells into the patient.
  • an in vivo delivery vehicle comprising
  • transplantable hematopoetic stem cells which express a therapeutically effective amount of a gene product encoded by a gene wherein the gene does not occur in hematopoetic stem cells or where the gene occurs in hematopoetic stem cells but is not expressed in the cells and wherein the gene can be modified to be expressed in hematopoetic stem cells is provided.
  • the transduced hematopoetic stem cells and compositions of hematopoetic stem cells encoding a gene of interest, hematopoetic stem cells transduced with a recombinant xenotropic retroviral vector, and hematopoetic stem cells characterized as CD34 + Thy-1 + Lin" transduced with recombinant retroviral particles are provided.
  • compositions are provided substantially free from contamination with replication competent retrovirus.
  • Event-specific promoter refers to transcriptional promoter/enhancer or locus defining elements, or other elements which control gene expression as discussed above, whose transcriptional activity is altered upon response to cellular stimuli.
  • Representative examples of such event-specific promoters include thymidine kinase or thymidylate syntheses promoters, alpha or beta interferon promoters and promoters that respond to the presence of hormones (either natural, synthetic or from other non-host organisms).
  • tissue-specific promoter refers to transcriptional promoter/enhancer or locus defining elements, or other elements which control gene expression as discussed above, which are preferentially active in a limited number of hematopoietic tissue types.
  • hematopoietic tissue-specific promoters include, but are not limited to, the IgG promoter, ⁇ - or ⁇ -globin promoters, T-cell receptor promoter, Granzyme A, Granzyme B, CD8, and CD11b.
  • Transduction involves the association of a replication defective, recombinant retroviral particle with a cellular receptor, followed by introduction of the nucleic acids carried by the particle into the cell.
  • Transfection refers to a method of physical gene transfer wherein no retroviral particle is employed.
  • Vector construct refers to a nucleic acid construct capable of directing the expression ofa gene of interest.
  • the retroviral vector must include at least one transcriptional
  • promoter/enhancer or locus defining element(s), or other elements which control gene expression by other means such as alternate splicing, nuclear RNA export, post-translational modification of messenger, or post-transcriptional modification of protein.
  • Such vector constructs must also include a packaging signal, long terminal repeats (LTRs) or portion thereof, and positive and negative strand primer binding sites appropriate to the retrovirus used (if these are not already present in the retroviral vector).
  • the vector construct may also include a signal which directs polyadenylation, as well as one or more restriction sites and a translation termination sequence.
  • such vectors will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second strand DNA synthesis, and a 3' LTR or a portion thereof
  • a gene of interest encoding the desired gene product is also included.
  • the present invention is based on the unexpected discovery that recombinant retroviral particles carrying a vector construct comprising a gene of interest can be used ex vivo to efficiently transduce hematopoietic stem cells.
  • recombinant retroviral particles according to the invention can be used for purposes of somatic gene therapy.
  • a more thorough description of such recombinant retroviral particles, their production and packaging, and uses therefore is provided below.
  • the present invention provides compositions and methods comprising recombinant retroviral particles, including recombinant xenotropic retroviral vector particles, for use in ex vivo somatic gene therapy.
  • recombinant retroviral vectors and particles are described in greater detail in an application entitled "Recombinant Retroviruses".
  • Production of transduction competent recombinant xenotropic retroviral particles is described in U.S.S.N. 08/156,789, and U.S.S.N. 07/965,084, which are hereby incorporated by reference in their entirety.
  • the recombinant vector constructs described herein are prepared by selecting a plasmid with a strong promoter, and appropriate restriction sites for insertion of DNA sequences of interest downstream from the promoter.
  • the recombinant vector construct is carried by a recombinant retrovirus.
  • Retroviruses are RNA viruses with a single positive strand genome which in general, are nonlytic. Upon infection, the retrovirus reverse transcribes its RNA into DNA, forming a provirus which is inserted into the host cell genome.
  • the retroviral genome can be divided conceptually into two parts
  • the "trans-acting" portion consists of the region coding for viral structural proteins, including the group specific antigen (gag) gene for synthesis of the core coat proteins; the pol gene for the synthesis of the reverse transcriptase and integrase enzymes; and the envelope (env) gene for the synthesis of envelope glycoproteins
  • the "cis-acting" portion consists of regions of the genome that is finally packaged into the viral particle These regions include the packaging signal, long terminal repeats (LTR) with promoters and polyadenylation sites, and two start sites for DNA replication
  • LTR long terminal repeats
  • the internal or "trans-acting" part of the cloned provirus is replaced by the gene of interest to create a "vector construct" When the vector construct is placed into a cell where viral packaging proteins are present, the transcribed RNA will be packaged as a viral particle which , in turn, will bud off from the cell.
  • the particles are used to transduce tissue cells, allowing the vector construct to integrate into the cell genome. Although the vector construct expresses its gene product, the virus carrying it is replication defective because the trans-acting portion of the viral genome is absent.
  • Various assays may be utilized in order to detect the presence of any replication competent in fectious retrovirus.
  • One preferred assay is the extended S + L- assay described in Example 4.
  • the retroviral vectors of the invention comprise a
  • Such vector constructs must also include a packaging signal, long terminal repeats (LTRs) or portion thereof, and positive and negative strand primer binding sites appropriate to the retrovirus used (if these are not already present in the retroviral vector).
  • the vector construct may also include a signal which directs polyadenylation, as well as one or more restriction sites and a translation termination sequence.
  • such vectors will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second strand DNA synthesis, and a 3' LTR or a portion thereof.
  • Such vectors do not contain one or more of a complete gag, pol, or env gene, thereby rendering them replication
  • nucleic acid molecules coding for a selectable marker are neither required nor preferred.
  • Preferred retroviral vectors contain a portion of the gag coding sequence, preferably that portion which comprises a splice donor and splice acceptor site, the splice acceptor site being positioned such that it is located adjacent to and upstream from the gene of interest.
  • the gag transcriptional promoter is positioned such that an RNA transcript initiated therefrom contains the 5' gag LTR and the gene of interest.
  • other suitable promoters some of which are described below, may be employed.
  • alternate enhancers may be employed in order to increase the level of expression of the gene of interest.
  • retroviral vectors are employed, particularly those based on Moloney murine leukemia virus (MoMLV)
  • MoMLV is a murine retrovirus which has poor infectivity outside of mouse cells.
  • the related amphotropic N2 retrovirus will infect cells from human, mouse and other organisms
  • retroviruses which may be used in the practice of the present invention include gibbon ape leukemia virus (GALV) (Todaro, et al, Virology, 67:335, 1975; Wilson, et al, J. Vir., 63:2374, 1989), feline immunodeficiency virus (FIV) (Talbatt, et al, Proc. Nat'l. Acad. Sci.
  • feline leukemia virus (Leprevette, et al, J. Vir., 50:884, 1984; Elder, et al., J. Vir., 46:871, 1983; Steward, et al., J. Vir., 58:825, 1986; Riedel, et al. , J. Vir., 60:242, 1986), although retroviral vectors according to the invention derived from other type C retroviruses (Weiss, RNA Tumor Viruses, vols. I and II, Cold Spring Harbor Laboratory Press, N.Y.) can also be generated.
  • promoters can be used in the vector constructs of the invention, including but not necessarily limited to the cytomegalovirus major immediate early promoter (CMV MIE), the early and late SV40 promoters, the adenovirus major late promoter, thymidine kinase or thymidylate synthase promoters, alpha or beta interferon promoters, event or tissue specific promoters, etc. Promoters may be chosen so as to potently drive high levels of expression or to produce relatively weak expression, as desired. As those in the art will appreciate, numerous RNA polymerase II and RNA polymerase III dependent promoters can be utilized in practicing the invention.
  • CMV MIE cytomegalovirus major immediate early promoter
  • adenovirus major late promoter thymidine kinase or thymidylate synthase promoters
  • alpha or beta interferon promoters alpha or beta interferon promoters
  • event or tissue specific promoters etc. Promoters
  • recombinant retroviral vectors comprising a gene of interest are under the transcriptional control of an event-specific promoter, such that upon activation of the event-specific promoter the gene is expressed.
  • event-specific promoters may be utilized within the context of the present invention, including for example, promoters which are activated by cellular proliferation (or are otherwise cell-cycle dependent) such as the thymidine kinase or thymidylate synthase promoters (Merrill, Proc. Nat I. Acad. Sci. USA, 86:4987, 1989; Deng, et al. , Mol.. Cell. Biol.
  • promoters such as the alpha or beta interferon promoters which are activated when a cell is infected by a virus (Fan and Maniatis, EMBO J., 5:101, 1989; Goodbourn, et al. , Cell, 45:601, 1986); and promoters which are activated by the presence of hormones, e.g., estrogen response promoters. See Toohey et al. , Mol.. Cell. Biol. , 6:4526, 1986; and promoters that are activated in response to cellualr stress or insult, e.g., electrophilic response elements (Friling, et al. , PNAS, 57:6258, 1990)
  • tissue-specific promoters which comprise a gene of interest under the transcriptional control of a tissue-specific promoter, such that upon activation of the tissue-specific promoter the gene is expressed
  • tissue-specific promoters include: B-cell specific promoters such as the IgG promoter; T-cell specific promoters such as the T-cell receptor promoter (Anderson, et al. , Proc. Natl. Acad Sci. USA, 55:3551, 1988; Winoto and Baltimore, EMBO J., 8:29, 1989); bone-specific promoters such as the osteocalcin promoter (Markose, et al., Proc. Natl Acad. Sci. USA, 87:1701, 1990; McDonnell, et al. , Mol. Cell Biol., 9:3517, 1989;
  • erythoid specific-transcription promoters which are active in erythroid cells, such as the porphobilinogen deaminase promoter (Mignotte, etal, Proc. Natl Acad. Sci. USA, 56:6458, 1990), ⁇ - or ⁇ -globin specific promoters (van Assendelft, et al. , Cell, 56:969,
  • endothelial cell specific promoters such as the vWf promoter, megakaryocyte specific promoters such as ⁇ - thromboglobulin, and many other tissue-specific promoters.
  • promoters that may be used to cause expression of the introduced sequence in specific cell types include Granzyme A and Granzyme B for expression in T-cells and NK cells, the CD34 promoter for expression in stem and progenitor cells, the CD8 promoter for expression in cytotoxic T- cells, and the CDI lb promoter for expression in myeloid cells.
  • Retroviral vectors according to the invention may also contain a non-LTR enhancer or promoter, e.g., a CMV or SV40 enhancer operably associated with other elements employed to regulate expression of the gene of interest. Additionally, retroviral vectors from which the 3' LTR enhancer has been deleted, thereby inactivating the 5' LTR upon integration into a host cell genome, are also contemplated by the invention. A variety of other elements which control gene expression may also be utilized within the context ofthe present invention, including, for example, locus-defining elements such as those from the ⁇ -globin gene and CD2, a T-cell marker. In addition, elements which control expression at the level of splicing, nuclear export, and/or translation may also be included in the retroviral vectors.
  • a non-LTR enhancer or promoter e.g., a CMV or SV40 enhancer operably associated with other elements employed to regulate expression of the gene of interest.
  • Representative examples include the ⁇ -globin intron sequences, the rev and rre elements from HTV-1, the constitutive transport element (CTE) from Mason-Pfizer monkey virus (MPMV), a 219 nucleotide sequence that allows rev-independent replication of rev- negative HIV proviral clones, and a Kozak sequence.
  • Rev protein functions to allow nuclear export of unspliced and singly spliced HIV RNA molecules.
  • the MPMV element allows nuclear export of intron-containing mRNA.
  • the CTE element maps to MPMV nucleotides 8,022-8,240 (Bray, et al. , Biochemistry, 91:1256, 1994).
  • the retroviral vector contains a splice donor (SD) site and a splice acceptor (SA) site, wherein the SA is located upstream of the site where the gene of interest is inserted into the recombinant retroviral vector.
  • SD splice donor
  • SA splice acceptor
  • the SD and SA sites will be separated by a short, i.e., less than 400 nucleotide, intron sequence.
  • Such sequences may serve to stabilize RNA transcripts.
  • stabilizing sequences typically comprise a SD-intron-SA configuration located 5' to the gene of interest.
  • the recombinant retroviral vectors of the invention will also preferably contain transcriptional promoters derived from the gag region operably positioned such that a resultant transcript comprising the gene of interest further comprises a 5' gag LTR
  • the present invention also provides for multivalent vector constructs, the
  • a second promoter may not be necessary.
  • an internal ribosome binding site (IRBS) or herpes simplex virus thymidine kinase (HSVTK) promoter is placed in conjunction with the second gene of interest in order to boost the levels of gene expression ofthe second gene.
  • IRBS internal ribosome binding site
  • HVTK herpes simplex virus thymidine kinase
  • the upstream untranslated region of the immunoglobulin heavy chain binding protein has been shown to support the internal engagement of a bicistronic message (Jacejak, et al. , Nature 353:90, 1991). This sequence is small, approximately 300 base pairs, and may readily be incorporated into a vector in order to express multiple genes from a multi-cistronic message whose cistrons begin with this sequence.
  • Retroviral vector constructs according to the invention will often be encoded on a plasmid, a nucleic acid molecule capable of propagation, segregation, and extra- chromosomal maintenance upon introduction into a host cell.
  • plasmids contain an origin of replication and typically are modified to contain a one or more multiple cloning sites to facilitate recombinant use.
  • plasmids used in accordance with the present invention will be capable of propagation in both eukaryotic and prokaryotic host cells.
  • a packaging cell is a cell comprising, in addition to its natural genetic com ple ment, additional nucleic acids coding for those retroviral structural polypeptides required to package a retroviral genome, be it recombinant (i.e., a retroviral vector) or otherwise.
  • the retroviral particles are made in packaging cells by combining the retroviral genome with a capsid and envelope to make a transduction competent, preferably replication defective, virion.
  • these and other packaging cells will contain one, and preferably two or more nucleic acid molecules coding for the various polypeptides, e.g., gag, pol, and ewv, required to package a retroviral vector into an infectious virion.
  • the packaging cells Upon introduction of a nucleic acid molecule coding for the retroviral vector, the packaging cells will produce infectious retroviral particles.
  • Packaging cell lines transfected with a retroviral vector according to the invention which produce infectious virions are referred to as "producer" cell lines.
  • a wide variety of animal cells may be utilized to prepare the packaging cells ofthe present invention, including without limitation, epithelial cells, fibroblasts, hepatocytes, endothelial cells, myoblasts, astrocytes, lymphocytes, etc..
  • cell lines are selected that lack genomic sequences which are homologous to the retroviral vector construct, gag/pol expression cassette and ewv expression cassette to be utilized. Methods for determining homology may be readily accomplished by, for example, hybridization analysis (Martin et al. , Proc. Natl. Acad. Sci., USA, 75:4892, 1981; and U.S.S.N.
  • PCLs packaging cell lines
  • PA12, PA317) are derived from murine cell lines.
  • murine cell lines are typically not the preferred choice to produce retroviral vectors intended for human therapeutic use because such cell lines are known to: contain endogenous retroviruses, some of which are closely related in sequence and retroviral type to the MLV vector system preferred for use in practicing the present invention, contain non-retroviral or defective retroviral sequences that are known to package efficiently; and cause deleterious effects due to the presence of murine cell membrane components.
  • An important consideration in developing packaging cell lines useful in the invention is the production therefrom of replication incompetent virions, or avoidance of generating replication-competent retrovirus (RCR) (Munchau, et al. , Virology, 176:262, 1991 ) This will ensure that infectious retroviral particles harboring the recombinant retroviral vectors of the invention will be incapable of independent repUcation in target cells, be they in vitro or in vivo. Independent replication, should it occur, may lead to the production of wild-type virus, which in turn could lead to multiple integrations into the chromosome(s) of a patient's cells, thereby increasing the possibility of insertional mutagenesis and its associated problems.
  • RCR replication-competent retrovirus
  • RER production can occur in at least two ways: (1) through homologous recombination between the therapeutic proviral DNA and the DNA encoding the retroviral structural genes ("gag/por and "ewv") present in the packaging cell line; and (2) generation of replication- competent virus by homologous recombination of the proviral DNA with the very large number of defective endogenous proviruses found in murine packaging cell lines.
  • packaging cell lines may be derived from various non-murine cell lines. These include cell lines from various mammals, including humans, dogs, monkeys, mink, hamsters, and rats. As those in the art will appreciate, a multitude of packaging cell lines can be generated using techniques known in the art (for instance, see U.S.S.N. 08/156,789 and U.S.S.N. 08/136,739). In preferred embodiments, cell lines are derived from canine or human cell lines, which are known to lack genomic sequences homologous to that of MoMLV by hybridization analysis (Martin et al. , supra). A particularly preferred parent dog cell line is D17 (ATCC. CRL 8543). HT-1080 (ATCC. CCL 121; Graham et al. , Vir., 52:456, 1973) and 293 cells
  • retroviral packaging cell lines incapable of producing replication competent, or "wild-type," virus.
  • packaging cell lines contain one or more nucleic acid molecules coding for the structural proteins required to assemble the retroviral vector into infectious retroviral particles
  • recombination events between these various constructs might produce replication competent virus, i.e., infectious retroviral particles containing a genome encoding all of the structural genes and regulatory elements, including a packaging signal, required for independent replication.
