+

WO1998012314A1 - Vecteurs retroviraux pouvant transduire des cellules ne se divisant pas - Google Patents

Vecteurs retroviraux pouvant transduire des cellules ne se divisant pas Download PDF

Info

Publication number
WO1998012314A1
WO1998012314A1 PCT/US1997/015934 US9715934W WO9812314A1 WO 1998012314 A1 WO1998012314 A1 WO 1998012314A1 US 9715934 W US9715934 W US 9715934W WO 9812314 A1 WO9812314 A1 WO 9812314A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
retrovirus
cell
viral
dividing
Prior art date
Application number
PCT/US1997/015934
Other languages
English (en)
Inventor
Didier P. Trono
Philippe A. Gallay
Original Assignee
The Salk Institute For Biological Studies
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Salk Institute For Biological Studies filed Critical The Salk Institute For Biological Studies
Priority to EP97940952A priority Critical patent/EP0970201A1/fr
Priority to AU42617/97A priority patent/AU4261797A/en
Priority to CA002265522A priority patent/CA2265522A1/fr
Priority to JP10514725A priority patent/JP2001501815A/ja
Publication of WO1998012314A1 publication Critical patent/WO1998012314A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13045—Special targeting system for viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13051—Methods of production or purification of viral material
    • C12N2740/13052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/60—Vectors comprising as targeting moiety peptide derived from defined protein from viruses
    • C12N2810/6045—RNA rev transcr viruses
    • C12N2810/6054—Retroviridae