  • replication competent virus i.e., infectious retroviral particles containing a genome encoding all of the structural genes and regulatory elements, including a packaging signal, required for independent replication.
  • constructions include: deletions in the 3' LTR and portions of the 5' LTR (see, Miller and Buttimore, Mol..
  • gag/pol and env function separately allows for manipulation of these functions independently.
  • a cell line that expresses ample amounts of gag/pol can be used, for example, to address questions of titer with regard to env.
  • one factor resulting in measured low titers is the density of appropriate receptor molecules on the target cell or tissue.
  • a second factor is the affinity of the receptor for the retroviral envelope protein.
  • xenotropic vector in the presence of replication-complement virus, may more effectively infect human hematopoietic progenitor cells (Eglitis, et al. , Biochem. Biophys. Res. Comm. 151:201, 1988).
  • vector-containing particles in the presence of replication-competent xenotropic virus, also infect cells from other species which are not easily infectable by amphotropic virus such as bovine, porcine, and equine cells (Delouis, et al. , Biochem. Biophys Res. Comm. 169:80, 1990).
  • packaging cell lines which express a xenotropic ewv gene are provided.
  • recombinant xenotropic retroviral particles produced from such packaging cell lines are substantially free from association with replication competent retrovirus ("RCR").
  • inhibitory molecule prevents production of RCR either by killing the producer cell (s) in which that event occurred or by suppressing production of the retroviral vectors therein.
  • inhibitory molecules may be used, including ribozymes, which cleave the RNA transcript of the replication competent virus, or a toxin such as ricin A, tetanus, or diphtheria toxin, herpes thymidine kinase, etc.
  • ribozymes which cleave the RNA transcript of the replication competent virus
  • a toxin such as ricin A, tetanus, or diphtheria toxin, herpes thymidine kinase, etc.
  • teachings therein may be readily adapted to the present invention.
  • the choice of host cell line for the packaging cell line is of importance because many of the biological properties (such as titer) and physical properties (such as stability) of retroviral particles are dictated by the properties of the host cell
  • the host cell must efficiently express (transcribe) the vector RNA genome. prime the vector for first strand synthesis with a cellular tRNA, tolerate and covalently modify the MLV structural proteins (proteolysis, glycosylation, myristylation, and phosphorylation), and enable virion budding from the cell membrane.
  • MLV structural proteins proteolysis, glycosylation, myristylation, and phosphorylation
  • Retroviruses of avian, murine (including MoMLV), feline, and simian origin are inactivated and lysed by normal human serum. See Welsh, et al. , Nature, 257:612, 1975; Welsh, et al. , Virology, 74:432, 1976; Banapour, et al. , Virology, 752:268, 1986; and Cooper, et al. , (1986) Immunology of the Complement System, Pub.
  • Retroviruses produced from various human packaging cell lines were resistant to inactivation by a component of human serum, presumably complement, but were sensitive to serum from baboons and macaques. See commonly owned U.S.S.N. 08/367,071, filed on December 30, 1994.
  • recombinant retroviral particles are produced in human packaging cell lines, with packaging cell lines derived from HT1080 or 293 cells being particularly preferred.
  • At least two other alternative systems can be used to produce recombinant retroviruses carrying the vector construct.
  • One such system (Webb, et al. , BBRC, 190:536, 1993) employs the insect virus, baculovirus, while the other takes advantage ofthe mammalian viruses vaccinia and adenovirus (Pavirani, et al. , BBRC, 145:234, 1987).
  • Each of these systems can make large amounts of any given protein for which the gene has been cloned. For example, see Smith, et al. (Mol. Cell. Biol.. , 3:12, 1983); Piccini, et al.
  • retroviral vectors can be used to produce proteins in tissue culture cells by insertion of appropriate genes and, hence, could be adapted to make retroviral vector particles from tissue culture
  • genes can be inserted into vectors and used to express proteins in mammalian cells either by in vitro construction (Ballay, et al. , 4:3861, 1985) or by recombination in cells (Thummel, et al. , J. Mol.. Appl. Genetics, 1:435, 1982).
  • retroviral structural proteins can be made in a baculovirus system (or other protein production systems, such as yeast or E. coli) in a similar manner as described in Smith et al. (supra).
  • Recombinant retroviral genomes are made by in vitro RNA synthesis (see, for example, Flamant and Sorge, J. Virol.., 62: 1827, 1988).
  • the structural proteins and RNA genomes are then mixed with tRNA, followed by the addition of liposomes with embedded env protein and cell extracts (typically from mouse cells) or purified components (which provide env and other necessary processing, and any or other necessary cell-derived functions).
  • the mixture is then treated (e.g., by sonication, temperature manipulation, or rotary dialysis) to allow encapsidation of nascent retroviral particles.
  • This procedure allows production of high titer, replication incompetent recombinant retroviruses without contamination with pathogenic retroviruses or replication-competent retroviruses.
  • viral titer Another important factor to consider in the selection of a packaging cell line is the viral titer produced therefrom following introduction of a nucleic acid molecule from which the retroviral vector is produced. Many factors can limit viral titer. One of the most significant limiting factors is the expression level of the packaging proteins gag, pol, and env In the case of retroviral particles, expression of retroviral vector RNA from the provirus can also significantly limit titer. In order to select packaging cells and the resultant producer cells expressing high levels of the required products, an appropriate titering assay is required. As described in greater detail below, a suitable PCR-based titering assay can be utilized.
  • packaging and producer cell lines which supply proteins for packaging that are homologous for the backbone of the viral vector, e.g., retroviral gag, pol, and env proteins for packaging of a retroviral vector
  • packaging and producer systems which result in chimeric viral particles, for instance a MoMLV-based retroviral vector packaged in a DNA virus capsid, may also be employed.
  • Many other packaging and producer systems based on viruses unrelated to that of the viral vector can also be utilized, as those in the art will appreciate.
  • Another aspect of the invention concerns recombinant xenotropic retroviral particles which have an altered host range as compared to retroviral particles containing amphotropic envelope proteins.
  • the host cell range specificity of a retrovirus is determined in part by the ewv gene products present in the lipid envelope.
  • envelope proteins from one retrovirus can often substitute, to varying degrees, for that of another retrovirus, thereby altering host range of the resultant vector.
  • packaging cell lines have been generated to express either amphotropic, ecotropic, xenotropic, polytropic, or other errveiope tropisms.
  • retroviruses according to the invention which contain "hybrid” or “chimeric” xenotropic envelope proteins can be similarly generated Retroviral particles produced from any of these packaging cell lines can be used to infect any cell which contains the corresponding distinct receptor (Rein and Schultz, Virology, 136:144, 1984).
  • retroviruses The assembly of retroviruses is characterized by selective inclusion of the retroviral genome and accessory proteins into a budding retroviral particle.
  • envelope proteins from non-murine retrovirus sources can be used for pseudotyping (i.e., the encapsidation of viral RNA from one species by viral proteins of another species) a vector to alter its host range. Because a piece of cell membrane buds off to form the retroviral envelope, molecules normally in the membrane may be carried along on the viral envelope.
  • a number of different potential ligands can be put on the surface of retroviral particles by manipulating the packaging cell line in which the vectors are produced or by choosing various types of cell lines with particular surface markers.
  • the present invention provides for enveloped retroviral particles comprising: a nucleocapsid including nucleocapsid protein having an origin from a first virus, which is a retrovirus; a packagable nucleic acid molecule encoding a gene of interest associated with the nucleocapsid; and a membrane-associated xenotropic protein which determines a host range.
  • the membrane-associated xenotropic protein of the vector particles is a chimeric or hybrid protein including an exterior receptor binding domain and a membrane-associated domain from a xenotropic envelope protein, at least a portion of the exterior receptor binding domain being derived from a different origin than at least a portion of the membrane-associated domain.
  • the chimeric protein is preferably derived from two origins, wherein no more than one of the two origins is retroviral.
  • Retroviral particles according to the invention may be targeted to a specific cell type by including in the retroviral particles a component, most frequently a polypeptide or carbohydrate, which binds to a cell surface receptor specific for that cell type. Such targeting may be accomplished by preparing a packaging cell line which expresses a chimeric env protein comprising a portion of the env protein required for viral particle assembly in conjunction with a cell-specific binding domain.
  • env proteins from more than one viral type may be employed, such that resultant viral particles contain more than one species of env proteins.
  • env proteins from more than one viral type may be employed, such that resultant viral particles contain more than one species of env proteins.
  • Yet another embodiment involves inclusion of a cell specific ligand in the retroviral capsid or xenotropic envelope to provide target specificity.
  • the xenotropic env gene employed encodes all or a portion of the xenotropic env protein required for retroviral assembly in conjunction with a receptor binding domain of a polypeptide ligand known to interact with a cell surface receptor whose tissue distribution is limited to the cell type(s) to be targeted, e.g., a hematopoietic stem cell.
  • a receptor binding domain which binds receptors expressed at high levels on the target cell's surface.
  • Non-viral membrane-associated proteins may also be used to enhance targeting of recombinant retroviral particles to hematopoietic stem cells.
  • Representative examples include polypeptides which act as ligands for hematopoietic stem cell surface receptors. Depending on the tissue distribution of the receptor for the protein in question, the recombinant retroviral particle could be targeted to a different subset of hematopoietic stem cells.
  • a ligand to be included within the xenotropic envelope is not a naturally occurring membrane-associated protein, it is necessary to associate the ligand with the membrane, preferably by making a "hybrid” or “chimeric" envelope protein. It is important to understand that such hybrid envelope proteins can contain extracellular domains from proteins other than other viral or retroviral env proteins. To accomplish this, the gene coding for the ligand can be functionally combined with sequences coding for a membrane- associated domain of the env protein.
  • naturally occurring membrane associated protein it is meant those proteins that in their native state exist in vivo in association with lipid membrane such as that found associated with a cell membrane or on a viral envelope
  • hybrid envelopes can be used to tailor the tropism (and effectively increase titers) of a retroviral vector according to the invention, as the extracellular component of env proteins is responsible for specific receptor binding.
  • the cytoplasmic domain of these proteins play a role in virion formation.
  • the present invention recognizes that numerous hybrid env gene products (i.e., specifically, retroviral env proteins having cytoplasmic regions and extracellular binding regions which do not naturally occur together) can be generated and may alter host range specificity.
  • this is accomplished by recombiming the gene coding for the ligand (or part thereof conferring receptor binding activity) proximate of the membrane- binding domain of the envelope proteins that stably assemble with a given capsid protein.
  • the resulting construct will code for a bifunctional chimeric protein capable of enhanced cell targeting and inclusion in a retroviral lipid envelope.
  • Vector particles having non-native membrane-associated ligands as described herein will, advantageously, have a host range determined by the ligand-receptor interaction ofthe membrane-associated protein.
  • a vector particle having altered host range can be produced using the methods of the present invention.
  • the ligand will be selected to provide a host range including hematopoietic stem cells.
  • Many different targeting strategies can be employed in connection with this aspect of the invention. For example, there are a number of progenitor cell types found in bone marrow that differentiate into blood cells. Many blood cells have relatively short life spans and therefore progenitor cells must continually divide and differentiate to replace the lost cells.
  • gene therapy targets hematopoietic progenitor cells, including pluripotent stem cells.
  • progenitor cells are known to have unique cellular determinants that permit histological identification, separation from other cell types by various techniques, including fluorescence activated cell sorting (FACS) and positive and negative selection [see U.S. Patent No. 5,061,620], and which can be used as cell receptors for the membrane-associated proteins of the vector particles of the present invention.
  • FACS fluorescence activated cell sorting
  • positive and negative selection see U.S. Patent No. 5,061,620
  • a hematopoietic stem cell is a primitive, or immature, cell capable of self-renewal and which is capable of differentiating into precursor cells of all hematopoietic lineages, i.e., they are said to be "pluripotent.”
  • Recombinant vectors according to the invention may be introduced into such cells or any their more differentiated progeny, such as the various primitive progenitors and the more lineage committed precursor cells that give rise to the various hematopoietic cell lineages.
  • One marker for such early hematopoietic cells is CD34, which can be identified using monoclonal antibodies.
  • WO 93/25216 describes a class of hematopoietic stem cells as having the phenotype CD34 + /CD38-/HLA-DR- and lacking the lineage committed antigens CD33, CD10, CD5, and CD71
  • Representative examples of anti- CD34 antibodies include 12.8 (Andrews, et al, Blood, 67:842, 1986) and My10 (Civin, et al. , J. Immunol.. , 133:157, 1984, commercially available from Becton Dickinson under the designation HPCA-2).
  • antibodies may be also utilized to target a selected cell type, such as anti-CD4 antibodies to target CD4+ T-cells and anti-CD8 antibodies to target CD8 + cells (see generally, Wilchek, et al., Anal. Biochem. , 171:1, 1988).
  • the vectors may be constructed to target these cell types for gene delivery by including an expressible gene which encodes a membrane-associated protein that binds to a unique cellular determinant of such hematopoietic progenitor cell types.
  • Examples of such progenitor cell types which could be targeted using recombinant retroviral particles ofthe present invention include pluripotent stem cells, erythroblasts, lymphoblasts, myeloblasts and megakaryocytes.
  • ligand molecules exogenously to the retroviral particles which are either incorporated into the lipid envelope or which can be linked chemically to the lipid or protein constituents thereof.
  • Targeting a retroviral vector carrying a gene of interest to a predetermined locus on a chromosome may also be employed. Clear advantages of such targeting include avoidance of insertional mutagenesis and assuring integration at sites known to be transcriptionally active. Techniques for targeting proviral integration to specific sites include integrase modification. See U.S.S.N. 08/156,789, supra.
  • the therapy of the present invention be performed in vitro.
  • in vitro therapy also referred to as "ex vivo therapy”
  • cells are removed and transduced in vitro.
  • the need to purify the cells to be targeted in vitro is optional because the vector would specifically transduce only the targeted cells.
  • bone marrow samples could be removed from a subject and the desired cell type transduced without the need to perform one or more cell sorting procedures. The transduced cells could then be returned to the same patient or one who is HLA matched.
  • binding pairs can be used as targeting elements.
  • Representative examples of include biotin/avidin with an affinity (Kp) of 10 -15 M (Richards, Meth. Enz., 184:3, 1990; Green, Adv. in Protein Chem., 29:85, 1985) and cystatin/papain with an affinity of 10 -14 M (Bjork, et al. , Biochemistry , 29: 1770, 1990).
  • a wide variety of other high affinity binding pairs may also be developed, for example, by preparing and selecting antibodies which recognize a selected hematopoietic stem cell antigen with high affinity (see generally, U.S. Patent Nos RE 32,01 1 , 4,902,614, 4,543,439, and 4,41 1,993; see also Monoclonal Antibodies, Hvbridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol, eds., 1980, and Antibodies: A Laboratory Manual, Harlow and Lane eds., Cold Spring Harbor Laboratory Press, 1988).
  • the binding pair for such antibodies may be produced by recombinant techniques (see Huse, et al. , Science, 246:1275, 1989; see also Sastry, et al. , Proc. Natl. Acad Sci. USA , 86:5728, 1989; and Michelle Akmg-Mees, et al. , Strategies in Molecular Biology , 3:1, 1990).
  • either member (or molecule) of the affinity binding pair may be coupled to the retroviral particle.
  • the larger of the two affinity binding pairs e.g., avidin of the avidin/biotin pair
  • the term “coupled” may refer to either noncovalent or covalent interactions, although generally covalent bonds are preferred.
  • a member of the high affinity binding pair is either expressed on, or included as an integral part of, a retroviral particle, e.g., in the retroviral lipid envelope.
  • a member of the high affinity binding pair may be co-expressed with the envelope protein as a hybrid protein or expressed from an appropriate vector which targets the member of the high affinity binding pair to the cell membrane in the proper orientation.
  • Retroviral Particles The efficient use of recombinant retroviral particles to infect cells is dependent on the tropism of the envelope protein expressed on the surface of the retroviral particle .
  • the use of a envelope enables infection of cells from different species.
  • the present invention provides methods for inhibiting the growth of a selected tumor ("cancer") in a human, comprising the step of transducing hematopoietic stem cells ex vivo with a vector construct which directs the expression of at least one anti-tumor agent
  • inhibiting the growth of a selected tumor refers to either (1) the direct inhibition of tumor cell division or metatstsis, or (2) immune cell mediated tumor cell lysis, or both, which leads to a suppression in the net expansion of tumor cells Inhibition of tumor growth by either of these two mechanisms may be readily determined by one of ordinary skill in the art based upon a number of well known methods, for example, by measuring the tumor size over time, such as by radiologic imaging methods (e.g., single photon and positron emission computerized tomography; see generally, "Nuclear Medicine in Clinical Oncology," Winkler, C.
  • radiologic imaging methods e.g., single photon and positron emission computerized tomography; see generally, "Nuclear Medicine in Clinical Onc
  • imaging agents including, for example, conventional imaging agents (e.g., Gallium-67 citrate) or specialized reagents for metabolite imaging, receptor imaging, or immunologic imaging.
  • conventional imaging agents e.g., Gallium-67 citrate
  • specialized reagents for metabolite imaging e.g., receptor imaging, or immunologic imaging.
  • non-radioactive methods such as ultrasound (see, "Ultrasonic Differential Diagnosis of Tumors", Kossoff and Fukuda, (eds ), Igaku-Shoin, New York, 1984), may also be utilized to estimate tumor size.
  • inhibition of tumor growth may be determined based upon a change in the presence of a tumor marker, e.g.
  • PSA prostate specific antigen
  • CEA carcino-embryonic antigen
  • inhibition of tumor growth may be determined based upon decreased numbers of leukemic cells in a representative blood cell count.
  • anti-tumor agent refers to a compound or molecule which inhibits tumor growth.
  • anti-tumor agents include immune activators and tumor proliferation inhibitors. Briefly, immune activators function by improving immune recognition of tumor-specific antigens such that the immune system becomes “primed.” Priming may consist of lymphocyte proliferation, differentiation, or evolution to higher affinity interactions. The immune system thus primed will more effectively inhibit or kill tumor cells. Immune activation may be subcategorized into immune modulators (molecules which affect the interaction between lymphocyte and tumor cell) and lymphokines, that act to proliferate, activate, or differentiate immune effector cells.
  • immune modulators include CD3, ICAM-1, ICAM-2, LFA-1, LFA-3, ⁇ -2-microglobulin, chaperones, alpha interferon and gamma interferon, B7/BB1 and major histocompatibility complex (MHC).
  • lymphokines include gamma interferon, tumor necrosis factor, IL-1, IL-2, IL-3, IL-4, DL-5, IL-6, IL-7, TL-8, IL-9, IL-10, IL-11, GM-CSF, CSF-1, and G-CSF.
  • RNA molecules having intrinsic biological activity may be utilized as anti-tumor agents.
  • Sequences which encode anti-tumor agents may be obtained from a variety of sources.
  • plasmids that contain sequences which encode anti-tumor agents may be obtained from a depository such as the American Type Culture Collection (ATCC, Rockville, MD), or from commercial sources such as British Bio-Technology Limited (Cowley, Oxford, England)
  • known cDNA sequences which encode anti- tumor agents may be obtained from cells which express or contain the sequences
  • cDNA or mRNA libraries from specific cell sources can be purchased from commercial sources from which the desired sequences can be readily cloned by conventional techniques, e.g., PCR amplification.
  • Sequences which encode anti-tumor agents may also be synthesized, for example, on an Applied Biosystems Inc. DNA synthesizer (e.g., ABI DNA synthesizer model 392, Foster City, CA).
  • anti-tumor agents which comprise a fusion protein of, for example, two or more cytokines, immune modulators, toxins or differentiation factors.
  • Preferred anti-tumor agents in this regard include alpha interferon - Interleukin-2, GM-CSF - IL-4, GM-CSF - IL-2, GM- CSF - IL-3 (see U.S. Patent Nos. 5,082,927 and 5,108,910), GM-CSF - gamma interferon, and gamma interferon - IL-4, with gamma interferon - interleukin-2 being particularly preferred.
  • the anti-tumor agent may further comprise a membrane anchor.
  • the membrane anchor may be selected from a variety of sequences, including, for example, the transmembrane domain of well known proteins.
  • membrane anchor sequences are regions of a protein that anchor the protein to a membrane.
  • Membrane anchors derived from integral membrane proteins are preferred.
  • Membrane spanning regions typically have a similar structure, with a 20 to 25 amino-acid residue portion consisting almost entirely of hydrophobic residues located inside the membrane (see Eisenberg et al. , Ann. Rev. Biochem. 53:595-623, 1984) Membrane spanning regions typically have an alpha helical structure (see Eisenberg et al. at 20; Heijne and Manoil at 109).
  • a membrane anchor is fused to the C- terminus of gamma interferon fusion protein, wherein the membrane anchor comprises the gamma-chain of the Fc receptor.
  • Tumorigenicity of an anti-tumor agent can be assessed by various assays.
  • Representative assays include tumor formation in nude mice or rats, colony formation in soft agar, and preparation of transgenic animals, such as transgenic mice. In addition to tumorigenicity studies, it is generally preferable to determine the toxicity of an anti-tumor agent. A variety of methods well known to those of skill in the art may be utilized to measure such toxicity, including for example, clinical chemistry assays which measure the systemic levels of various proteins and enzymes, as well as blood cell volume and number
  • an anti-tumor agent Once an anti-tumor agent has been selected, it is placed into a vector construct according to the invention.
  • Such a vector construct can then be packaged into a recombinant retroviral vector and be used to transduce ex vivo hematopoietic stem ceils which are then re- introduced into the patient.
  • the removed cells may not only be returned to the same patient, but may also be utilized to inhibit the growth of selected tumor cells in another allogeneic human.
  • Recombinant retroviral particles can be produced in a variety of ways, as those in the art will appreciate.
  • producer cells i.e., cells containing all necessary components for retroviral vector packaging (including a nucleic acid molecule encoding the retroviral vector)
  • Cells can be maintained either on a solid support in liquid medium, or grown as suspensions.
  • a wide variety of bioreactor configurations and sizes can be used in the practice of the present invention.
  • Cell factories typically contain 2, 10, or 40 trays, are molded from virgin polystyrene, treated to provide a Nuclon DTM surface, and assembled by sonic welding one to another Generally, these factories have two port tubes which allow access to the chambers for adding reagents or removing culture fluid.
  • a 10-layer factory provides 6000 cm 2 of surface area for growing cells, roughly the equivalent of 27 T-225 flasks Cell factories are available from a variety of manufacturers, including for example Nunc (Baxter, Sanform, ME).
  • Most cell types are capable of producing high titer vector for 3 to 6 days, allowing for multiple harvests Each cell type is tested to determine the optimal harvest time after seeding and the optimal number of harvest days
  • Cells are typically initially grown in DMEM supplemented with 2 to 20% FBS in roller bottles until the required number of cells for seeding a cell factory is obtained Cells are then seeded into the factories and 2 liters (L) of culture supernatant containing vector is harvested later at an appropriate time. Fresh media is used to replenish the cultures.
  • Hollow fiber culture methods may also be used Briefly, high titer retroviral production using hollow fiber cultures is based on increasing viral concentration as the cells are being cultured to a high density in a reduced volume of media.
  • Cells are fed nutrients and waste products are diluted using a larger volume of fresh media which circulates through the lumen of numerous capillary fibers.
  • the cells are cultured on the exterior spaces ofthe capillary fibers in a bioreactor chamber where cell waste products are exchanged for nutrients by diffusion through 30 kD pores in the capillary fibers.
  • Retroviruses which are produced from the cell lines are too large to pass through the pores, and thus concentrate in the hollow fiber bioreactor along side of the cells.
  • the volume of media being cultured on the cell side is approximately 10 to 100 fold lower then volumes required for equivalent cell densities cultured in tissue culture dishes or flasks. This decrease in volume inversely correlates with the fold induction of titer when hollow fiber retroviral titers are compared to tissue culture dishes or flasks. This 10 to 100 fold induction in titer is seen when an individual retroviral producer cell line is amiable to hollow fiber growth conditions. To achieve maximum cell density, the individual cells must be able to grow in very close proximity and on top of each other. Many cell lines will not grow in this fashion and retroviral packaging cell lines based on these types of cell lines may not achieve 10 fold increases in titer.
  • Non-adherent cell line which would grow very well would be non-adherent cell line and it is believed that a retroviral producer line based on a non-adherent cell line may reach 100 fold increases in titer compared to tissue culture dishes and flasks.
  • high titer from about 10 7 -10 11 cfu/mL
  • high titer stocks can be prepared that will cause high level expression of the desired products upon introduction into appropriate cells.
  • high titer stocks are preferred, retroviral preparations having titers ranging from about 10 3 to 10 6 cfu/mL may also be employed, although retroviral titers can be increased by various purification methods, as described below
  • the infectious recombinant retroviral particles may be preserved in a crude or purified form.
  • Cmde retroviral particles are produced by cultivated infected cells, wherein retroviral particles are released from the cells into the culture media.
  • the virus may be preserved in crude form by first adding a sufficient amount of a formulation buffer to the culture media containing the recombinant virus to form an aqueous suspension.
  • Recombinant retroviral particles can also be preserved in a purified form. More specifically, prior to the addition of formulation buffer, the crude retroviral preparation described above is clarified by passing it through a filter, and then concentrated, such as by a cross flow concentrating system (Filtron Technology Corp.. Nortborough. MA) Within one embodiment, DNase is added to the concentrate to digest exogenous DNA. The digest is then diafiltrated to remove excess media components and establish the recombinant virus in a more desirable buffered solution. The diafiltrate is then passed over a gel filtration column, such as a Sephadex S-500 gel column, and the purified recombinant virus is eluted.
  • a cross flow concentrating system Frtron Technology Corp.. Nortborough. MA
  • Cmde recombinant retroviral preparations can also be purified by ion exchange column chromatography, such as is described in more detail in U.S.S.N. Serial
  • the crude preparation is clarified by passing it through a filter, and the filtrate loaded onto a column containing a highly sulfonated cellulose matrix, wherein the amount of sulfate per gram of cellulose ranges from about 6 - 15 ⁇ g.
  • the recombinant retrovirus is eluted from the column in purified form by using a high salt buffer.
  • the high salt buffer is then exchanged for a more desirable buffer by passing the eluate over a molecular exclusion column.
  • the purified preparation may then be formulated or stored, preferably at -70°C.
  • the preparations containing recombinant retroviruses according to the invention can be concentrated during purification in order to increase the titer of recombinant retrovirus.
  • a wide variety of methods may be utilized for increasing retroviral concentration, including for example, precipitation of recombinant retroviruses with ammonium sulfate, polyethylene glycol ("PEG”) concentration, concentration by centrifiigation (either with or without gradients such as PERCOLLTM, or "cushions” such as sucrose, use of concentration filters (e.g., Amicon filtration, Chicago, IL), and 2-phase separations.
  • PEG polyethylene glycol
  • concentration filters e.g., Amicon filtration, Chicago, IL
  • ammonium sulfate is added slowly to an appropriate concentration, followed by centrifiigation and removal ofthe ammonium sulfate either by dialysis or by separation on a hydrophobic column.
  • recombinant retroviruses may be concentrated from culture medium with PEG (Green, et al. , PNAS 67:385, 1970; Syrewicz, et al. , Appl. Micro. 24:488, 1972)
  • PEG Green, et al. , PNAS 67:385, 1970; Syrewicz, et al. , Appl. Micro. 24:488, 1972
  • PEG ammonium sulfate precipitation
  • use of PEG also concentrates other proteins from solution.
  • recombinant retroviruses may be concentrated by centrifugation, and more particularly, low speed centrifiigation, which avoids difficulties associated with pelleting that accompanies high speed centrifugation (e.g., virus destruction or inactivation).
  • Recombinant retrovi ruses according to the invention may also be concentrated by an aqueous two-phase separation method.
  • polymeric aqueous two-phase systems may be prepared by dissolving two different non-compatible polymers in water
  • Many pairs of water-soluble polymers may be utilized in the construction of such two-phase systems, including for example PEG or methylcellulose, and dextran or dextran sulfate (see Walter and Johansson, Anal. Biochem. 755:215, 1986; Albertsson, "Partition of Cell Particles and Macromolecules” Wiley, New York, 1960).
  • an aqueous two-phase system may be established suitable for purifying recombinant retroviruses. Utilizing such procedures, approximate 100-fold concentration can be achieved with yields of
  • recombinant retroviruses may be prepared either from roller bottles, cell factories, or bioreactors prior to concentration.
  • Removed media containing the recombinant retrovirus may be frozen at -70°C, or more preferably, stored at 2°C to 8°C in large pooled batches prior to processing.
  • the recombinant retrovirus pool is first clarified through a 0.8 ⁇ m filter (1.2 ⁇ m glass fiber pre-filter, 0.8 ⁇ m cellulose acetate) connected in series with a 0.65 ⁇ m filter.
  • This filter arrangement provides approximately 2 square feet (sq. ft.)of filter, and allows processing of about 15-20 L of pooled material before clogging.
  • a single 0.65 ⁇ m cartridge (2 sq. ft.) normally suffices for volumes up to 40 L.
  • a 5 sq. ft. filter may be required.
  • the filter is rinsed with buffer (e.g., 150 mM NaCl, 25 mM Tris, pH 7.2-7.5).
  • buffer e.g. 150 mM NaCl, 25 mM Tris, pH 7.2-7.5.
  • recombinant retroviruses are concentrated by tangential flow ultrafiltration utilizing cassettes with a 300,000 mw cut off.
  • bioreactor material containing 12% to 16% FBS
  • 4 to 5 L of material may be concentrated per cassette.
  • 5 to 6 L of material may be concentrated per cassette.
  • 8 to 9 L of material may be concentrated per cassette. Utilizing such procedures at an appropriate pressure differential between filtrate and retentate, up to 80 L of material may be
  • DNAse may be added to a concentration of 50 U/mL, and recirculated at a lower pump speed with the filtrate line closed for 30 minutes Discontinuous diafiltration is then accomplished by adding additional buffer and utilizing the same cross differential pressure as before. Generally, recovery after this step is
  • Tangential flow filtration may once again be utilized to further reduce the volume of the preparation, after which the concentrated material is sterilized by filtration through a 0.2 ⁇ m Millipore filter (Philadelphia, PA).
  • the retroviral packaging proteins may be produced, together or separately, from appropriate cells.
  • an in vitro packaging reaction is conducted comprising the gag, pol, and xeno env proteins, the retroviral vector, tRNA, and other necessary components.
  • the resulting retroviral particles can then purified and, if desired, concentrated.
  • compositions comprising recombinant retroviral vectors as described above, in combination with a pharmaceutically acceptable carrier or diluent, while another aspect is directed toward a method for preserving an infectious recombinant retroviruses for subsequent reconstitution such that the recombinant retrovirus is capable of infecting mammalian cells upon reconstitution.
  • the methods described can be used to preserve a variety of different viruses, including recombinant type C retroviruses such as gibbon ape leukemia virus, feline leukemia virus and xeno-, poly- and amphotropic murine leukemia virus (Weiss, et al. , RNA Tumor Viruses, 2d ed. 1985). See U.S.S.N. 08/153,342.
  • compositions are nontoxic to recipients at the dosages and concentrations employed.
  • Representative examples of carriers or diluents for injectable solutions include water, isotonic saline solutions, preferably buffered at a physiological pH (such as phosphate-buffered saline or Tris-buffered saline), mannitol, dextrose, glycerol, and ethanol, as well as polypeptides or proteins such as human serum albumin (HSA)
  • HSA human serum albumin
  • a particularly preferred composition comprises a recombinant retrovirus in 10 mg/mL mannitol, 1 mg/mL HSA, 20 mM Tris, pH 7.2, and 150 mM NaCl.
  • the recombinant retroviral particle since the recombinant retroviral particle represents approximately 1 ⁇ g of material, it may be less than 1% of high molecular weight material, and less than 1/100,000 of the total material (including water). This composition is stable at -70°C for at least six months.
  • compositions of the present invention may also additionally include factors which stimulate hematopoietic stem cell division, and hence, uptake and
  • recombinant retroviruses to transduce hematopoietic stem cells useful in treating patients requires that the product be able to be transported and stored for long periods at a desired temperature such that infectivity and viability of the recombinant retrovirus is retained.
  • the difficulty of preserving recombinant retroviruses absent low temperature storage and transport presents problems in Third World countries, where adequate refrigeration capabilities are often lacking.
  • sugars such as sucrose, raffinose, glucose and trehalose were added in various combinations as stabilizing agents prior to lyophilization of viruses.
  • sugars such as sucrose, raffinose, glucose and trehalose were added in various combinations as stabilizing agents prior to lyophilization of viruses.
  • the use of sugars enhanced recovery of viable viruses, for research purposes which require that only some virus survive for later propagation.
  • Recombinant retroviruses according to the invention can be stored in liquid, or preferably, lyophilized form. Factors influencing stability include the formulation (liquid, freeze dried, constituents thereof, etc.) and storage conditions, including temperature, storage container, exposure to light, etc. Alternatively, retroviral particles according to the invention can be stored as liquids at low temperatures. In a preferred embodiment, the recombinant retroviruses of the invention are formulated to preserve infectivity in a lyophilized form at elevated temperatures, and for this form to be suitable for injection into patients following reconstitution.
  • Recombinant retroviral particles comprising retroviral vector constructs according to the invention can be formulated in crude or, preferably, purified form.
  • Cmde retroviral preparations may be produced by various cell culture methods, where retroviral particles are released from the cells into the culture media.
  • Recombinant retroviral particles may be preserved in crude form by adding a sufficient amount of formulation buffer.
  • the formulation buffer is an aqueous solution containing various components, such as one or more saccharides, high molecular weight structural additives, buffering components, and/or amino acids.
  • the recombinant retroviruses described herein can also be preserved in a purified form.
  • crude preparations as described above may be clarified by filtration, and then concentrated, such as by a cross flow concentrating system (Filtron Technology Corp., Nortborough, MA).
  • DNase may be added to the concentrate to digest exogenous DNA, followed by diafiltration to remove excess media components and substitute in a more desirable buffered solution.
  • the diafiltrate may then passed over a gel filtration column, such as a SephadexTM S-500 gel column, and the eluted retroviral particles retained.
  • a sufficient amount of formulation buffer may then be added to the eluate to reach a desired final concentration of the constituents and to minimally dilute the retroviral preparation.
  • the aqueous suspension can then be stored, preferably at - 70°C, or immediately formulated.