Definitions

  • the present invention relates generally to the field of viral vectors.
  • the invention relates to novel recombinant retroviruses which are useful for the transfer and expression of nucleic acid sequences in non-dividing cells.
  • Retroviral vectors based on simple retroviruses, such as the Moloney Leukemia Virus (MLV) , are often selected because they efficiently integrate into the genome of the target cell. Integration is thought to be a prerequisite for long-term expression of the transduced gene.
  • MMV Moloney Leukemia Virus
  • retroviral vectors can only integrate in actively dividing cells. This feature severely limits the use of retroviral vectors for in vivo gene transfer. Non-dividing cells are the predominant, long-lived cell type in the body, and account for most desirable targets of gene transfer, including liver, muscle, and brain.
  • retroviruses deliver their nucleoprotein core into the cytoplasm of the target cell.
  • reverse transcription of the viral genome takes place while the core matures into a preintegration complex.
  • the complex must reach the nucleus to achieve integration of the viral DNA into the host cell chromosomes.
  • retroviruses oncoretroviruses
  • this step re-quires the dissolution of the nuclear membrane at itotic prophase, most likely because the bulky size of the preintegration complex prevents its passive diffusion through the nuclear pores.
  • retroviral vectors are useful for many kinds of in vitro gene transfer studies, problems including low titers limit their use for some in vitro and most . in vivo studies. Further, another problem is that integration of retroviral vectors into the host genome was thought to be restricted to cells undergoing DNA replication. Thus, although retroviral vectors capable of infecting a broad class of cell types are known, cell division is necessary for the proviral integration of these vectors. This effectively restricts the efficient use of retrovirus vectors to replicating cells. Thus, retroviruses have not been utilized to introduce genes into non-dividing or post- itotic cells.
  • HIV and other lentiviruses have the ability to infect non-dividing cells, such as terminally differentiated macrophages and quiescent T lymphocytes. This property is likely crucial for HIV transmission, spread and persistence in the body, as well as for AIDS induction.
  • Matrix (MA) and Vpr govern the import of the HIV-1 nucleoprotein complex (NPC) through the nucleopore of the target cells in the absence of the breakdown of the nuclear envelope, allowing integration of the viral genome into the host cell chromosome.
  • MA has intrinsic karyophilic properties conferred by a highly conserved stretch of basic residues which acts as a nuclear localization signal (NLS) .
  • NSC nuclear localization signal
  • Tyrosine-phosphorylated MA then binds to integrase (IN) and becomes a component of the NPC in which its karyophilic potential is revealed by interacting with one component of the cell nuclear import machinery, karyopherin oi .
  • Vpr is another component of the viral NPC that exhibits karyophilic properties.
  • Vpr does not contain a canonical NLS. Instead, the domain critical for nuclear localization of Vpr has recently been mapped to the two putative N-terminal ⁇ -helices thereof. Vpr could therefore play a crucial role in HIV-1 nuclear import through a pathway independent from MA; an NLS-independent pathway .
  • NLS peptide affects MA, but not Vpr, nuclear localization, and interferes with MA, but not Vpr-mediated HIV-1 nuclear import (see Gallay et al . in J. Virology 20:1027-1032 (1996)).
  • the availability of these two distinct yet convergent pathways most probably ensures the ability of HIV-1 to infect non-dividing cells under a variety of conditions.
  • HIV-1-based vector containing the same mutations displayed lower in vivo levels of transduction of heterologous sequences in nonmitotic cells, such as neurons, compared with the wild-type vector, confirming the key roles of MA and Vpr in HIV-1 infection of non-dividing cells .
  • a retroviral vector system was recently derived from HIV (Naldini et al . , Science 272 -263-267 (1996)).
  • the HIV vector could transduce heterologous sequences into a variety of growth-arrested cells in vitro, as well as into human primary macrophages.
  • HIV vector could mediate stable in vivo gene transfer into terminally differentiated neurons.
  • the ability of HIV-based viral vectors to deliver genes in vivo into non-dividing cells could increase the applicability of retroviral vectors in gene therapy.
  • a major limitation of these vectors is in their potential biohazard, as HIV is a major pathogen in humans .
  • retroviral vectors derived from viruses which are not known to be pathogenic in humans e.g., MLV
  • retroviruses can be rendered capable of infecting non-dividing cells by introducing into the viral particle one of several specifically defined modifications.
  • an element which is recognized by the nuclear import machinery of a non-dividing cell and which can associate with the nucleoprotein complex of the retrovirus can be introduced into a retrovirus .
  • integrase the enzyme responsible for inserting the viral DNA into the host cell chromosomes
  • IN the enzyme responsible for inserting the viral DNA into the host cell chromosomes
  • NPC HIV-1 nucleoprotein complex
  • IN facilitates the migration of the viral genome to the nucleopore by interacting with one component of the cell nuclear import machinery, karyopherin a (presumably through the IN NLS(s)).
  • IN has been identified as a preferred "element" for use in the practice of the present invention.
  • At least one protein encoded by viral gag or pol nucleic acid is modified so as to be recognized by the nuclear import machinery of a non-dividing cell.
  • Figure 1 illustrates the ability of HIV-1 integrase to enhance the transduction of non-dividing cells by an MLV vector .
  • retroviruses capable of infecting non-dividing cell(s).
  • invention retroviruses comprise the following components: a viral GAG, a viral POL, a viral ENV, an element which associates with the nucleoprotein complex of said retrovirus, wherein said element is recognized by the nuclear import machinery of said non- dividing cell, an heterologous nucleic acid operably linked to a regulatory sequence, and cis-acting nucleic acid necessary for reverse transcription and integration, and optionally, packaging of said retrovirus.
  • viral particles produced by the above-described recombinant retroviruses.
  • inventions comprise transfecting a suitable packaging host cell with one or more vectors comprising: a nucleic acid encoding a viral gag, a nucleic acid encoding a viral pol, a nucleic acid encoding a viral env, a nucleic acid encoding an element which associates with the nucleoprotein complex of said retrovirus, wherein said element is recognized by the nuclear import machinery of said non- dividing cell, and a nucleic acid encoding a packaging signal flanked by cis-acting nucleic acids necessary for reverse transcription and integration, and a cloning site for introduction of a heterologous nucleic acid, operably linked to a regulatory nucleic acid.
  • invention method further comprises recovering the recombinant virus produced by the above- described transfected host cell.
  • Retroviruses are RNA viruses wherein the viral genome is RNA.
  • the genomic RNA is reverse transcribed into a DNA intermediate which is integrated very efficiently into the chromosomal DNA of infected cells.