  • the crude preparation can be purified by ion exchange column chromatography, as described in co-owned U.S.S.N. 08/093,436, filed July 16, 1993. Briefly, the crude recombinant retrovirus is clarified by filtration and then loaded onto a column comprising a highly sulfonated cellulose matrix. Highly purified recombinant retrovirus is eluted from the column using a high salt buffer, which is then exchanged for a more desirable buffer by passing the eluate over a molecular exclusion column. After recovery, formulation buffer may then added to adjust the final concentration, as discussed above, followed by low temperature storage, preferably at -70°C, or immediate formulation.
  • an aqueous preparation containing a crude or purified retroviral preparation can be prepared by lyophilization or evaporation
  • Lyophilization involves cooling the aqueous preparation below the glass transition temperature or below the eutectic point temperature of the solution, and removing water by sublimation.
  • a multistep freeze drying procedure as described by Phillips et al. (Cryobiology, 18:414, 1981) can be used to lyophilize the formulated recombinant virus, preferably from a temperature of -40°C to -45°C
  • the resulting composition should contain less than 10% water by weight
  • lyophilized such a preparation is stable and may be stored at -20°C to 25°C.
  • water is removed by evaporation from the retroviral preparation aqueous suspension at ambient temperature.
  • Evaporation can be accomplished by various techniques, including spray drying (see EP 520,748), where the preparation is delivered into a flow of preheated gas, usually air, whereupon water rapidly evaporates from droplets of the suspension.
  • Spray drying apparatus are available from a number of manufacturers (e.g., Drytec, Ltd., Tonbridge, England; Lab-Plant, Ltd., Huddersfield, England).
  • the recombinant retroviral preparation is stable and may be stored at -20°C to 25°C.
  • the resulting moisture content of the dried or lyophilized preparation may be determined through use of a Karl-Fischer apparatus (EM Science Aquastar' VIB volumetric titrator, Cherry Hill, NJ), or through a gravimetric method.
  • the recombinant retrovirus is stable and may be stored at -20°C to 25°C.
  • aqueous preparations comprising retroviruses according to the invention used for formulation are typically composed of one or more saccharides, high molecular weight structural additives, buffering components, and water, and may also include one or more amino acids. It has been found that the combination of these components acts to preserve the activity of the recombinant retrovirus upon freezing and lyophilization, or drying through evaporation. See co-owned U.S.S.N. 08/153,342, filed November 15, 1993.
  • Various saccharides may be used alone or in combination, including sucrose, mannitol, glucose, trehalose, inositol, fructose, maltose, and galactose, with lactose being particularly preferred.
  • the concentration of the saccharide can range from 0.1% to 30% by weight, preferably from about 1% to 12% by weight. A particularly preferred concentration of lactose is 3% to 4% by weight.
  • saccharide combinations can also be employed, including lactose and mannitol or sucrose and mannitol. It will also be evident to those skilled in the art that it may be preferable to use certain saccharides in the aqueous solution when the lyophilized formulation is intended for room temperature storage Specifically, disaccharides, such as lactose or trehalose, are preferred for such formulations
  • One or more high molecular weight structural additives may be used to aid in preventing retroviral aggregation during freezing and provides structural support in the lyophilized or dried state.
  • structural additives are considered to be of "high molecular weight” if they are greater than 5000 daltons.
  • a preferred high molecular weight structural additive is human semm albumin (HSA), although other substances may also be used, such as hydroxyethyl-cellulose, hydroxymethyl-cellulose. dextran, cellulose, gelatin, povidone, etc.
  • the concentration of the high molecular weight structural additive can range from 0.05% to 20%, with 0.1% to 10% by weight being preferred, and a concentration of 0.1% by weight HSA being particularly preferred
  • Amino acids if present, tend to further preserve retroviral infectivity.
  • amino acids function to further preserve retroviral infectivity during sublimation ofthe cooled aqueous suspension and while in the lyophilized state.
  • a preferred amino acid is arginine, but other amino acids such as lysine, ornithine, serine, glycine, glutamine, asparagine, glutamic acid or aspartic acid can also be used.
  • the amino acid concentration ranges from 0.1% to 10% by weight.
  • a particularly preferred arginine concentration is 0.1% by weight.
  • buffering components may be used to maintain a relatively constant pH, depending on the pH range desired, preferably between 7.0 and 7.8.
  • Suitable buffers include phosphate buffer and citrate buffer.
  • a particularly preferred formulation pH is 7.4, and a preferred buffer is tromethamine.
  • Suitable neutral salts include sodium chloride, potassium chloride, and magnesium chloride, with sodium chloride being preferred.
  • a particularly preferred method of preserving recombinant retroviruses in a lyophilized state for subsequent reconstitution comprises: (a) preparing an aqueous recombinant retroviral preparation comprising, in addition to the recombinant retrovirus, about (i) 4% by weight of lactose, (ii) 0.1% by weight of HSA, (iii) 0.03% or less by weight of NaCl, (iv) 0.1% by weight of arginine, and a sufficient amount of tromethamine to provide a pH of approximately 7.4; (b) cooling the preparation to a temperature of about -40°C to -45°C to form a frozen preparation; and (c) removing water from the frozen preparation by sublimation to form a lyophilized composition having less than 2% water by weight. It is preferred that the recombinant retrovirus be replication defective and suitable for
  • the lyophilized or dehydrated retroviruses of the subject invention may be reconstituted using a variety of substances, but are preferably reconstituted using water.
  • dilute salt solutions which bring the final formulation to isotonicity may also be used.
  • aqueous solutions containing components known to enhance the activity of the reconstituted virus include cytokines, such as IL-2, polycations, such as protamine sulfate, or other components which enhance the transduction efficiency of the reconstituted virus.
  • Lyophilized or dehydrated recombinant virus may be reconstituted with any convenient volume of water or the reconstituting agents noted above that allow substantial, and preferably total
  • methods are provided for treating human patients afflicted with a variety of diseases, including a genetic disease, cancer, an infectious disease, an autoimmune disease, and inflammatory disease, a cardiovascular disease, and a degenerative disease.
  • diseases include but are not limited to;
  • cancers include but are not limited to; solid tumors, leukemias and lymphomas. Representative examples include melanomas, colorectal carcinomas, lung carcinomas (including large cell, small cell, squamous and adeno-carcinomas), renal cell carcinomas, cervical cancer, adult T-cell lymphoma leukemia, and breast adeno-carcinomas.
  • Infectious diseases include but not limited to; hepatitis, tuberculosis, malaria, human immunodeficiency virus, herpes virus, tetanus, dysentery, shigella, FeLV, and FIV.
  • Degenerative diseases include but are not limited to: Alzheimer's disease, multiple sclerosis, muscular dystrophy, amyotrophic lateral sclerosis, Inflammatory diseases include rheumatoid arthritis, spinal meningitis, and pancreatitis.
  • Autoimmune diseases include diabetes, uveitis, HIV, and SCID.
  • Cardiovascular diseases include, chronic rheumatic heart disease, arteriosclerosis, mitral valve and aortic stenosis, myocarditis, pericarditis, Marfan's syndrome, Ehlers-Danlos syndrome, Churg-Strauss syndrome, and scleroderma.
  • a "therapeutically effective amount" of a gene product expressed from a vector construct according to the invention is an amount that achieves a desired therapeutic benefit in a patient to an extent greater than that observed when the patient was not treated with the gene product.
  • a “therapeutically effective amount” refers to the amount of factor VIII needed to produce therapeutically beneficial clotting and will thus generally be determined by each patient's attending physician, although semm levels of about 0.2 ng/mL (about 0.1% of "normal” levels) or more will be therapeutically beneficial.
  • a “therapeutically effective amount” is an amount sufficient to achieve a clinically relevant change in the patient's condition through reduced expression of the harmful gene product, most often a protein.
  • the RNA molecule with intrinsic biological activity will be expressed in transduced hematopoietic stem cells in molar excess to the targeted RNA molecule.
  • Expression levels of the heterologous and targeted RNAs can be determined by various assays, e.g., by PCR analysis.
  • the dosage for ex vivo gene modified hematopoietic stem cells will be in the range of 10 5 to 10 8 cells per kilogram pateint body weight depending upon the purity of stem cells in the starting population.
  • 10 to 10 8 cells will be transduced ex vivo for re-infusion into a patient; for more highly enriched stem cell populations, such as CD34 + Thy-1 L + in- selected cells, usually from 10 5 to 10 7 cells will be transduced and re-infused.
  • the stem cell population will usually be transduced at mutiplicity of infection (MOI) of 10 to 1000, typically around 100 infectious recombinant retroviral particles per cell.
  • MOI mutiplicity of infection
  • high titer preparations of retroviral vectors are used for ex vivo transduction.
  • the volume that the high titer preparation is delivered in ex vivo is preferably not greater than 10% of the culture medium volume of the cell culture. More preferably, the volume of the high titer preparation is less than 1%, still more preferably less than 0 1%, and still more preferably less than 0.001% of the total cell culture volume.
  • the retrovirus is delivered in a medium that is free of agents that disturb or are toxic to the transduced cells in culture (eg. in an aqueous liquid with a composition similar to that of cell culture medium).
  • a pluripotent hematopoietic stem cell may be defined as follows: (1) a cell which gives rise to progeny in all defined hematolymphoid lineages; and (2) a cell which is capable of fully reconstituting a seriously immunocompromised host of all blood cell types and their progenitors, including the pluripotent hematopoietic stem cell by self renewal.
  • Hematopoietic stem cells refers to a population of hematopoietic cells having all of the long-term engrafting potential in vivo.
  • Animal models for long-term engrafting potential of candidate human hematopoietic stem cell populations include the SClD-hu bone model (Kyoizumi et al. (1992) Blood 79: 1704; Murray et al. (1995) Blood 85(2) 368-378) and the in utero sheep model (Zanjani et al. (1992) J. Clin. Invest. 89: 1179.
  • LTCIC long-term culture-initiating cell
  • a highly enriched stem cells population is preferred in order to maximize efficiency of gene transfer into the desired target cells.
  • an enriched stem cell population is exemplified by a population of cells selected by expression of the CD34 marker.
  • a population enriched in CD34+ cells will have an LTCIC frequency in the range of 1/50 to 1/500, more usually in the range of 1/50 to 1/200.
  • the stem cell population will be more highly enriched for stem cells than that provided by a population selected on the basis of CD34 + expression alone.
  • a highly enriched stem cell population will typically have an LTCIC frequency in the range of 1/5 to 1/100, more usually in the range of 1/10 to 1/50. Preferably it will have an LTCIC frequency of at least 1/50.
  • Exemplary of a highly enriched stem cell population is a population having the CD34 + Thyl + Lin- phenotype as described in U.S. Patent No. 5,061,620. A population of this phenotype will typically have an average LTCIC frequency of approximately 1/20. Murray et al. (1995); Lansdorp et al. (1993) J. Exp. Med. 177: 1331.
  • Hematopoietic stem cells may be isolated from any known source of stem cells, including bone marrow, mobilized peripheral blood (MPB), and umbilical cord blood.
  • stem cells including bone marrow, mobilized peripheral blood (MPB), and umbilical cord blood.
  • MPB mobilized peripheral blood
  • umbilical cord blood may be isolated from any known source of stem cells, including bone marrow, mobilized peripheral blood (MPB), and umbilical cord blood.
  • bone marrow cells may be obtained from a source of bone marrow, including ileum (e.g., from the hip bone via the iliac crest), tibia, femur, spine, or other bone cavities.
  • Other sources of hematopoietic stem cells include embryonic yolk sac, fetal liver, and fetal spleen
  • an appropriate solution may be used to flush the bone, including saline solution, conveniently supplemented with fetal calf semm (FCS) or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from about 5 to 25 mM.
  • Convenient buffers include HEPES, phosphate buffers and lactate buffers. Otherwise bone marrow may be aspirated from the bone in accordance with conventional techniques.
  • cytokines e.g., GM-CSF, G-CSF or IL-3
  • cytokines e.g., GM-CSF, G-CSF or IL-3
  • apheresis for total white cells begins when the total white cell count reaches 500-2000 cells/ml and the platelet count reaches 50,000/ml.
  • daily aphoresis samples may be monitored for cells expressing CD34 and/or Thy-1.
  • the collection of peripheral blood leukocytes begins when an increase in CD34 + and/or Thy-1 + cells is detected in order to obtain a sample during the peak of stem cell mobilization. While CD34 expression normally correlates with stem cell mobilization, in some cases it does not. Therefore, it is preferable to use the Thy-1 marker alone or in combination with CD34 to monitor stem cell mobilization (Murray, et al. 1995).
  • Monoclonal antibodies and monoclonal antibody fragments are particularly useful for identifying markers associated with particular cell lineages and/or stages of differentiation.
  • the antibodies (or antibody fragments) may be attached to a solid support to allow for crude separation. The separation techniques employed should maximize the viability of the fraction to be collected.
  • separation techniques include those based on differences in physical (density gradient centrifugation and counter-flow centrifugal elutriation), cell surface (lectin and antibody affinity), and vital staining properties (mitochondria-binding dye rhodamine 123 and DNA-binding dye Hoechst 33342).
  • Procedures for separation may include 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 complement and cytotoxins, and "panning" with antibody attached to a solid matrix or any other convenient technique.
  • Techniques providing accurate separation include 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.
  • a large proportion of the differentiated cells may be removed by initially using a relatively crude separation, where major cell population lineages of the hematopoietic system, such as lymphocytic and myelomonocytic, are removed, as well as minor populations, such as megakaryocytic, mast cells, eosinophils and basophils Usually, at least about 70 to 90 percent of the hematopoietic cells will be removed.
  • Positive selection refers to a selection procedure whereby the cell population of interest expresses the marker used as the basis for selection and, therefore, cells expressing the marker are retained and those not expressing the marker are discarded
  • Negative selection refers to a selection procedure whereby the cell population of interest does not express the marker or have the characteristic that is used as the basis for selection and, therefore, cells having the specified marker or characteristic are discarded and those not having the specified marker or characteristic are retained
  • positive selection techniques may be employed to select stem cells based on, e.g., CD34 expression.
  • One technique providing positive selection for CD34+ cells with high purity is described in PCT patent application No. WO94/02016, in which CD34+ cells are selected using a hapten conjugated anti-CD34 antibody and a hapten competition release system.
  • a negative selection maybe carried out, where antibodies to lineage-specific markers present on dedicated cells are employed and the lineage-committed removed, e.g., by magnetic bead depletion or flow cytometer.
  • these markers include CD2-, CD3-, CD7-, CD8-, CD10-, CD14-, CD15-, CD16-, CD19-, CD20-, CD33- and glycophorin A-.
  • negative selection will yield a cell population which is at least CD14- and CD15-, and preferably which is at least CD2-, CD14-, CD15-, CD16-, CD19- and glycophorin A-.
  • Lin- refers to a cell population lacking at least one lineage-specific marker.
  • the hematopoietic cell composition may then be further separated using positive selection for the Thy-1 marker and/or selection for rhodamine 10 , whereby a highly enriched hematopoietic stem cell population is achieved. See Table 1.
  • the purified hematopoietic stem cells have low side scatter and low to medium forward scatter profiles by FACS analysis.
  • Cytospin preparations show the enriched stem cells to have a size between mature lymphoid cells and mature granulocytes.
  • Cells may be selected based on light-scatter properties as well as their expression of various cell surface antigens.
  • cells are initially separated by a crude separation, followed by a fine separation, with positive selection of one or more markers associated with stem cells and negative selection for one or more markers associated with lineage committed cells.
  • Compositions highly enriched in hematopoietic stem cells may be achieved in this manner.
  • the desired hematopoietic stem cells are exemplified by a population with the CD34 + Thy-1 + Lin- phenotype.
  • a hematopoietic stem cell composition is characterized by being able to be maintained in culture for extended periods of time, being capable of selection and transfer to secondary and higher order cultures, and being capable of differentiating into the various lymphocytic and myelomonocytic lineages, particularly B and T lymphocytes, monocytes, macrophages, neutrophils, erythrocytes and the like
  • the stem cells may be grown in culture in an appropriate nutrient medium, including conditioned medium, a co-culture with an appropriate stromal cell line or a medium comprising a combination of growth factors sufficient to maintain the growth of hematopoietic cells.
  • an appropriate nutrient medium including conditioned medium, a co-culture with an appropriate stromal cell line or a medium comprising a combination of growth factors sufficient to maintain the growth of hematopoietic cells.
  • various stromal cell lines may be used. Since human stromal cell lines are not required, other stromal cell lines may be employed, including rodentiae, particularly murine stromal cell lines. Suitable murine stromal cell lines include AC3 and AC6, which are described in Whitlock, et al. Cell 48: 1009, 1987.
  • the stromal cell line used is a passage of AC6, AC6.21 (otherwise referred to as SySl).
  • Cytokines may also be added, including, e.g., leukemia inhibitory factor (LIF), interleukins, colony stimulating factors, and stem cell factor (SCF, also known as steel factor, c-kit ligand, MGF).
  • LIF leukemia inhibitory factor
  • SCF stem cell factor
  • MGF steel factor
  • LJT leukemia inhibitory factor
  • stem cell factor JL-3, JL-6, GM- CSF, G-CSF, MlP-1 ⁇
  • the flk2/flt3 ligand the flk2/flt3 ligand
  • TPO/npl ligand TPO/npl ligand.
  • the factors which are employed may be naturally occurring or synthetic, e.g., prepared recombinantly, and preferably are human. The amount of the factors used will generally be in the range of about 1 ng/ml to 100 ng/ml.
  • the concentration will be in the range of about 1 ng/ml to 100 ng/mg, more usually 5 ng/ml to 30 ng/ml; for lL-3, the concentration will be in the range of about 5 ng/ml to 100 ng/ml, more usually 5 ng/ml to 50 ng/ml; for JL-6, the concentration will be in the range of about 5 ng/ml to 50 ng/ml, more usually 5 ng/ml to 20 ng/ml, and for GM-CSF, the concentration will generally be 5 ng/ml to 50 ng/ml, more usually 5 ng/ml to 20 ng/ml, and for SCF the concentration will generally be 10 ng/ml to 150 ng/ml, and usually 50 ng/ml to 100 ng/ml.
  • the hematopoietic stem cells are optionally expanded prior to or after retroviral transduction.
  • the growth factors may be present only during the initial course of the stem cell growth and expansion, usually at least 24 hours, more usually at least about 48 hours to 4 days, or may be maintained during the course of the expansion.
  • the stem cells are cultured with or without cytokines in an appropriate medium, transduced with the
  • hematopoietic stem cells in a given hematopoietic cell population are transduced with a recombinant retroviral particle according to the invention.
  • At least about 1% of the hematopoietic stem cells in a given hematopoietic cell population are transduced with a recombinant retroviral particle according to the invention
  • at least about 5% of the hematopoietic stem cells in a given hematopoietic cell population are transduced with a recombinant retroviral particle according to the invention
  • at least about 10% of the hematopoietic stem cells in a given hematopoietic cell population are transduced with a recombinant retroviral particle as described herein.
  • At least about 25% ofthe hematopoietic stem cells in a given hematopoietic cell population are transduced.
  • at least about 50% of the hematopoietic stem cells in a given hematopoietic cell population are transduced.
  • at least about 60%, 70%, 75%, 80%, 90%, or 95% of the hematopoietic stem cells in a given hematopoietic cell population are transduced with a recombinant retroviral particle as described herein.
  • Transduction may be accomplished by the direct co-culture of stem cells with recombinant retroviral particle producer cells, e.g., by the method of Bregni, et al. , Blood 50:1418, 1992. For clinical applications, however, transduction by culturing the
  • hematopoietic stem cells with recombinant retroviral supernatant alone or with purified retroviral preparations as described herein, in the absence of stromal cells, is preferred.
  • Transductions may be performed by culturing the hematopoietic stem cells with the virus for from about four hours to six days. Preferably, transduction is carried out for three days, with the media replaced daily with media containing fresh recombinant retrovirus particles.
  • the stem cells may be cultured in the presence of the retrovirus for several hours, e.g., four hours, daily for three to four days, with fresh media replacing the virus- containing media each day.
  • the cell/virus preparations may be centrifuged.
  • growth factors will be included in such amounts and concentration to maintain cell viability and induce cell cycling.
  • the cultures will include at least stem cell factor (SCF, also known as steel factor, MGF, and c-kit ligand), lL-3, and lL-6.
  • SCF stem cell factor
  • Other cytokines of interest include leukemia inhibitory factor (LlF), G-CSF, GM-CSF, MlP, flk2/flt3 ligand, and TPO.
  • Polycations such as protamine sulfate, polybrene and the like, will generally be included to promote binding. Protamine sulfate and polybrene are typically used preferably at a concentration of 4 ng/ml).
  • Gene transfer into hematopoietic stem cells may be used to treat a variety of neoplastic, infectious or genetic diseases. For example, one may introduce genes that confer resistance to chemotherapeutic agents, thereby protecting the progeny hematopoietic cells, allowing higher doses of chemotherapy and thereby improving the therapeutic benefit.
  • the mdrl gene (see U.S Patent No. 5,206,352) may be introduced into
  • hematopoietic stem cells to provide increased resistance to a wide variety of
  • chemotherapeutic drugs which are exported by the mdrl gene product, in combination with the administration of chemotherapeutics such as taxol, e.g., for breast cancer treatment.
  • chemotherapeutics such as taxol, e.g., for breast cancer treatment.
  • genes that provide increased resistance to alkylating agents, such as melphalan may be introduced into hematopoietic stem cells in conjunction with high dose
  • stem cells may be modified to endow the progeny with resistance to the infectious agent.
  • HAV human immunodeficiency virus
  • specific, sense, antisense or ribo zyme sequences may be introduced that interfere with viral infection or replication in the target cells.
  • the introduced gene products may serve as "decoys" by binding essential viral proteins, thereby interfering with the normal viral life cycle and inhibiting replication.
  • hematopoietic stem cells may be modified to produce a product to correct a genetic deficiency, or where the host has acquired a genetic deficiency through a subsequent disease.
  • Genes that may correct a genetic deficiency include adenosine deaminase for the treatment of ADA- severe combined immunodeficiency;
  • glucocerebrosidase for the treatment of Gaucher's disease
  • beta-globin for the treatment of sickle cell anemia
  • factor VM or factor IX for the treatment of hemophilia.
  • cell immunotherapy involves removal of bone marrow or other source of hematopoietic stem cells from a human host, isolating the stem cells from the source and optionally expanding the stem cells. Meanwhile, the host may be treated to partially, substantially or completely ablate native hematopoietic capability.
  • the isolated hematopoietic stem cells may be modified prior to or during this period of time, so as to provide hematopoietic stem cells having the desired genetic modification.
  • the modified hematopoietic stem cells are reintroduced to the host to provide for expression of the foreign gene (s), and to reconstitute a functional hematopoietic system, if necessary.
  • the methods of stem cell removal, host ablation and hematopoietic stem cell repopulation are known in the art. If necessary, the process may be repeated to ensure substantial repopulation of the modified stem cells.
  • a vector-specific probe or PCR using vector-specific primers, may be used to verify the presence of the vector construct in the transduced stem cells or their progeny.
  • the cells may be grown under various conditions to ensure that they are capable of maturation to all of the hematopoietic lineages while maintaining the capability, as appropriate, of the introduced DNA Various tests in vitro and in vivo may be employed to ensure that the pluripotent capability of the stem cells has been maintained
  • compositions comprising hematopoietic stem cells provide for production of myeloid cells and lymphoid cells in appropriate cultures
  • mouse or human stromal cells are provided, which may come from various sources, including, but not limited to, AC3, AC6 or stromal cells derived from mouse or human bone marrow by selection for the ability to maintain hematopoietic stem cells, and the like.
  • the stromal cells are AC6.21 and the ability to produce B lymphocytes and myeloid cells is determined in cultures supplied with LIF and JL-6.
  • the cells are analyzed by FACS for expression of CD19 (a B cell marker) andCD33 (a myeloid cell marker).
  • the hematopoietic stem cells can be analyzed for the ability to give rise to B cells, T cells and myelomonocytic cells in in vivo assays, as described below.
  • fetal thymus is isolated and cultured from 4 to 7 days at 25°C, so as to deplete substantially the lymphoid population.
  • the cells to be tested for T-cell activity are then microinjected into the thymus tissue, where the HLA ofthe population which is injected is mismatched with the HLA of the thymus cells.
  • the thymus tissue may then be transplanted into a scid/scid mouse as described in US Patent No.
  • mice are sacrificed 6 to 7 weeks after transplantation and the thymus graft recovered and reduced to a single cell suspension.
  • Donor-derived cells are detected by HLA staining and thymus differentiation analyzed by CD4 and CD8 staining and FACS analysis.
  • human fetal bone is isolated and a longitudinally sliced portion of this bone is transferred into the mammary fat pad of a SClD/SClD animal.
  • the bone cavity is diminished in endogenous cells by whole body irradiation of the mouse host prior to injection of the test donor hematopoietic cell population.
  • the HLA of the population which is injected is mismatched with the HLA of the recipient bone cells.
  • Stem cells from human hematopoietic sources will sustain B lymphopoiesis and myelopoiesis in such a SCID-hu bone model.
  • BFU-E units for example, methylcellulose culture to show that the cells are capable of developing the erythroid lineage See Metcalf ( 1977) In Recent Results in Cancer Research 61. Springer- Verlag, Berlin, pp. 1-227. Examples
  • Vector KT-1 differs from KT-3B and KT-3C in that the former lacks a selectable marker which in KT-3B is neomycin resistance, whereas KT-3C confers phleomycin resistance.
  • the N2R5 construct is mutated by site-directed in vitro mutagenesis to change the ATG start codon to ATT preventing gag expression.
  • This mutagenized fragment is 200 base pairs (bp) in length and flanked by Pst I restriction sites.
  • the Pst I-Pst I mutated fragment is purified from the SK + plasmid and inserted into the Pst I site of N2 MoMLV 5' LTR in plasmid pUC31 to replace the non-mutated 200 bp fragment.
  • the plasmid pUC31 is derived from pUCl9 (Stratagene, La Jolla, CA) in which additional restriction sites Xho I, Bgl II, BssH II and Nco I are inserted between the EcoR I and Sac I sites of the polylinker. This construct is designated pUC31/N2R5gM.
  • a 1.0 kilobase (Kb) MoMLV 3' LTR EcoR I-EcoR I fragment from N2 is cloned into plasmid SK + resulting in a construct designated N2R3-.
  • a 1.0 Kb Cla I-Hind III fragment is purified from this construct.
  • the Cla I-Cla I dominant selectable marker gene fragment from pAFVXM retroviral vector (Kriegler et al. , Cell 35:483, 1984; St. Louis et al. , PNAS 85:3150, 1988), comprising a SV40 early promoter driving expression of the neomycin (neo) phosphotransferase gene, is cloned into the SK + plasmid.
  • This construct is designated SK + SV 2 -neo
  • This construct is designated SK + SV 2 -neo
  • Kb Cla I- BstB I gene fragment is purified from the SK + SV 2 -ne
  • KT-3B orKT-1 vectors are constructed by a three part ligation in which the Xho I- Cla I fragment containing the gene of interest and the 1.0 Kb MoMLV 3' LTR Cla I-Hind III fragment are inserted into the Xho I-Hind lll site of pUC31/N2R5gM plasmid. This gives a vector designated as having the KT-1 backbone.
  • the 1.3 Kb Cla I-BstB I neo gene fragment from the pAFVXM retroviral vector is then inserted into the Cla I site of this plasmid in the sense orientation to yield a vector designated as having the KT-3B backbone.
  • phleomycin resistance is used to make the retroviral backbone KT-3C as follows.
  • the plasmid pUT507 (Mulsant, et al. , supra) is digested with Nde I and the ends blunted with Klenow polymerase I.
  • the sample is then further digested with Hpa I, Cla I linkers ligated to the mix of fragments, followed by digestion with Cla I to remove excess Cla I linkers.
  • the 1.2 Kb Cla I fragment carrying the RSV LTR and the phleomycin resistance gene is isolated by agarose gel electrophoresis followed by purification using Gene Clean (BiolOl, San Diego, CA). This fragment is used in place of the 1.3 Kb Cla I-BstB I neomycin resistance fragment to give the backbone KT- 3C.
  • part (A) describes the production of a vector construct encoding the marker gene ⁇ galactosidase from E. coli
  • part (B) human interferon (hlFN) part (C) a retroviral vector construct encoding human interleukin-2 (hlL-2)
  • part (D) the production of two retroviral vector constructs coding for human factor VIII.
  • the first factor VIII construct codes for the B domain deleted form ofthe protein, while the second construct codes for full length factor VIII.
  • A. Preparation of C ⁇ -gal. ⁇ -gal is obtained from the plasmid pSP65 as a Hindlll-Smal fragment.
  • the murine homologue is first cloned as follows: A m ⁇ -IFN cDNA is cloned into the EcoR I site of pUC1813 essentially as set forth below. Briefly, pUC1813 (containing a sequence encoding ⁇ -IFN) is prepared as essentially described by Kay et al., Nucleic Acids Research 15:211%, 1987; and Gray et al. , PNAS 80.5842, 1983) ( Figure 1A).
  • the m ⁇ -IFN cDNA is retrieved by EcoR I digestion of pUC1813, and the isolated fragment is cloned into the EcoR I site of phosphatase-treated pSP73 (Promega; Madison, WI). This construct is designated SP m ⁇ -IFN.
  • the orientation ofthe cDNA is verified by appropriate restriction enzyme digestion and DNA sequencing. In the sense orientation, the 5' end of the cDNA is adjacent to the Xho I site of the pSP73 polylinker and the 3' end adjacent to the Cla I site.
  • the Xho I-Cla I fragment containing the m ⁇ -IFN cDNA in either sense or antisense orientation is retrieved from SP m ⁇ -IFN construct and cloned into the Xho I-Cla I site of the KT-3 retroviral backbone. This construct is designated KT m ⁇ -IFN.
  • RNA isolation (b) RNA Isolation
  • the PHA stimulated Jurkat cells are resuspended in 22.0 ml guanidinium solution (4 M guanidinium thiocyanate; 20 mM sodium acetate, pH 5.2; 0.1 M dithiothreitol, 0.5% sarcosyl).
  • This cell-guanidinium suspension is then passed through a 20 gauge needle six times in order to disrupt cell membranes.
  • a CsCl solution (5.7 M CsCl, 0.1 M EDTA) is then overlaid with 11.0 mL of the dismpted cell-guanidinium solution.
  • the solution is centrifuged for 24 hours at 28,000 rpm in a SW28.1 rotor (Beckman, Fullerton, CA) at 20°C.
  • RNA is resuspended in a guanidinium-HCl solution (7.4 M guanidinium-HCl; 25 mM Tris-HCl, pH 7.5; 5 mM dithiothreitol) to a final volume of 500.0 ⁇ l.
  • a guanidinium-HCl solution (7.4 M guanidinium-HCl; 25 mM Tris-HCl, pH 7.5; 5 mM dithiothreitol) to a final volume of 500.0 ⁇ l.
  • This solution is transferred to a microcentrifuge tube. Twelve and one-half microliters of concentrated Glacial acetic acid (HAc) and 250 ⁇ l of 100% EtOH are added to the microfuge tube.
  • the solution is mixed and stored for several days at -20°C to precipitate RNA.
  • RNA is determined by measuring optical densities at 260 and 280 nm.
  • the PCR reaction mixture contains 100.0 ⁇ l reverse transcription reaction; 356.0 1 DI H 2 O; 40.0 ⁇ l lOx PCR buffer; 1.0 ⁇ l (137 nmol/mL) V-OLI #5 (Sequence ID No. 2); 0.5 ⁇ l (320 nmol/mL) V-OLI #6 (Sequence ID No. 3), and 2.5 ⁇ l, 5,000 U/ml, Taq polymerase (EC 2.7.7.7, Perkin-Elmer Cetus, CA). One hundred microliters of this mixture is aliquoted into each of 5 tubes.
  • This primer is complementary to a sequence of the m ⁇ -IFN cDNA 30 base pairs downstream of the stop codon.
  • This primer is complementary to the 5' coding region of the m ⁇ -lFN gene, beginning at the ATG start codon.
  • the 5' end of the primer contains a Xho I restriction site.
  • This primer is complementary to the 3' coding region of the m ⁇ -JFN gene, ending at the TAA stop codon.
  • the 5' end of the primer contains a Cla I restriction site.
  • reaction vessel first at 95°C for 1 minute, next at 67°C for 2 minutes and finally at 72°C for 7 minutes.
  • the completed PCR amplification reactions are stored at 4°C for 1 month in preparation for PCR DNA isolation.
  • the aqueous phase from the PCR amplification reactions are transferred into a single microfuge tube Fifty microliters of 3 M sodium acetate and 500 0 ⁇ l of chloroform isoamyl alcohol (24:1) is added to the solution. The solution is vortexed and then centrifuged at 14,000 rpm at room temperature for 5 minutes. The upper aqueous phase is transferred to a fresh microfuge tube and 1.0 mL of 100% EtOH is added. This solution is incubated for 4.5 hours at -20°C and then centrifuged at 14,000 rpm for 20 minutes. The supernatant is decanted, and the pellet is rinsed with 500.0 ⁇ l of 70% EtOH. The pellet is dried by centrifugation under a vacuum. The isolated h ⁇ -IFN PCR DNA is resuspended in 10.0 ⁇ l DI H 2 O.
  • the h ⁇ -INF PCR DNA is blunt ended using T4 DNA polymerase.
  • T4 DNA polymerase buffer 0.33 M Tris-acetate, pH 7.9, 0.66 M potassium acetate, 0.10 M magnesium acetate, 5 mM dithiothreitol, 1 mg/mL bovine semm albumin (BSA)
  • BSA bovine semm albumin
  • 1.0 ⁇ l, 2.5 mM dNTP a mixture containing equal molar concentrations of dATP, dGTP, dTTP and dCTP
  • 7.0 ⁇ l DI H 2 O 1.0 ⁇ l, 5000 U/mL
  • Klenow fragment (EC 2.7.7.7, New England Biolabs, MA); and 1.0 ⁇ l, 3000 U/ml, T4 DNA polymerase (EC 2.7.7.7, New England Biolabs, MA) are mixed together and incubated at
  • reaction mixture is then incubated at room temperature for 40 minutes and followed by an incubation at 68°C for 15 minutes.