  • This integrated DN A intermediate is referred to as a provirus.
  • Transcription of the provirus and assembly into infectious virus occurs in a cell line containing appropriate sequences enabling encapsidation, or, if necessary, in the presence of an appropriate helper virus.
  • a helper virus is not required for the production of the recombinant retrovirus of the present invention, since the sequences required for encapsidation are provided by co-transfection with appropriate vectors.
  • the retroviral genome and the proviral DNA have three genes: the gag, the pol, and the env, which are flanked by two long terminal repeat (LTR) sequences.
  • the gag gene encodes the internal structural (matrix, capsid, and nucleocapsid) proteins; the pol gene encodes integrase and the RNA-directed DNA polymerase (reverse transcriptase) and the env gene encodes viral envelope glycoproteins .
  • the 5' and 3' LTRs serve to promote transcription and p- olyadenylation of the virion RNAs .
  • the LTRs contain all other cis-acting sequences necessary for viral replication.
  • Lentiviruses have additional genes including vif, vpr, tat, rev, vpu, ne , and vpx (in HIV-1, HIV-2 and/or SIV) .
  • Adjacent to the 5' LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient encapsidation of viral RNA into particles (the Psi site) . If the sequences necessary for encapsidation (or packaging of retrovirual RNA into infectious virions) are missing from the viral genome, the result is a cis defect which prevents encapsidation of genomic RNA. However, the resulting mutant is still capable of directing the synthesis of all virion proteins.
  • Invention retroviruses can be produced by a variety of methods, e.g., by transfecting a suitable host cell with one or more vectors comprising: a nucleic acid encoding a viral gag; a nucleic acid encoding a viral pol; a nucleic acid encoding a viral env; a nucleic acid encoding an element which associates with the nucleoprotein complex of said retrovirus, wherein said element is recognized by the nuclear import machinery of said non-dividing cell, and a nucleic acid sequence encoding a packaging signal flanked by cis-acting nucleic acid sequences for reverse transcription and integration, and providing a cloning site for introduction of a heterologous gene, operably linked to a regulatory nucleic acid sequence.
  • a presently preferred method for the production of retroviruses according to the invention involves the use of a combination of a minimum of four vectors in order to produce a recombinant virion or recombinant retrovirus .
  • a first vector provides a nucleic acid encoding a viral gag and a viral pol. These sequences encode a group specific antigen, reverse transcriptase, integrase and protease- enzymes necessary for reverse transcription, integration and maturation.
  • Such sequences can be obtained from a variety of viral sources, e.g., Moloney murine leukemia virus (MoMuLV) , Harvey murine sarcoma virus (HaMuSV) , murine mammary tumor virus (MuMTV) , gibbon ape leukemia virus (GaLV) , human immunodeficiency virus (HIV) , Rous Sarcoma Virus (RSV) , and the like.
  • MoMuLV Moloney murine leukemia virus
  • HaMuSV Harvey murine sarcoma virus
  • MoMTV murine mammary tumor virus
  • GaLV gibbon ape leukemia virus
  • HAV human immunodeficiency virus
  • RSV Rous Sarcoma Virus
  • a second vector employed in the practice of the present invention provides a nucleic acid encoding a viral envelope (env) .
  • the env gene can be derived from any virus, including retroviruses.
  • the env may be amphotropic envelope protein which allows transduction of cells of human and other species, or may be ecotropic envelope protein, which is able to transduce only mouse and rat cells.
  • it may be desirable to target the recombinant virus by linkage of the envelope protein with an antibody or a particular ligand for targeting to a receptor of a particular cell-type.
  • a sequence (including regulatory region) of interest into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is now target specific.
  • Retroviral vectors can be made target specific by inserting, for example, a glycolipid, or a protein. Targeting is often accomplished by using an antibody to target the retroviral vector. Those of skill in the art will know of, or can readily ascertain without undue experimentation, specific methods to achieve delivery of a retroviral vector to a specific target.
  • retroviral-derived env genes examples include Moloney murine leukemia virus (MoMuLV) , Harvey murine sarcoma virus (HaMuSV) , murine mammary tumor virus (MuMTV) , gibbon ape leukemia virus (GaLV) , human immunodeficiency virus (HIV), Rous Sarcoma Virus (RSV) , and the like.
  • MoMuLV Moloney murine leukemia virus
  • HaMuSV Harvey murine sarcoma virus
  • MuMTV murine mammary tumor virus
  • GaLV gibbon ape leukemia virus
  • HV human immunodeficiency virus
  • RSV Rous Sarcoma Virus
  • Other env genes such as Vesicular stomatitis virus (VSV) (Protein G) can also be used.
  • VSV Vesicular stomatitis virus
  • the vector providing the viral env nucleic acid sequence is operably associated with regulatory sequence, e.g. , a promoter or enhancer.
  • the regulatory sequence is a viral promoter .
  • the regulatory sequence can be any eukaryotic promoter or enhancer, including for example, the Moloney murine leukemia virus promoter- enhancer element, the human cytomegalovirus enhancer (as used in the illustrative example), or the vaccinia P7.5 promoter. In some cases, such as the Moloney murine leukemia virus promoter-enhancer element, these promoter- enhancer elements are located within or adjacent to the LTR sequences.
  • a third vector contemplated for use in the practice of the present invention provides the cis-acting viral sequences necessary for the viral life cycle.
  • Such sequences include the ⁇ packaging sequence, reverse transcription signals, integration signals, viral promoter, enhancer, and polyadenylation sequences.
  • Such sequences can be obtained from a variety of viral sources, e.g., MoMuLV, HaMuSV, MuMTV, GaLV, HIV, RSV, and the like.
  • the third vector also contains a cloning site for introduction of a heterologous nucleic acid sequence.
  • the cloning site (either containing the heterologous nucleic acid sequence therein, or absent any insert therein) can then be transferred to a non-dividing cell.
  • the heterologous nucleic acid sequence is typically incorporated into this vector prior to incorporation of products encoded thereby into a viral particle.
  • this third vector can be used for the preparation of a viral particle containing the heterologous nucleic acid sequence, thereby facilitating the direct introduction of the heterologous nucleic acid sequence into a non-dividing cell.
  • a fourth vector contemplated for use in the practice of the present invention provides the element which associates with the nucleoprotein complex of said retrovirus.
  • HIV-1 integrase is expressed under the control of the HIV-1 promoter .
  • recombinant retroviruses produced by standard methods in the art are defective, they require assistance in order to produce infectious vector particles.
  • this assistance is provided, for example, by using a helper cell line that provides the missing viral functions.
  • helper cell lines which have deletions of the packaging signal include ⁇ , PA317, PA12 , and the like. Suitable cell lines produce empty virions, since no genome is packaged. If a retroviral vector is introduced into such cells in which the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virion produced.