  • the blunt ended h ⁇ -INF is isolated by agarose gel electrophoresis. Specifically, 2 0 ⁇ l of loading dye (0.25% bromophenol blue; 0.25% xylene cyanol; and 50% glycerol) is added to reaction mixture and 4.0 ⁇ l is loaded into each of 5 lanes of a 1% agarose/Tris- borate-EDTA (TBE) gel containing ethidium bromide. Electrophoresis of the gel is performed for 1 hour at 100 volts The desired DNA band containing h ⁇ -INF, approximately
  • This band is removed from the gel by electrophoretic transfer onto NA 45 paper (Schleicher and Schuell, Keene, NH.
  • NA 45 paper Schoeller and Schuell, Keene, NH.
  • the paper is incubated at 68°C for 40 minutes in 400.0 ⁇ l of high salt NET buffer (1 M NaCl; 0.1 mM EDTA; and 20 mM Tris, pH 8.0) to elute the
  • the NA 45 paper is removed from solution and 400.0 ⁇ l of
  • the upper aqueous phase is transferred, a second time, to a fresh tube and 700.0 ⁇ l of 100% EtOH is added
  • the tube is incubated at -20°C for 3 days Following incubation, the DNA is precipitated from the tube by centrifugation for 20 minutes at 14,000 rpm The supernatant is decanted and the pellet is rinsed with 500 0 ⁇ l of 70% EtOH
  • the pellet, containing blunt ended h ⁇ -lFN DNA is dried by centrifugation under vaeuum and resuspended in 50.0 ⁇ l of DI H 2 O.
  • the isolated blunt ended h ⁇ -IFN DNA is phosphorylated using polynucleotide kinase.
  • 10x kinase buffer 0.5 M Tris- HCl, pH 7.6, 0 1 M MgCl 2 ; 50 mM dithiothreito
  • SK + plasmid is digested with Hinc II restriction endonuclease and purified by agarose gel electrophoresis as described below. Specifically, 5.9 ⁇ l (1 .7 mg/mL) SK + plasmid DNA (Stratagene, San Diego, CA); 4.0 ⁇ l 10x Universal buffer (Stratagene, San Diego, CA); 30.1 ⁇ l DI H 2 O, and 4.0 ⁇ l Hinc II, 10,000 U/mL, are mixed in a tube and incubated for 7 hours at 37°C.
  • Dephosphorylation of the Hinc ll cleavage site of the plasmid is performed using calf intestine alkaline phosphatase. Specifically, 50.0 ⁇ l digested SK + plasmid, 5.0 ⁇ l 1 M Tris, pH 8.0; 2.0 ⁇ l 5 mM EDTA, pH 8.0; 43.0 ⁇ l H 2 O and 2.0 ⁇ l, 1,000 U/mL, calf intestinal phosphatase ("CIP") (Boehringer Mannheim, Indianapolis, IN) are mixed in a tube and incubated at 37°C for 15 minutes. Following incubation, 2.0 ⁇ l CIP is added. and the solution is incubated at 55°C for 90 minutes.
  • CIP calf intestinal phosphatase
  • the h ⁇ -INF PCR DNA is ligated into the SK + plasmid using T4 DNA ligase.
  • DNA is isolated from white bacterial colonies using the procedure described by Sambrook et al. (Molecular Cloning, Cold Springs Harbor Press, 1989). The presence of the h ⁇ -lFN gene is determined by restriction endonuclease cleavage with Xho I, Cla I, Ava ll, Dra I, and Ssp I. The expected endonuclease restriction cleavage fragment sizes for plasmids containing the h ⁇ - IFN gene are presented in Table 2. The isolated DNA plasmid is designated SK h ⁇ -lFN and used in constructing the retroviral vectors.
  • the interferon gene is removed from SK h ⁇ -IFN vector by digestion with Xho I and Cla I restriction endonucleases.
  • the resulting fragment containing the h ⁇ -IFN gene is approximately 500 bp in length, and is isolated in a 1% agarose/TBE gel electrophoresis .
  • the Xho I-Cla I h ⁇ -IFN fragment is then ligated into the KT-3 retroviral backbone. This construct is designated KT h ⁇ -IFN.
  • the structure and presence expression of h ⁇ -IFN is determined by transforming DH5a bacterial strain with the KT h ⁇ -lFN construct.
  • the bacteria is transformed with 15.0 ⁇ l of ligation reaction mixture.
  • the transformed bacterial cells are plated on LB plates containing ampicillin. The plates are incubated overnight at 37°C and bacterial colonies are selected .
  • the DNA is isolated as described in (b) above, and digested with Xho I, Cla I, Dra I, Nde I, and Ssp I.
  • the expected endonuclease restriction cleavage fragment sizes for plasmids containing the h ⁇ - lFN gene are presented in Table 3.
  • This primer is the anti-sense sequence complimentary to codons 106 to 120 of the h ⁇ -IFN gene, and is designated h ⁇ -IFN Rep B.
  • This primer is the sense sequence compUmentary to codons 106 to 120 of the h ⁇ -IFN gene, and is designated h ⁇ -IFN Rep A.
  • This primer is the anti-sense sequence complimentary to the stop codon ATT region and extending seven codons upstream of the h ⁇ -IFN gene, and is designated h ⁇ -lFN 3b.
  • oligonucleotide h ⁇ -lFN 1b 15.0 ⁇ l, 24.6 nmol/ml, oligonucleotide h ⁇ - IFN RepB; and 2.5 ⁇ l, Taq polymerase is mixed in a microfuge tube and 50 ⁇ l is aliquoted into 10 tubes Similarly, a solution of 1 x 10 6 KT h ⁇ -IFN plasmid molecules in 395.0 ⁇ l, DI H 2 O; 50.0 ⁇ l, 10x PCR buffer (500 mM KCl, 200 mM Tris-HCl, pH 8.4, 25 mM MgCl 2 , 1 mg/ml BSA); 5.0 ⁇ l, 2.5 mM
  • each reaction tube is mixed with 25.0 ⁇ l loading dye (0.25% bromophenol blue, 0.25% xylene cyanol and 50% glycerol, agarose gel loading dye) and loaded into the wells of a 2% agarose gel containing ethidium bromide.
  • the gel is electrophoresed at approximately 90 volts for 1 hour.
  • Ultraviolet light is used to visualize the DNA band separation. Two bands are isolated, one fragment of 250 bp in size and the other of 150 bp in size by electrophoretic transfer onto NA 45 paper. Following precipitation, each of the two DNA pellets is resuspended in 20.0 1 DI H 2 O and prepared for further PCR amplification.
  • Each tube is placed in a PCR machine (Model 9600, Perkin-Elmer-Cetus, CA).
  • the PCR program regulates the temperature ofthe reaction vessel for 5 cycles at 94°C for 0.5 minutes.
  • the next cycle is regulated at 55°C for 1 minute.
  • 1.2 ⁇ l h ⁇ -IFN lb and 1.5 ⁇ l h ⁇ -IFN 3b are added to the reaction mixture.
  • the tubes are then PCR amplified using program 10.
  • the product is designated rh ⁇ -IFN.
  • the PCR amplified h ⁇ -lFN DNA is blunt ended using T4 polymerase.
  • 120.0 ⁇ l rh ⁇ -IFN PCR solution is mixed with 1.25 ⁇ l, 2.5 mM dATP; 1.25 ⁇ l, 2.5 mM dGTP; 1.25 ⁇ l, 2.5 mM dCTP; 1.25 1, 2.5 mM dTTP; 1 1, T4 DNA polymerase; and 1.0 ⁇ l Klenow fragment.
  • This mixture is incubated at room temperature for 10 minutes.
  • 13.0 ⁇ l of agarose gel loading dye is added to the mixture and this solution is loaded into a 1% agarose gel.
  • the gel is electrophoresed at approximately 90 volts for 1 hour. Ultraviolet light is used to visualize the DNA banding.
  • a 500 bp band is isolated by electrophoretic transfer onto NA 45 paper as described above. Following precipitation, the DNA pellet is resuspended in 12.0 1 DI H 2 O.
  • the isolated 500 bp fragment is blunt ended using T4 polynucleotide kinase.
  • 1.0 mg of this fragment is mixed with 1.5 ⁇ l 10x kinase buffer (0.5 mM Tris- HCl, pH 7.6; 0.1 mM MgCl 2 ; 50 mM dithiothreitrol; 1 mM spermidine; 1 mM EDTA); 1.5 ⁇ l, 10 mM ATP; and 1.0 ⁇ l, T4 polynucleotide kinase, and incubated at 37°C for 30 minutes
  • KT h ⁇ -IFN Ligation of h ⁇ -IFN gene into retroviral vector is performed as described above.
  • the new vector is designated KT h ⁇ -IFN.
  • the method for cloning hlL-2 into KT-3 retroviral vector is essentially identical to the procedure for cloning hg-lFN into KT-3, with the exception that different primers are required for amplification of the hlL-2 DNA sequence.
  • the following hlL-2 PCR primer sequences are used:
  • V-OLI #55 (Sequence ID No. 8)
  • V-OLI #1 (Sequence ID No. 9)
  • This primer is the sense sequence of the hlL-2 gene complimentary to the 5' coding region beginning at the ATG start codon.
  • the 5' end of the primer contains a Xho I restriction site.
  • V-OLI #2 (Sequence ID No. 10)
  • the primer is the anti-sense sequence of the hlL-2 gene complimentary to the 3' coding region ending at the TAA stop codon.
  • the 5' end of the primer contains the Cla I restriction site.
  • retroviral vectors encoding factor VIII. Due to the size of the full length factor Vlll gene (7,056 bp), packaging constraints of retroviral vectors and because selection for transduced cells is not a requirement for ex vivo hematopoietic stem cell therapy, a retroviral backbone, e.g., KT-1, lacking a selectable marker gene is employed.
  • a gene encoding full length factor VIII can be obtained from a variety of sources.
  • One such source is the plasmid pCIS-F8 (EP 0 260 148 A2, published March 3, 1993), which contains a full length factor Vlll cDNA whose expression is under the control of a CMV major immediate-early (CMV MlE) promoter and enhancer.
  • CMV MlE CMV major immediate-early
  • the factor VIII cDNA contains approximately 80 bp of 5' untranslated sequence from the factor VIII gene and a 3' untranslated region of about 500 bp.
  • the CMV promoter and the factor Vlll sequence lies a CMV intron sequence, or "cis" element.
  • the cis element spanning about 280 bp, comprises a splice donor site from the CMV major immediate-early promoter about 140 bp upstream of a splice acceptor from an immunoglobulin gene, with the intervening region being supplied by an Ig variable region intron.
  • a plasmid, pJW-2, encoding a retroviral vector for expressing full length factor VIII is constructed using the KT-1 backbone from pKT-1.
  • the unique Xho I site is converted to a Not I site by site directed mutagenesis.
  • the resultant plasmid vector is then opened with Not I and Cla I.
  • pCIS-F8 is digested to completion with Cla I and Eag I, for which there are two sites, to release the fragment encoding full length factor VM. This fragment is then ligated into the Not I/Cla I restricted vector to generate a plasmid designated pJW-2.
  • Plasmid Encoding Retroviral Vector ND-5 A plasmid vector encoding a truncation of about 80% (approximately 370 bp) ofthe
  • 3' untranslated region of the factor Vlll cDNA, designated pND-5 is constructed in a pKT-1 vector as follows: As described for pJW-2, the pKT-1 vector employed has its Xho I restriction site replaced by that for Not I.
  • the factor Vlll insert is generated by digesting pCIS-F8 with Cla I and Xba I, the latter enzyme cutting 5' of the factor VIII stop codon The approximately 7 kb fragment containing all but the 3' coding region of the factor Vlll gene is then purified.
  • pCIS-F8 is also digested with Xba I and Pst I to release a 121 bp fragment containing the gene's termination codon.
  • This fragment is also purified and then ligated in a three way ligation with the larger fragment encoding the rest of the factor Vlll gene and Cla I/Pst I restricted BLUESCPJPT® KS + plasmid (Stratagene, San Diego, CA) to produce a plasmid designated pND-2.
  • the unique Sma I site in pND-2 is then changed to a Cla I site by ligating Cla I linkers (New England Biolabs, Beverly, MA) under dilute conditions to the blunt ends created by a Sma I digest. After recircularization and ligation, plasmids containing two Cla 1 sites are identified and designated pND-3.
  • the factor Vlll sequence in pND-3 bounded by Cla I sites and containing the full length gene with a truncation of much of the 3' untranslated region, is cloned as follows into a plasmid backbone derived from a Not I/Cla I digest of pJW-1 [a pKT-1 derivative by cutting at the Xho I site, blunting with Klenow, and inserting a Not I linker (New England Biolabs)], which yields a 5.2 kb Not I/Cla I fragment.
  • pCIS-F8 is cleaved with Eag I and Eco RV and the resulting fragment of about 4.2 kb, encoding the 5' portion of the full length factor VIII gene, is isolated.
  • pND-3 is digested with Eco RV and Cla I and a 3.1 kb fragment is isolated. The two fragments containing portions of the factor Vlll gene are then ligated into the Not I/Cla I digested vector backbone to produce a plasmid designated pND- 5.
  • the precursor DNA for the B-deleted FVIII is obtained from Miles Laboratory.
  • This expression vector is designated p25D and has the exact backbone as pCISF8 above.
  • the Hpa I site at the 3' of the FVllI8 cDNA in p25D is modified to Cla-I by oligolinkers.
  • An Ace I to Cla I fragment is clipped out from the modified p25D plasmid. This fragment spans the B-domain deletion and includes the entire 3' two-thirds of the cDNA.
  • An Ace I to Cla I fragment is removed from the pJW-2 above, and replaced with the modified B-domain deleted fragment just described. This construct is designated B-del-1.
  • Plasmid clones containing the the multi-drug resistance- 1 (MDR-1) gene were obtained from ATCC (ATCC No. 61360 and 65704). The gene is isolated and purified using methods provided in Sambrook et al. (Molecular Cloning. A Laboratory Manual, 2d., 1989). Appropriate endonuclease restriction sites, Xho I and Cla I, (Molecular Cloning: A
  • CD34 + Pluripotent hematopoeitic stem cells, CD34 + are collected from the bone marrow of a patient by a syringe evacuation performed by known techniques. Alternatively, CD34 + cells may also be obtained from the cord blood of an infant if the patient is diagnosed before birth. Generally, 20 bone-marrow aspirations are obtained by puncturing femoral shafts or from the posterior iliac crest under local or general anesthesia. Bone marrow aspirations are then pooled and suspended in Hepes-buffered Hanks' balanced salt solution containing heparin sulfate at 100 Units/ml and deoxyribonuclease I at 100 ⁇ g/ml and then subjected to a Ficoll gradient separation.
  • the buffy coated marrow cells are then collected and washed according to CEPRATETM LC (CD34) Separation system (Cellpro, Bothell, WA).
  • the washed buffy coated cells are then stained sequentially with anti-CD34 monoclonal antibody, washed, then stained with biotinylated secondary antibody supplied with the CEPRATETM system.
  • the cell mixture is then loaded onto the CEPRATETM avidin column.
  • the biotin- labeled cells are adsorbed onto the column while unlabeled cells pass through
  • the column is then rinsed according to the CEPRATE system directions and CD34 + cells eluted by agitation of the column by manually squeezing the gel bed.
  • the purified stem cells are counted and plated at a concentration of 1 x 10 5 cells/ml in Iscove's modified Dulbecco's medium, IMDM (Irvine Scientific, Santa Ana, CA), containing 20% pooled non-heat inactivated human AB semm (hAB semm).
  • IMDM Irvine Scientific, Santa Ana, CA
  • CD34 + cells After purification of CD34 + cells, several methods of transducing purified stem cells may be performed.
  • One approach involves transduction of the purified stem cell population with vector containing supernatant cultures derived from vector producing cells.
  • a second approach involves co-cultivation of an irradiated monolayer of vector producing cells with the purified population of non-adherent CD34 + cells.
  • a third approach involves a similar co- cultivation approach, however the purified CD34 + cells are pre-stimulated with various cytokines and cultured 48 hours prior to the co-cultivation with the irradiated vector producing cells.
  • Pre-stimulation prior to transduction increases effective gene transfer (Nolta et al. , Exp. Hematol. 20:1065; 1992). The increased level of transduction is attributed to increased proliferation of the stem cells necessary for efficient retroviral transduction.
  • Stimulation of these cultures to proliferate also provides increased cell populations for re- infusion into the patient.
  • a MLV structural gene expression vectors To decrease the possibility of replication-competent virus being generated by genetic interactions between the MLV proviral vector DNA and the structural genes of the packaging cell line ("PCL"), separate expression vectors, each lacking the viral LTR, were generated to express the gag/pol and env genes independently. To further decrease the possibility of homologous recombination with MLV vectors and the resultant generation of replication- competent virus, minimal sequences other than the protein coding sequences were used. In order to express high levels of the MLV structural proteins in the host cells, strong
  • MoMLV gag/pol expression vector pSCVIO An example ofthe construction of a MoMLV gag/pol expression vector pSCVIO follows:
  • the CMV promoter-MoMLV gag/pol-SV40 termination signal was ligated into the bluescript vector SK + (Stratagene, San Diego, CA).