  • the invention method for production of recombinant retrovirus employs different constructs than are employed in the standard helper virus/packaging cell line method described above.
  • the four or more individual vectors used to co-transfect a suitable packaging cell line collectively contain all of the required genes for production of a recombinant virus for infection and transfer of nucleic acid to a non-dividing cell. Consequently, there is no need for use of a helper virus.
  • packaging cell lines can be prepared in accordance with the present invention.
  • a stable packaging cell line containing several of the above-described vectors can be prepared, such that one only need introduce a vector containing the heterologous nucleic acid sequence in order to produce a virion which is capable of infecting non-dividing cells and hence delivering heterologous nucleic acid sequences thereto.
  • stable packaging cell lines containing:
  • nucleic acid encoding a viral gag a nucleic acid encoding a viral pol, a nucleic acid encoding a viral env, and a nucleic acid encoding an element which associates with the nucleoprotein complex of a retrovirus, wherein said element is recognized by the nuclear import machinery of a non-dividing cell.
  • the above-described cell lines are especially useful for the rapid introduction of heterologous nucleic acid sequences into a host.
  • a vector encoding a nucleic acid encoding a packaging signal flanked by cis-acting nucleic acids necessary for reverse transcription and integration, and a heterologous nucleic acid operably linked to a regulatory nucleic acid is introduced into the above-described stable packaging cell line, which is then induced to produce viral particles.
  • the resulting viral particles are useful for the introduction of heterologous nucleic acid sequences into non-dividing cells.
  • stable packaging cell lines containing:
  • a first nucleic acid encoding a viral gag a second nucleic acid encoding a viral pol, wherein at least one protein encoded by said first or second nucleic acid is modified so as to be recognized by the nuclear import machinery of a non-dividing cell, and a nucleic acid encoding a viral env.
  • Elements which associate with the nucleoprotein complex of a retrovirus (either directly or indirectly) , and which are recognized by the nuclear import machinery of a non-dividing cell, as contemplated for use in the practice of the present invention include viral proteins which are directly recognized by the nuclear import machinery of a non-dividing cell, such as, for example, matrix protein (MA) , integrase (IN) , and the like, as well as viral proteins which are indirectly recognized by the nuclear import machinery of a non-dividing cell (by associating with the nucleoprotein complex and an agent which is recognized by the nuclear import machinery of a non-dividing cell) , such as, for example, reverse transcriptase (RT) , nucleocapsid, protease, and the like.
  • a presently preferred viral protein contemplated for use herein is a lentiviral integrase, with HIV
  • NLS nuclear localization signals
  • Those of skill in the art can readily identify NLSs suitable for use herein. See, for example, the numerous NLS sequences described by Dingwall and Laskey in TIBS 16:478-481 (1991) and Goerlich and Mattaj in Science 271:1513-1518 (1996) .
  • a suitable NLS can be derived from HIV-1 integrase.
  • Another protein that exhibits karyophilic properties, and hence is useful herein, is Vpr.
  • consensus NLSs have also been identified in the art, characterized as comprising a contiguous se-quence of seventeen amino acids, wherein the first two amino acids are basic amino acids, followed by a spacer region of any ten amino acids, followed by a basic cluster in which at least three of the next five amino acids are basic.
  • numerous specific NLSs have been identified in the art, e.g.:
  • amino acid sequence KRKQ (SEQ ID NO:l), the amino acid sequence KELQKQ (SEQ ID NO : 2 ) , the amino acid sequence KRKGGIG (SEQ ID NO:
  • yeast GAL4 targeting signal the yeast GAL4 targeting signal, and the like.
  • Heterologous nucleic acid sequence (s) employed in the practice of the present invention are operably linked to a regulatory nucleic acid sequence.
  • the term “heterologous” nucleic acid sequence refers to a sequence that originates from a foreign species, or, if from the same species, it may be substantially modified from its original form. Alternatively, an unchanged nucleic acid sequence that is not normally expressed in a cell is a heterologous nucleic acid sequence.
  • the term “operably linked” refers to a functional linkage between the regulatory sequence and the heterologous nucleic acid sequence.
  • the heterologous sequence is linked to a promoter, resulting in a chimeric gene.
  • the heterologous nucleic acid sequence is preferably under control of either the viral LTR promoter- enhancer signals or of an internal promoter. Retained signals within the retrovirual LTR can still bring about efficient integration of the vector into the host cell genome .
  • the promoter sequence may be homologous or heterologous to the desired gene sequence.
  • a wide range of promoters are suitable for use in the practice of the present invention, including viral or mammalian promoters. Cell or tissue specific promoters can be utilized to target expression of gene sequences in specific cell populations. Mammalian and viral promoters suitable for use in the practice of the present invention are well knwn and readily available in the art .
  • the nucleic acid construct referred to as the transfer vector having the packaging signal and the heterologous cloning site, will also contain a selectable marker gene.
  • Marker genes are utilized to assay for the presence of the vector, and thus, to confirm infection and integration. The presence of this marker gene ensures the growth of only those host cells which express the inserted DNA.
  • Typical selection genes encode proteins that confer resistance to antibiotics and other toxic substances, e.g., histidinol, puromycin, hygromycin, neomycin, methotrexate, and the like.
  • Other marker systems commmonly used in the art include ⁇ -galactosidase (LacZ) and luciferase reporter or marker systems, which can conveniently be monitored visually.
  • nucleic acid sequence refers to any nucleic acid molecule, preferably DNA.
  • the nucleic acid molecule may be derived from a variety of sources, including DNA, cDNA, synthetic DNA, RNA, or combinations thereof .
  • Such nucleic acid sequences may comprise genomic DNA which may or may not include naturally occurring introns .
  • genomic DNA may be obtained in association with promoter regions, introns, or poly A sequences.
  • Genomic DNA may be extracted and purified from suitable cells by means well known in the art .
  • messenger RNA (mRNA) can be isolated from cells and used to produce cDNA by reverse transcription or other means .
  • non-dividing cell refers to a cell that does not go through mitosis. Non-dividing cells may be blocked at any point in the cell cycle, (e.g. , G 0 /G 1# G.,/S, G 2 /M) , as long as the cell is not actively dividing.
  • a dividing cell can be treated to block cell division by standard techniques used by those of skill in the art, such as, for example, irradiation, aphidocolin treatment, serum starvation, contact inhibition, and the like.
  • ex vivo infection is often performed without blocking the cells since many cells are already arrested (e.g., stem cells) .
  • Recombinant retrovirus vectors according to the present invention are capable of infecting any non-dividing cell, regardless of the mechanism used to block cell division or the point in the cell cycle at which the cell is blocked.
  • Examples of pre-existing non-dividing cells in the body include neurons, myocytes, hepatocytes, hematopoietic stem cells, lymphocytes, dendritic cells, epithelial cells, macrophages, and the like, as well as derivatives thereof.
  • Recombinant retrovirus vectors according to the present invention can be used for both in vivo gene delivery (for example, by injection) , as well as ex vivo gene delivery .
  • the method of the present invention contemplates the use of at least three vectors which provide all of the functions re-quired for packaging of recombinant virions as discussed above, plus a fourth vector which provides the element which is capable of associating with the nucleoprotien complex, thereby facilitating infection of non-dividing cells.
  • Invention method also contemplates transfection of vectors including viral genes such as vpr, vif, nef, vpx, tat, rev, and vpu. Some or all of these genes can be included, for example, on the packaging construct vector, or, alternatively, they may reside on individual vectors.
  • vectors which can be utilized there is no limitation to the number of vectors which can be utilized, as long as they are co- transfected to the packaging cell line in order to produce a single recombinant retrovirus.
  • the env nucleic acid sequence on the same construct as the gag and pol .
  • Viral vectors contemplated for use herein are introduced via transfection or infection into a packaging cell line.
  • the packaging cell line produces viral particles that contain the vector genome. Methods for transfection or infection are well known by those of skill in the art. After co-transfection of the at least four vectors to the packaging cell line, the recombinant virus is recovered from the culture media and titered by standard methods used by those of skill in the art .
  • retroviruses which are capable of infecting non- ividing cell(s) .
  • This class of invention retroviruses comprises the following components: a viral GAG, a viral POL, a viral ENV, an heterologous nucleic acid operably linked to a regulatory sequence, and cis-acting nucleic acid necessary for reverse transcription and integration, wherein a protein associated with the nucleoprotein complex of said retrovirus is modified so as to be recognized by the nuclear import machinery of said non- dividing cell.
  • viral particles produced by the above-described recombinant retroviruses.
  • invention infection method comprises transfecting a suitable packaging host cell with one or more vectors comprising: a first nucleic acid encoding a viral gag, a second nucleic acid encoding a viral pol, wherein at least one protein encoded by said first or second nucleic acid is modified so as to be recognized by the nuclear import machinery of said non-dividing cell, a nucleic acid encoding a viral env, and a nucleic acid encoding a packaging signal flanked by cis-acting nucleic acids necessary for reverse transcription and integration, and a cloning site for introduction of a heterologous nucleic acid, operably linked to a regulatory nucleic acid.
  • invention method further comprises recovering the recombinant virus produced by the above- described transfected host cell.
  • proteins associated with the nucleoprotein complex of said retrovirus are contemplated for use herein, such as, for example, by mutation of the protein associated with the nucleoprotein complex of said retrovirus so as to be recognized by the nuclear import machinery of said non-dividing cell.
  • the protein associated with the nucleoprotein complex of the retrovirus can be modified by the addition of a karyophilic agent thereto.
  • Examplary karyophilic agents contemplated for use herein include reverse transcriptase, matrix protein, nucleocapsid, protease, integrase, and the like.
  • retrovirus according to the invention can be prepared employing protein associated with the nucleoprotein complex thereof which has been modified by the addition thereto of a nuclear localization signal, as described hereinabove.
  • inventions for the introduction and expression of heterologous nucleic acids in non-dividing cell(s).
  • invention methods comprise infecting non-dividing cell(s) with any of the recombinant viruses described herein, and expressing the heterologous nucleic acid in said non-dividing cell.
  • a gene regulating molecule in a cell by the introduction of a molecule by the method of the invention.
  • modulate envisions the suppression of expression of a gene when it is over-expressed, or augmentation of expression when it is under-expressed.
  • nucleic acid sequences that interfere with the gene's expression at the translational level can be used. This approach utilizes, for example, antisense nucleic acid, ribozymes, or triplex agents to block transcription or translation of a specific mRNA, either by masking that mRNA with an antisense nucleic acid or triplex agent, or by cleaving it with a ribozyme.
  • Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, Scientific American. 262 :40. 1990) . In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule. The antisense nucleic acids interfere with the translation of the mRNA, since the cell will not translate a mRNA that is double-stranded. Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced into the target cell. The use of antisense methods to inhibit the in vitro translation of genes is well known in the art (Marcus- Sakura, Anal .Biochem.. 272:289, 1988).
  • the antisense nucleic acid can be used to block expression of a mutant protein or a dominantly active gene product, such as amyloid precursor protein that accumulates in Alzheimer's disease. Such methods are also useful for the treatment of Huntington' s disease, hereditary Parkinsonism, and other diseases. Antisense nucleic acids are also useful for the inhibition of expression of proteins associated with toxicity.
  • triplex strategy Use of an oligonucleotide to stall transcription is known as the triplex strategy since the oligomer winds around double-helical DNA, forming a three-strand helix. Therefore, these triplex compounds can be designed to recognize a unique site on a chosen gene (Maher, et al .. Antisense Res, and Dev., 1(3) :227, 1991; Helene, C, Anticancer Drug Design, 6 (6) :569, 1991) .
  • Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases . Through the modification of nucleotide sequences which encode these RNAs, it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, J. Amer. Med. Assn., 260:3030, 1988) . A major advantage of this approach is that, because they are sequence-specific, only mRNAs with particular sequences are inactivated.
  • nucleic acid encoding a biological response modifier.
  • immunopotentiating agents including nucleic acids encoding a number of the cytokines classified as "interleukins” . These include, for example, interleukins 1 through 12.
  • interferons include gamma interferon ( ⁇ -IFN), tumor necrosis factor (TNF) and granulocyte-macrophage- colony stimulating factor (GM-CSF) . It may be desirable to deliver such nucleic acids to bone marrow cells or macrophages to treat enzymatic deficiencies or immune defects.
  • Nucleic acids encoding growth factors, toxic peptides, ligands, receptors, or other physiologically important proteins can also be introduced into specific non-dividing cells.