  • Host Cell Selection Host cell lines were screened for their ability to efficiently (high titer) rescue a drug resistance retroviral vector A alpha N2 (Armentano, et al. , J. Vir. 61: 1641, 1987; and Eglitas, et al. , Science 230: 1395, 1985) using replication competent retrovirus to produce the gag/pol and env structural genes ("MA" virus). Titer was measured from confluent monolayers 16 h after a medium change by adding filtered supernatants (0 45 urn filters) to 5x10 4 NTH 3T3 TK- cells on a 6 cm tissue culture plate in the presence of 4 ug/ml polybrene followed by selection in G418.
  • MA gag/pol and env structural genes
  • Non-murine cell lines which demonstrated the ability to package MoMLV-based vector with high titre were the cell lines CF2 (canine), D17 (canine), 293 (human), and HT1080 (human). These cell lines are preferred for production of packaging and producer cell lines, although many other cells may be tested and selected by such means.
  • C. Generation of Packaging Cell Lines CF2 (canine), D17 (canine), 293 (human), and HT1080 (human). These cell lines are preferred for production of packaging and producer cell lines, although many other cells may be tested and selected by such means.
  • gag/pol in the cell line and env from the vector allowed efficient packaging of the vector as determined by cell-free transfer of G418 resistance to 3T3 cells (titer). Titer was measured from confluent monolayers 16 h after a medium change by adding filtered supematants (0.45 ⁇ m filters) to 5x10 4 NlH353 TK + cells on a 6 cm tissue culture plate in the presence of 4 ug/ml polybrene followed by selection in G418. Significantly, the vector titers from the cell lines correlated with the levels of p30 gag (Table 4).
  • the gag/pol over-expressors for D17 (4-15) and HT1080 (SCV21) were co-transfected by the same techniques described above except that 1 ⁇ g of either the phleomycin resistance vector, pUT507 (for SCV21), or the hygromycin B resistance marker, pY3 (for 4-15, see Blochlinger and Diggelmann, Mol. Cell Biol. 4:2929, 1984), and 10 ⁇ g of the xenotropic envelope expression vector, pCMVxeno (above) was used.
  • vector particles containing other viral envelopes may be generated in the following manner.
  • 10 ⁇ g ofthe plasmid DNA encoding the retroviral vector construct to be packaged is co-transfected into a cell line which expresses high levels of gag/pol with 10 ⁇ g of DNA from which a VSV-g protein is expressed.
  • the resultant vector, containing VSV-g protein is produced transiently in the co-transfected cells.
  • cell free supematants are added to prospective xenotropic packaging cell lines (which express gag, pol, and env). Cell free supematants are then collected from the confluent monolayers and titered by PCR. Cell clones producing the highest titers are selected as packaging cell lines. This procedure is sometimes referred to "G-hopping " VII Alternative Viral Vector Packaging Techniques
  • Adenovirus vectors are derived from nuclear replicating viruses and can be defective Genes can be inserted into vectors and used to express proteins in mammalian cells either by in vitro construction (Ballay, et al. , EMBO J. 4:3861, 1985) or by recombination in cells (Thummel, et al. , J. Mol. Appl. Genetics 7 435, 1982).
  • One preferred method is to construct plasmids using the adenovirus Major Late
  • MLP Promoter driving (1) gag/pol, (2) env, (3) a modified viral vector construct.
  • a modified viral vector construct is possible because the U3 region of the 5' LTR, which contains the viral vector promoter, can be replaced by other promoter sequences (see, for example, Hartman, Nucl. Acids Res. 76:9345, 1988). This portion will be replaced after one round of reverse transcriptase by the U3 from the 3' LTR.
  • plasmids can then be used to make adenovirus genomes in vitro (Ballay, et al., supra), which are then transfected into 293 cells (a human cell line making adenovirus ElA protein), for which the adenoviral vectors are defective, to yield pure stocks of gag/pol, env and retroviral vector carried separately in defective adenovirus vectors Since the titers of such vectors are typically 10 7 -10 11 /ml, these stocks can be used to infect tissue culture cells simultaneously at high multiplicity The cells will then be programmed to produce retroviral proteins and retroviral vector genomes at high levels Since the adenovirus vectors are defective, no large amounts of direct cell lysis will occur and retroviral vectors can be harvested from the cell supernatants .
  • viral vectors such as those derived from unrelated retroviral vectors (e.g., RSV, MMTV or HlV) can be used in the same manner to generate vectors from primary cells .
  • these adenoviral vectors are used in conjunction with primary cells, giving rise to retroviral vector preparations from primary cells.
  • retroviral vector preparations from primary cells e.g., RSV, MMTV or HlV
  • retroviral vector preparations from primary cells e.g., MMTV or HlV
  • Another alternative for making recombinant xenotropic retroviral particles is an in vitro packaging system.
  • such a system can be employ the following components:
  • gag/pol and env proteins made in the baculovirus system in a similar manner as described in Smith, et al. , supra, or in other protein production systems, such as yeast or E. coli);
  • tRNA made as in (2) or purified from yeast or mammalian cells made as in (2) or purified from yeast or mammalian cells;
  • liposomes preferably with embedded env protein
  • cell extract or purified components typically from mouse cells to provide env processing, and any or other necessary cell-derived functions.
  • the components of (1), (2), and (3) are mixed.
  • the env protein, cell extract and pre-liposome mix (in a suitable solvent) is then added.
  • the env protein is embedded in the liposomes prior to adding the resulting liposome-embedded env to the mixture of (1), (2), and (3).
  • the mix is treated (e.g., by sonication, temperature manipulation, or rotary dialysis) to allow encapsidation of the nascent viral particles with lipid plus embedded env protein in a manner similar to that for liposome encapsidation of pharmaceuticals, as described in Gould-Fogerite, et al. , Anal.
  • the extended S + L- assay determines whether replication competent, infectious virus is present in the supernatant of the cell line of interest.
  • the assay is based on the empirical observation that infectious retroviruses generate foci on the indicator cell line MiCl 1 (ATCC No. CCL 64. 1).
  • the MiCl 1 cell line is derived from the MvlLu mink cell line (ATCC No. CCL 64) by transduction with Murine Sarcoma Virus (MSV). It is a non-producer, non- transformed, revertant clone containing a replication defective murine sarcoma provirus, S + , but not a replication competent murine leukemia provirus, L-.
  • Infection of MiCl 1 cells with replication competent retrovirus "activates" the MSV genome to trigger "transformation" which results in foci formation.
  • Supernatant is removed from the cell line to be tested for presence of replication competent retrovirus and passed through a 0.45 ⁇ m filter to remove any cells.
  • MvlLu cells are seeded at 1.0 x 10 5 cells per well (one well per sample to be tested) on a 6 well plate in 2 mL Dulbecco's Modified Eagle Medium (DMEM), 10% FBS and 8 ⁇ g/mL polybrene.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS FBS
  • 8 ⁇ g/mL polybrene 8 ⁇ g/mL polybrene.
  • MvlLu cells are plated in the same manner for positive and negative controls on separate 6 well plates. The cells are incubated overnight at 37°C, 10% CO 2 .
  • 1.0 mL of test supernatant is added to the MvlLu cells.
  • the negative control plates are incubated with 1.0 mL of media.
  • the positive control consists of three dilutions (200 focus forming units (ffu), 20 ffu and 2 ffu each in 1.0 mL media) of MA virus (Miller, et al. , Molec. and Cell Biol, 5:431, 1985) which is added to the cells in the positive control wells.
  • the cells are incubated overnight.
  • the media is aspirated and 3.0 mL of fresh DMEM and 10% FBS is added to the cells.
  • the cells are allowed to grow to confluency and are split 1 :10 on day 6 and day 10, amplifying any replication competent retrovirus.
  • the media on the MvlLu cells is aspirated and 2.0 mL DMEM and 10% FBS is added to the cells.
  • the MiCli cells are seeded at 1.0 x 10 5 cells per well in 2.0 mL
  • the test article is determined to be contaminated with replication competent retrovirus if foci appear on the MiCli cells.
  • producer cells are cocultivated with an equivalent number of Mus dunni cells (NlH NIAlD Bethesda, MD).
  • Small scale co-cultivations are performed by mixing of 5 0 x 10 5 Mus dunni cells with 5.0 x 10 5 producer cells and seeding the mixture into 10 cm plates (10 mL standard culture media/plate, 4 ⁇ g/mL polybrene) at day 0. Every 3-4 days the cultures are split at a 1:10 ratio and 5.0 x 10 5 Mus dunni cells are added to each culture plate to effectively dilute out the producer cell line and provide maximum amplification of RCR.
  • culture supernatants are harvested, passed through a 0.45 ⁇ m cellulose- acetate filter, and tested in the MdH marker rescue assay.
  • Large scale co-cultivations are performed by seeding a mixture of 1.0 x 10 8 Mus dunni cells and 1.0 x 10 8 producer cells into a total of twenty T-150 flasks (30 mL standard culture media/flask, 4 ⁇ g/mL polybrene). Cultures are split at a ratio of 1 :10 on days 3, 6, and 13 and at a ratio of 1 :20 on day 9.
  • the final supematants are harvested, filtered and a portion of each is tested in the MdH marker rescue assay.
  • the MdH marker rescue cell line is cloned from a pool of Mus dunni cells transduced with LHL, a retroviral vector encoding the hygromycin B resistance gene (Palmer, et al. , Proc. Nat'l. Acad. Sci. USA, 84:1055, 1987).
  • the retroviral vector can be rescued from MdH cells upon infection of the cells with RCR.
  • One mL of test sample is added to a well of a 6-well plate containing 1 x 10 5 MdH cells in 2 mL standard culture medium (DMEM with 10% FBS, 1% 200 mM L-glutamine, 1% non-essential amino acids) containing 4 ⁇ g/mL polybrene.
  • the packaging cell line HX or DX is seeded at 5.0 x 10 5 cells on a 10 cm tissue culture dish on day 1 with DMEM and 10% fetal bovine serum (FBS). On day 2, the media is replaced with 5.0 mL fresh media 4 hours prior to transfection. Standard calcium phosphate-DNA co-precipitations are performed by mixing 40.0 ⁇ l 2.5 M CaCl 2 , 10 ⁇ g of the plasmid encoding the vector to be packaged, and deionized H 2 O to a total volume of 400 ⁇ l.
  • the DNA-CaCl 2 solutions are then added dropwise with constant agitation to 400 ⁇ l of precipitation buffer (50 mM HEPES-NaOH, pH 7.1; 0.25 M NaCl and 1.5 mM Na 2 HPO 4 - NaH 2 PO 4 ). These mixtures are incubated at room temperature for 10 minutes. The resultant fine precipitates are added to different culture dishes of cells. The cells are incubated with the DNA precipitate overnight at 37°C. On day 3, the media is aspirated and fresh media is added. Supematants are removed on day 4, passed through 0.45 ⁇ m filters, and stored at -80°C.
  • precipitation buffer 50 mM HEPES-NaOH, pH 7.1; 0.25 M NaCl and 1.5 mM Na 2 HPO 4 - NaH 2 PO 4 .
  • DX or HX packaging cells are seeded at 1.0 x 10 5 cells/3 cm tissue culture dish in 2 mL DMEM and 10% FBS, 4 ⁇ g/mL polybrene (Sigma, St. Louis, MO) on day 1. On day 2, 3.0 mL, 1.0 mL and 0.2 mL of each of a freshly collected supernatant containing VSV-g pseudotyped retroviral particles carrying the desired vector are added to the HX cells. The cells are incubated overnight at 37°C.
  • the pools of cells are cloned by limiting dilution by removing the cells from the plate and counting the cell suspension, diluting the cells suspension down to 10 cells/mL and adding 0.1 mL to each well (1 cell/well) of a 96 well plate (Corning, Corning, NY). Cells are incubated for 14 days at 37°C, 10% CO 2 .
  • Several clones producing the desired recombinant xenotropic retrovirus are selected and expanded up to 24 well plates, 6 well plates, and finally to 10 cm plates, at which time the clones are assayed for expression of the appropriate retroviral vector and the supematants are collected and assayed for retroviral titer.
  • DX and HX cell lines may be derived that produce recombinant xenotropic retroviral vectors with titers greater than or equal to 1 x 10 6 cfu/mL in culture.
  • telomere titers are determined by transduction of target cells such as HT1080, with appropriate dilutions of a vector preparation, followed by antibiotic selection and counting of surviving colonies (WO 91/02805).
  • recombinant retroviral vectors carrying a desired vector construct may not include a gene coding for setectable marker, as may be the case when the vector construct encodes a large gene of interest, for instance, full length factor VIII, titering assays other than those based on selection of drug resistant colonies are required.
  • antibody and PCR assays the latter of which is described below, may be employed to determine retroviral vector titer, i.e., the number of infectious particles comprising the retroviral vectors of the invention. While such a PCR assay may be required in the context of a vector lacking a selectable marker, it is understood that such an assay can be employed for any given vector.
  • primers can readily be designed by those skilled in the art and will depend on the retroviral vector backbone employed and the components thereof, the particular region(s) desired to be amplified, etc.
  • Representative examples of particular primer pairs include those specific for LTR sequences, packaging signal sequences or other regions of the retroviral backbone, and also include primers specific for the gene of interest in the vector. Additional advantages in using such a PCR titering assay include the ability to assay for genome rearrangement, etc.
  • the PCR titering assay is performed by growing a known number of HT 1080 cells, typically 1 x 10 5 cells, transduced with a retroviral vector capable of directing expression of the gene of interest on 6-well plates for at least 16 hr. before harvest.
  • the retroviral vectors used for these transductions are preferably obtained from cell culture supematants. One well per plate is reserved for cell counting. Cells from the other wells are lysed and their contents isolated.
  • DNA is prepared using a QIAmp Blood Kit for blood and cell culture PCR (QIAGEN, Inc., Chatsworth, CA). DNAs are resuspended at 5 x 10 6 cell equivalents/mL, where one cell equivalent is equal to the DNA content of one cell .
  • HT1080 cells negative control
  • HT1080 cells transduced with a known vector and having one copy of that vector per cell genome positive control
  • DNA is resuspended at 5 x 10 6 cell equivalents/mL.
  • the standard curve is generated by combining different amounts of the positive and negative control DNA, while keeping the total amount of DNA constant, and amplifying specific sequences therefrom by PCR using primers specific to a particular region of the retroviral vector.
  • a representative group of mixtures for generating a standard curve is:
  • PCR reactions 50 ⁇ L total volume are then initiated by adding 45.0 ⁇ L of a reaction mix containing the following components per tube to be tested: 24.5 ⁇ L water, 5 ⁇ L 10X reaction PCR buffer, 4 ⁇ L of 25 mM MgCl 2 , 4 ⁇ L dNTPs (containing 2.5 mM of each of dATP, dGTP, dCTP, and dTTP), 5 ⁇ L of primer mix (100 ng or each primer), 0.25 ⁇ L Taq Start monoclonal antibody (Clontech Laboratories, Inc., Palo Alto, CA), 1.00 ⁇ L Taq Start buffer (Clontech Labs, Inc.), and 0.25 ⁇ L AmpliTaq DNA polymerase (Perkin-Elmer, Inc.
  • thermocycler Just prior to aliquoting the reaction mix to the reaction tubes, 1 ⁇ L of a- 32 P dCTP (250 ⁇ Ci; 3000 C/mmol, 10 mCi/mL, Amersham Corp., Arlington Heights, JL) is added into the reaction mix. After aliquoting 45.0 ⁇ L the reaction mix into each of the reaction tubes, the tubes are capped and placed into a thermocycler. The particular denaturation, annealing, elongation times and temperatures, and number of thermocycles will vary depending on size and nucleotide composition of the primer pair used. 20 to 25 amplification thermocycles are then performed.
  • the phosphor screen is scanned, whereby light is emitted in proportion to the radioactivity on the original filter
  • the scanning results are then downloaded and plotted on a log scale as cpm (ordinate) versus percent positive control DNA (abscissa).
  • Titers infectious units/mL
  • Titers are calculated by multiplying the number of cells from which DNA was isolated by the percentage (converted to decimal form) determined from the standard curve based on the detected radioactivity, divided by the volume of retroviral vector used to transduce the cells.
  • other methods of detection such as colorimetric methods, may be employed to label the amplified products.
  • the recombinant retroviruses of the invention can be cultivated in a variety of modes, such as in a batch or continuous mode.
  • various cell culture technologies can be employed to produce commercial scale quantities of the recombinant retroviruses according to the invention. Several such techniques are described below, although others known to those in the art may likewise be employed.
  • the hollow fiber bioreactor e.g., HFB; Cellco, Inc., Germantown, MD
  • HFB Cellco, Inc., Germantown, MD
  • the growth media preferably is that to which the cell line has been adapted. All liquids in the HFB when originally shipped should be aspirated and replaced with the complete growth media.
  • the cells When seeding the bioreactor, the cells should not have been split more than 48 hours earlier and should be in log growth phase at the time of harvest for the seeding of the HFB.
  • the cells typically are harvested by trypsinazation and pelleted by centrifugation.
  • the cell pellet is then resuspended in 4 mL of 25% pre-conditioned media and delivered to the extra-capillary space by syringe using the side syringe ports found on the HFB.
  • the cells are allowed to adhere for 20 to 30 minutes before starting the circulation pump During this time, the media used to condition the HFB is replaced with 100-200 mL of 25% preconditioned media.
  • the circulation feed pump is initiated with the starting flow rate set at 25 mL/min. (setting 5 with 2 long pump pins). After 1 hour from the time of switching the pump on, a one mL sample of media is collected in order to record the initial levels of lactate and ammonia.