  • the recombinant retrovirus of the invention can be used to treat an HIV infected cell (e.g. , T-cell or macrophage) with an anti-HIV molecule.
  • respiratory epithelium for example, can be infected with a recombinant retrovirus of the invention having a gene for cystic fibrosis transmembrane conductance regulator (CFTR) for treatment of cystic fibrosis.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • the method of the invention may also be useful for neuronal or glial cell transplantation, or "grafting", which involves transplantation of cells infected with the recombinant retrovirus of the invention ex vivo, or infection in vivo into the central nervous system or into the ventricular cavities or subdurally onto the surface of a host brain.
  • grafting Such methods for grafting will be known to those skilled in the art and are described in Neural Grafting in the Mammalian CNS, Bjorklund and Stenevi, eds . , (1985) , incorporated by reference herein. Procedures include intraparenchymal transplantation, (i.e. , within the host brain) achieved by injection or deposition of tissue within the host brain so as to be apposed to the brain parenchyma at the time of transplantation.
  • Administration of the cells or virus into selected regions of the recipient subject's brain may be made by drilling a hole and piercing the dura to permit the needle of a microsyringe to be inserted.
  • the cells or recombinant retrovirus can alternatively be injected intrathecally into the spinal cord region.
  • a cell preparation infected ex vivo, or the recombinant retrovirus of the invention permits grafting of neuronal cells to any predetermined site in the brain or spinal cord, and allows multiple grafting simultaneously in several different sites using the same cell suspension or viral suspension and permits mixtures of cells from different anatomical regions .
  • Cells infected with a recombinant retrovirus of the invention, in vivo, or ex vivo, used for treatment of a neuronal disorder for example, may optionally contain an exogenous gene, for example, a gene which encodes a receptor or a gene which encodes a ligand.
  • a gene which encodes a receptor or a gene which encodes a ligand include receptors which respond to dopa ine, GABA, adrenaline, noradrenaline, serotonin, glutamate, acetylcholine and other neuropeptides, as described above.
  • Examples of ligands which may provide a therapeutic effect in a neuronal disorder include dopamine, adrenaline, noradrenaline, acetylcholine, gamma-aminobutyric acid and serotonin.
  • a cell genetically modified to secrete a neurotrophic factor such as nerve growth factor (NGF)
  • NGF nerve growth factor
  • cells be grafted into a subject with a disorder of the basal ganglia such as Parkinson's disease
  • Parkinson's disease can be modified to contain an exogenous gene encoding L-DOPA, the precursor to dopamine. Parkinson's disease is characterized by a loss of dopamine neurons in the substantia-nigra of the midbrain, which have the basal ganglia as their major target organ.
  • Alzheimer's disease is characterized by degeneration of the cholinergic neurons of the basal forebrain.
  • the neurotransmitter for these neurons is acetylcholine, which is necessary for their survival.
  • Engraftment of cholinergic cells infected with a recombinant retrovirus of the invention containing an exogenous gene for a factor which would promote survival of these neurons can be accomplished by the method of the invention. Following a stroke, there is selective loss of cells in the CA1 of the hippocampus as well as cortical cell loss which may underlie cognitive function and memory loss in these patients.
  • molecules responsible for CA1 cell death can be inhibited by the methods of this invention.
  • antisense sequences, or a gene encoding an antagonist can be transferred to a neuronal cell and implanted into the hippocampal region of the brain.
  • the method of transferring nucleic acid also contemplates the grafting of neuroblasts in combination with other therapeutic procedures useful in the treatment of disorders of the CNS.
  • the retroviral infected cells can be co-administered with agents such as growth factors, gangl ios ides , antibiotics, neurotransmitters, neurohormones, toxins, neurite promoting molecules and antimetabolites and precursors of these molecules such as the precursor of dopamine, L-DOPA.
  • lysosomal storage diseases such as those involving ⁇ -hexosaminidase or glucocerebrosidase,- deficiencies in hypoxanthine phosphoribosyl transferase activity (the "Lesch-Nyhan” syndrome) ; amyloid polyneuropathies (prealbumin) ; Duchenne's muscular dystrophy, and retinoblastoma, for example.
  • gene transfer could introduce a normal gene into the affected tissues for replacement therapy, as well as to create animal models for the disease using antisense mutations.
  • a Factor IX encoding nucleic acid into a retrovirus for infection of a muscle or liver cell.
  • Integrase contains multiple putative nuclear localization signals (NLSs) .
  • NPC HIV-1 nucleoprotein complex
  • ⁇ IN HIV-1 mutant lacking IN
  • RT reverse transcriptase
  • cytoplasmic and nuclear extracts were analyzed for the presence of nucleocapsid (NC) and RT, other components of the NPC.
  • NC or RT were predominantly associated with the cytoplasmic compartment at 1 hour, whereas they were found in the nucleus at 8 hours (about 50% of molecules) .
  • Example 2 IN is a karyophilic protein
  • IN contains karyophilic properties
  • cells were transfected with a construct expressing IN alone. Nuclear and cytoplasmic extracts were then analyzed for their IN content. IN was found essentially associated with the nucleus of transfected cells.
  • fluorescein-isothiocyanate (FITC) - labeled IN microinjected in the cytoplasm of cells was also detected predominantly in the nucleus of these cells, whereas FITC-BSA used as control remained associated with the cytoplasm.
  • FITC fluorescein-isothiocyanate
  • Example 4 directly binds karyopherin ⁇ in an
  • the C-terminal domain of IN contains two NLSs recognized by karyopherin
  • NLS were noted; NLS.,, located around positions 186-188
  • KRKQ KRKQ
  • NLS 2 NLS 2 , around positions 211-216 (KELQKQ) .
  • NLSs were mutated and the resulting GST- ⁇ NLS IN proteins were tested for their affinity for Rchl.l. Mutations introduced in NLS 1 completely abrogated the IN-Rchl complex formation, whereas mutations in NLS 2 decreased (about 50%) the interaction between the two moles .
  • NLS 1 IN (consensus: KRKGGIG) is highly conserved among lentiviruses (HIV-1, HIV-2, SIV, BIV, FIV, Visna, CAEV, EIAV) , but is absent in non-lentiviruses (MLV, RSV, HTLV) , suggesting an important role for this NLS motif in lentiviral infection.
  • NLS-substrate such as IN or MA
  • Example 7 governs HIV-1 nuclear import in ⁇ - irradiated CD4+ HeLa cells through its NLSs
  • ⁇ -irradiated CD4+ HeLa cells were acutely infected with a vpr-defective strain mutated in both MA NLS and IN NLS 1 ( ⁇ vpr MA ⁇ NLS IN ⁇ NLS1) .
  • cytoplasmic and nuclear extracts were analyzed for the presence of components of HIV-1 NPC; NC and RT.
  • an MLV-based vector (encoding the marker, beta- galactosidase) , pseudotyped with the VSV G envelope, an HIV-based vector (encoding the marker, beta- galactosidase) , pseudotyped with the VSV G envelope, or a "chimeric" vector according to the invention, wherein the above-described MLV-based vector is supplemented with an additional vector encoding HIV-1 integrase.
  • non-arrested (i.e., dividing) HeLa cells were infected with each of the above-described vectors.

Landscapes

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

Abstract

Selon la présente invention, on a mis au point des procédés destinés à modifier des vecteurs rétroviraux dérivés de virus qui ne sont pas connus comme étant pathogènes chez l'homme (par exemple le virus de la leucémie murine de Moloney), afin que ces vecteurs puissent transduire des séquences hétérologues dans des cellules ne se divisant pas. Ainsi, on a découvert que l'on pouvait rendre des rétrovirus capables d'infecter des cellules ne se divisant pas, en introduisant dans la particule virale l'une de ces modifications déterminées bien précisément. Par exemple, on peut associer un élément reconnu par le mécanisme d'importation nucléaire d'une cellule ne se divisant pas, au complexe de nucléoprotéines du rétrovirus. Dans un autre mode de réalisation, on modifie au moins une protéine codée par l'acide nucléique viral gag ou pol, de façon que cette protéine soit reconnue par le mécanisme d'importation nucléaire d'une cellule ne se divisant pas.
PCT/US1997/015934 1996-09-17 1997-09-08 Vecteurs retroviraux pouvant transduire des cellules ne se divisant pas WO1998012314A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP97940952A EP0970201A1 (fr) 1996-09-17 1997-09-08 Vecteurs retroviraux pouvant transduire des cellules ne se divisant pas
AU42617/97A AU4261797A (en) 1996-09-17 1997-09-08 Retroviral vectors capable of transducing non-dividing cells
CA002265522A CA2265522A1 (fr) 1996-09-17 1997-09-08 Vecteurs retroviraux pouvant transduire des cellules ne se divisant pas
JP10514725A JP2001501815A (ja) 1996-09-17 1997-09-08 非分裂細胞への形質導入が可能なレトロウイルスベクター

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US71531896A 1996-09-17 1996-09-17
US08/715,318 1996-09-17

Publications (1)

Publication Number Publication Date
WO1998012314A1 true WO1998012314A1 (fr) 1998-03-26

Family

ID=24873552

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1997/015934 WO1998012314A1 (fr) 1996-09-17 1997-09-08 Vecteurs retroviraux pouvant transduire des cellules ne se divisant pas

Country Status (5)