  • one HFB was seeded with 1.3 x 10 7 cells (representing the low seed culture), the other with 1.6 x 10 8 cells.
  • the cell line 2x- ⁇ -GAL 17-14 was able to initiate a good hollow fiber run under both seeding conditions. Initiating a run with fewer cells is primarily convenient for reducing the effort required for generating the number of cells required to start a culture, although fewer cells initially extends the time it takes to reach optimal cell densities, which usually yield the highest titers.
  • 2x- ⁇ -GAL 17-14 adapted well to hollow fiber culture, eventually requiring daily media changes of 500 mL in order to avoid accumulation of toxic levels of lactate.
  • ⁇ -gal titers for the above experiment were determined from frozen samples on 293 cells assayed 48 or 72 hours after transduction. The transduced cells were stained for ⁇ -gal activity and counted on a hemocytometer to yield a titer based on the number of blue cells /mL (BCT/mL). Optimum titers were generally obtained on day 7 of a high seed cal ture at 1.8 x10 8 BCT/mL from a 72 hour blue cell titer on 293 cells.
  • Previously seed cultures (from tissue culture dishes or flasks) titered using 48 hour blue cell titers on HT1080 cells (calculated to be about 5 x 10 6 BCT/mL) the increase in titer by using hollow fiber systems is approximately ten fold higher. These maximum titers observed were reached prior to hitting 20 mmol/L lactate levels, which appeared to reduce titers produced the following week.
  • Cmde supematants can be harvested every 9 hours with out any loss of titer and three harvests per day should be possible with minimum titre loss.
  • continuous hollow fiber cultures can be maintained for several weeks. When titers were compared between the low and the high seed culture, there was little differences by day 11 between the two seed cultures, both of which averaged 4 x 10 7 BCT/mL.
  • the bottom layer (approximately 20 mL) is carefully eluted, and the interphase (approximately 1 mL) is collected in a 15 mL conical FALCON tube.
  • the interphase containing vector is diluted to 10 mL by addition of PBS, and incubated at 37°C in order to bring the solution to room temperature and destabilize the micelles.
  • KCl is added to a final concentration 0.4 M, and mixed well.
  • the tube is then placed on ice for ten minutes, and spun for 2 minutes at 2,000 rpm in a bench-top centrifuge. The supernatant is removed and filtered through a 0.45 ⁇ m syringe filter.
  • the other half of the interphase containing vector is separated by S-500 Sephadex chromatography in IX PBS. The results of these concentration processes, as determined in a BCFU assay, are shown below in Table 6:
  • the vector- containing solution may be further subjected to concentration utilizing an MY-membrane Amicon filter, thereby reducing the volume from 10 to 14 mL, down to less than 1 mL.
  • concentration utilizing an MY-membrane Amicon filter thereby reducing the volume from 10 to 14 mL, down to less than 1 mL.
  • DX/ND7 ⁇ -gal Clone 87 Utilizing a Cell Factory DX/ND7 bgal clone 87, an expression vector, was grown in cell factories. Cells were grown in DMEM supplemented with Fetal Bovine Serum in roller botttes until enough cells to seed 20 10-layer cell factories (NUNC) at a 1:3 dilution were obtained. Each 10-layer cell factory is seeded with approximately 0.8 liters of cell medium.
  • NUNC 10-layer cell factories
  • Cells were seeded into the cell factory by pouring media containing cells into the factory so that the suspensions evenly fill the 10 layers. The factory is then carefully tilted away from the port side to prevent the suspension from redistribution in the common tube. Finally, the cell factory is rotated into its final upright position. A hepavent filter is attached to each port. The factory was then placed in a CO 2 incubator.
  • Verification of the vector was performed by transduction of HT1080 cells. These cells were harvested 2 days later and stained for b-gal protein. The titer of the supernatant was determined to be 2 x 10 7 /ml.
  • Producer cell lines DA/ ⁇ gal and HX/DN-7 were cultured in a culture flask and a roller bottle, respectively, containing Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum plus lmM L-Glutamine, Sodium pymvate, non- essential amino acids and antibiotics. Viral supernatant was harvested from the flask and roller bottle, and were filtered through a 0.45 urn syringe filter. The filtered supematants were stored either at 4°C (HX/ND7), or frozen at -70°C (DA ⁇ -gal).
  • DMEM Dulbecco's Modified Eagle's Medium
  • Viral supernatant was aliquoted into 50 ml sterile OAKRBDGE screw cap tubes, and placed into an SS34 rotor for use in a Sorvall centrifuge. The tubes were spun for 1 hour at 16,000 rpm (25,000g-force) at 4°C. Upon completion of the spin, the tubes were removed, the supernatant decanted and a small opaque pellet resuspended in the DMEM media described above.
  • Concentrated virus was titered on HT1080 cells plated 24 hours earlier at a cell density of 2x10 5 cells per well in a six well plate + 4 ⁇ g/ml polybrene. Briefly, virus preps were diluted from 1/10 to 1/10,000 and 50 ⁇ l of each dilution was used to infect one well from the six well plate. Plates were incubated overnight at 37°C. Forty-eight hours later, cells were fixed and stained with X-gal. The results are set forth below in Table 7.
  • virus recovery ranged from 30% to 99%, with the best recovery being obtained from human producer cells (HX/ND7; recovery ranged from 91% to 99%).
  • the resultant titer was determined by assaying HT1080 target cells set up at a concentration of 1 x 10 5 cells per well 24 hours prior to transduction of the viral sample. Cells were transduced in the presence of 8 ⁇ g/ml polybrene and 2 mL growth media (DMEM plus 10% FBS) per well. As shown in Table 8 below, approximately one hundred percent of the virus was recovered utilizing this procedure (note that titers are in BCFU/ml).
  • Freezing protocol Producer cells are frozen in DMEM media containing 10% to 20% FBS, and 5 to 15% DMSO, at a concentration of 1 x 10 7 cells/ml/vial. Cells are frozen in a controlled rate freezer (Series PC, Controlled Rate Freezing System, Custom Biogenic Systems, Warren MI) at a rate of from 1 to 10°C per minute. Frozen cells are stored in liquid nitrogen.
  • Cells are thawed from frozen vials at 37°C, washed once with media to remove DMSO, and expanded into 850 cm 2 "FALCON" roller bottles (Corning, Corning, NY) Expanded cell culture is used to inoculate a "CELLIGEN PLUS" bioreactor (5 liter working volume; New Brunswick, Edison, NJ). The cells are grown on microcarriers (i.e., Cytodex 1 or Cytodex 2; Pharmacia, Piscataway, N.J.) at a concentration of 3 to 15 g/L microcarrier. Initial inoculation densities are from 4 to 9 cells/bead at half to full volume for 2 to 24 hours.
  • microcarriers i.e., Cytodex 1 or Cytodex 2; Pharmacia, Piscataway, N.J.
  • the media constituents for virus production are DMEM-high glucose (Irvine Scientific, Santa Ana, CA.) basal media supplemented with FBS (10 to 20%), Glutamine (8 to 15mM), glucose (4.5 to 6.5 g/L), Nonessential amino acids (IX), RPMI 1640 amino acids ( 0.2 to 9.6X), 10 mM HEPES, RPMI 1640 Vitamins (0.2 to 5X).
  • FBS 10 to 20%
  • Glutamine 8 to 15mM
  • glucose 4.5 to 6.5 g/L
  • Nonessential amino acids IX
  • RPMI 1640 amino acids 0.2 to 9.6X
  • 10 mM HEPES RPMI 1640 Vitamins (0.2 to 5X).
  • the bioreactor is monitored for viable cells, titer, glucose, lactate, ammonia levels, and lack of contamination.
  • Viable cells and titer range from 1 x 10 5 cells/ml to 1 x 10 7 cells/ml.
  • Glucose ranges from 6 to 0.25 g/L, Lactate from 1 to 25 mM, and
  • Ammonia ranges from 0.5 to 30 mM. Cells are incubated in the bioreactor for 5 to 25 days.
  • GM-CSF granulocyte macrophage colony stimulating factor
  • Antibodies to CD 14 and CD 15 were obtained as FITC conjugates from Becton-
  • Thy-1 Thy-1
  • Dr. Wolfgang Rettrg Ludwig Institute, New York
  • Antibody to CD34 T ⁇ k 3
  • Dr. Andreas Ziegler Universality of Berlin
  • an anti- ⁇ 3 -Texas Red conjugate Southern Biotechnologies
  • Anti-CD34 antibody or an IgG3 isotype matched control were added to cells in staining buffer (HBSS, 2% FCS, 10 mM HEPES) for 20 minutes on ice, together with anti- Thy-1 antibody at 5 mg/ml.
  • Cells were washed with a FCS underlay, and then incubated with Texas Red conjugated goat anti-mouse IgG3 antibody and phycoerythrin-conjugated goat anti-mouse IgGl antibody for 20 minutes on ice. Blocking IgGl was then added for 10 minutes. After blocking, the FITC-conjugated lineage antibody panel (CD 14 and CDI 5) was added, and incubated for another 20 minutes on ice. After a final washing, cells were resuspended in staining buffer containing propidium iodide (PI).
  • PI propidium iodide
  • Cells were sorted either on the FACSTAR Plus cell sorter equipped with dual argon ion lasers, the primary laser emitting at 488 nm and a dye laser (Rhodamine 6G) emitting at 600 nm (Coherent Innova 90, Santa Cmz, CA) or on a high speed cell sorter as described in PCT patent application number PCT/US93/08205.
  • Residual erythrocytes, debris and dead cells were excluded by light scatter gating plus an FL3 (PI) low gate.
  • PI FL3
  • Methylcellulose assay In order to determine transduction frequency of the stem cells, the following experiment was performed. 5 x 10 3 or 2.5 x 10 3 cells from each transduction were added to 5 ml of methylcellulose (Stem Cell Technologies) containing the following cytokines: c-kit ligand 10 ng/ml; GM-CSF 25 ng/ml; G-CSF 25 ng/ml; lL-3 10 ng/ml; rhEPO 2 units/ml. 1.1 ml of the cell/cytokine methylcellulose mixture was plated onto four 3 cm gridded plates using a 5 ml syringe and 16.5 gauge needle, and the plates were placed in a 37°C incubator for 2 weeks.
  • the lysates were amplified by PCR to determine the presence of the vector in the transduced cells.
  • the PCR assay amplified a 134 bp fragment of the vector psi packaging sequence.
  • a 40 cycle amplification (25 ul total volume) in Perkin-Elmer 9600 Cycler using 10 ul lysate and 0.20 uM each FI and B5 primers (Genset) was performed as follows: one cycle 95°C 30 sec.; 40 cycles 95°C 10 sec., 64°C 15 sec, 72°C 15 sec.; final extension 72°C 5 min. PCR products were visualized on ethidium bromide agarose gels. The results appear in Table 9.
  • CD34+Thy+Lin- (MPB) viable cells were counted and transduced for 72 hours as described in Example 13 herein.
  • Viable cells seeded in 2-fold serial dilutions in a 96- well plate on pre-formed AC6.21 monolayers in Whitlock-Witte media (50/50 RPMI/IMDM, 10% Fetal Calf Serum, Pen-Strep. L-Glutamine, Sodium Pyruvate, and 2-ME) supplemented with LIF at 50 ng/ml and IL-6 at 10 ng/ml.
  • Final cell concentrations, set up in duplicate were 100, 50, 25, 12.5, 6.25, 3.125, 1.56, and 0.78 cells/well respectively.
  • the plates were cultured at 37°C, 5% CO 2 for 5 weeks, with the cells fed weekly by replacement of one-half the spent media with fresh media and cytokines.
  • CAFC cobblestone area forming cells
  • LTCIC plates were prepared by harvesting wells containing multiple cobblestones and filtering the cultures through Nitex filters into 24-well plates. Cutlures were overlaid with approximately 0.6 mis methylcellulose containing 10 ng/ml c-kit ligand, 25 ng/ml GM-CSF, 25 ng/ml G-CSF, 10 ng/ml H-3, and 1.2 U/ml rhEPO, and cultured for 10-14 days at 37°C, 5% CO 2 (i.e., approximately 7 weeks culture from the time of transduction). Individual methylcellulose colonies were picked and analyzed by PCR for the presence of the vector psi packaging sequence as described in Example 14 herein. The results are shown in Table 10 below.
  • the numbers represent the number of bulk LTCIC wells with at least one PCR positive colony/total number of bulk wells analyzed by PCR.
  • the results show that the xenotropic vector did transduce stem cells as measured by LTCIC.
  • the numbers do not represent a transduction frequency of LTCIC since the wells that were harvested contained multiple cobblestone areas, and the resulting methylcellulose colonies could have been derived from different cobblestone area forming cells and hence LTCIC.
  • the clonal LTCIC assay was performed as described above for the bulk LTCIC assay with the exception that individual wells were harvested where, based on limiting dilution, the wells harvested were most likely colonal (>95% probability based on Poisson distribution, i. e. , ⁇ 37% of the wells at a given dilution contained a CAFC).
  • the results are shown in Table 10 below.
  • the numbers represent the number of clonogenic LTCIC wells with at least one positive colony by PCR/total number of clonal wells analyzed by PCR.
  • the results from one experiment show that the xenotropic vector transduced 2 of 9 LTCIC. Since the cells from each well harvested were derived from a single cell, the results ofthe clonal LTCIC assay represents a true LTCIC transduction frequency.

Abstract

L'invention porte sur des compositions, ainsi que sur des techniques, destinées à l'introduction ex vivo d'acide nucléique dans des cellules souches hématopoïétiques à médiation par particules rétrovirales recombinées. Les produits de recombinaison de vecteur recombiné transportés par les particules rétrovirales recombinanées codent pour la production d'un produit génique désiré ayant une application thérapeutique à partir d'un gène présentant un intérêt. Lors de leur réintroduction chez un patient, les cellules souches hématopoïétiques ayant subi une transduction produisent un produit génique désiré en quantité suffisante pour traiter un état pathologique particulier.
PCT/US1996/005432 1995-04-20 1996-04-19 Transduction ex vivo a productivite elevee de cellules souches hematopoietiques au moyen de preparations retrovirales xenotropes recombinees WO1996033281A1 (fr)

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WO2000049141A2 (fr) * 1998-06-08 2000-08-24 Acgt Corporation Procede de production et d'administration d'une proteine in vivo
US7291604B2 (en) 2003-09-03 2007-11-06 The General Hospital Corporation Methods of treating restenosis
US7390652B2 (en) 1997-01-31 2008-06-24 Schering Corporation Device for cultivating cells and propagating viruses
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US7994144B2 (en) * 2001-07-10 2011-08-09 Johnson & Johnson Research Pty, Limited Process for the preparation of a composition of genetically modified hematopoietic progenitor cells
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JP6212039B2 (ja) * 2011-07-27 2017-10-11 ブクタリ 真核細胞の形質導入に有用なウイルスベースのベクター組成物

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WO1997012049A1 (fr) * 1995-09-28 1997-04-03 Medical Research Council Materiels et procedes se rapportant au transfert d'acide nucleique dans des cellules quiescentes
US6723561B2 (en) 1995-09-28 2004-04-20 Mayo Foundation For Medical Education And Research Materials and methods relating to the transfer of nucleic acid into quiescent cells
EP0951533A1 (fr) * 1996-12-31 1999-10-27 Nura, Inc. Vecteurs et procedes de mutagenese de genes mammaliens
US8389795B2 (en) 1996-12-31 2013-03-05 Omeros Corporation Vectors and methods for the mutagenesis of mammalian genes
EP0951533A4 (fr) * 1996-12-31 2004-08-18 George A Gaitanaris Vecteurs et procedes de mutagenese de genes mammaliens
US7390652B2 (en) 1997-01-31 2008-06-24 Schering Corporation Device for cultivating cells and propagating viruses
JP2009297044A (ja) * 1998-05-04 2009-12-24 Canji Inc ウイルス産生プロセス
JP2013165736A (ja) * 1998-05-04 2013-08-29 Canji Inc ウイルス産生プロセス
EP1078095B2 (fr) † 1998-05-04 2016-09-07 CANJI, Inc. Processus de production de vecteurs viraux
WO2000049141A3 (fr) * 1998-06-08 2000-12-14 Acgt Corp Procede de production et d'administration d'une proteine in vivo
WO2000049141A2 (fr) * 1998-06-08 2000-08-24 Acgt Corporation Procede de production et d'administration d'une proteine in vivo
US7776595B2 (en) 2001-07-10 2010-08-17 Johnson & Johnson Research Pty, Limited Methods for genetic modification of hematopoietic progenitor cells and uses of the modified cells
US7994144B2 (en) * 2001-07-10 2011-08-09 Johnson & Johnson Research Pty, Limited Process for the preparation of a composition of genetically modified hematopoietic progenitor cells
US7291604B2 (en) 2003-09-03 2007-11-06 The General Hospital Corporation Methods of treating restenosis
US8133878B1 (en) 2003-09-03 2012-03-13 The General Hospital Corporation Methods for treating restenosis
EP2441770A1 (fr) 2006-02-10 2012-04-18 The University of Cincinnati Protéine 1 d'inhibiteur de phosphatase en tant que régulateur de la fonction cardiaque

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EP0821740A1 (fr) 1998-02-04
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