Country Link
EP (1) EP0970201A1 (fr)
JP (1) JP2001501815A (fr)
AU (1) AU4261797A (fr)
CA (1) CA2265522A1 (fr)
WO (1) WO1998012314A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999058701A1 (fr) * 1998-05-13 1999-11-18 Genetix Pharmaceuticals, Inc. Nouvelles cellules lentivirales d'encapsidation
EP1103615A1 (fr) * 1999-11-25 2001-05-30 Universite De Geneve Vecteurs capables d'immortaliser les cellules ne se divisant pas et les cellules immortalisées par ces vecteurs
WO2001091801A2 (fr) * 2000-05-26 2001-12-06 Chiron Corporation Procedes de transduction de cellules neuronales cerebelleuses par le biais de vecteurs de lentivirus
EP1171624A1 (fr) * 1999-04-29 2002-01-16 Cell Genesys, Inc. Procede et moyens de production de vecteurs lentiviraux recombinants surs et a titre eleve
US6808905B2 (en) 2001-05-14 2004-10-26 Cell Genesys, Inc. Lentiviral vectors encoding clotting factors for gene therapy
US6924144B2 (en) 1997-12-12 2005-08-02 Cell Genesys, Inc. Method and means for producing high titer, safe, recombinant lentivirus vectors

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NATURE, 14 October 1993, Vol. 365, BUKRINSKY et al., "A Nuclear Localization Signal within HIV-1 Matrix Protein that Governs Infection of Non-Dividing Cells", pages 666-669. *
SCIENCE, 12 April 1996, Vol. 272, NALDINI et al., "In Vivo Gene Delivery and Stable Transduction of Nondividing Cells by a Lentiviral Vector", pages 263-267. *

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6924144B2 (en) 1997-12-12 2005-08-02 Cell Genesys, Inc. Method and means for producing high titer, safe, recombinant lentivirus vectors
US8846385B2 (en) 1997-12-12 2014-09-30 Gbp Ip, Llc Method and means for producing high titer, safe recombinant lentivirus vectors
US7083981B2 (en) 1997-12-12 2006-08-01 Cell Genesys, Inc. Method and means for producing high titer, safe, recombinant lentivirus vectors
US7311907B2 (en) 1998-05-13 2007-12-25 Genetix Pharmaceuticals, Inc. Lentiviral packaging cells
WO1999058701A1 (fr) * 1998-05-13 1999-11-18 Genetix Pharmaceuticals, Inc. Nouvelles cellules lentivirales d'encapsidation
US8034620B2 (en) 1998-05-13 2011-10-11 Bluebird Bio, Inc. Lentiviral packaging cells and uses therefor
US7250299B1 (en) 1999-04-29 2007-07-31 Cell Genesys, Inc. Method and means for producing high titer, safe, recombinant lentivirus vectors
EP1171624A1 (fr) * 1999-04-29 2002-01-16 Cell Genesys, Inc. Procede et moyens de production de vecteurs lentiviraux recombinants surs et a titre eleve
EP1171624A4 (fr) * 1999-04-29 2002-06-26 Cell Genesys Inc Procede et moyens de production de vecteurs lentiviraux recombinants surs et a titre eleve
US8652837B2 (en) 1999-04-29 2014-02-18 Gbp Ip, Llc Method and means for producing high titer, safe, recombinant lentivirus vectors
EP1849873A1 (fr) * 1999-04-29 2007-10-31 Cell Genesys, Inc. Procédé et supports de production de vecteurs de lentivirus recombinant sécurisé à fort dosage
WO2001038548A3 (fr) * 1999-11-25 2001-10-18 Univ Geneve Vecteurs capables d'immortaliser des cellules ne se divisant pas, et cellules immortalisees au moyen de ces vecteurs
WO2001038548A2 (fr) * 1999-11-25 2001-05-31 Universite De Geneve Vecteurs capables d'immortaliser des cellules ne se divisant pas, et cellules immortalisees au moyen de ces vecteurs
EP1103615A1 (fr) * 1999-11-25 2001-05-30 Universite De Geneve Vecteurs capables d'immortaliser les cellules ne se divisant pas et les cellules immortalisées par ces vecteurs
JP2003514565A (ja) * 1999-11-25 2003-04-22 ユニヴェルシテ・ドゥ・ジュネーブ 非分裂細胞を不死化する能力をもつベクター及び前記ベクターで不死化された細胞
WO2001091801A2 (fr) * 2000-05-26 2001-12-06 Chiron Corporation Procedes de transduction de cellules neuronales cerebelleuses par le biais de vecteurs de lentivirus
WO2001091801A3 (fr) * 2000-05-26 2003-02-06 Chiron Corp Procedes de transduction de cellules neuronales cerebelleuses par le biais de vecteurs de lentivirus
US7179903B2 (en) 2001-05-14 2007-02-20 Cell Genesys, Inc Liver specific transcriptional enhancer
US7745179B2 (en) 2001-05-14 2010-06-29 Gbp Ip, Llc Lentiviral vectors featuring liver specific transcriptional enhancer and methods of using same
US6808905B2 (en) 2001-05-14 2004-10-26 Cell Genesys, Inc. Lentiviral vectors encoding clotting factors for gene therapy

Also Published As

Publication number Publication date
EP0970201A1 (fr) 2000-01-12
JP2001501815A (ja) 2001-02-13
AU4261797A (en) 1998-04-14
CA2265522A1 (fr) 1998-03-26

Similar Documents

Publication Publication Date Title
EP0871459B1 (fr) Vecteur et procede permettant d'alimenter en acide nucleique des cellules non divisant
WO1997012622A9 (fr) Vecteur et procede permettant d'alimenter en acide nucleique des cellules ne subissant pas de division
JP4418536B2 (ja) レトロウイルスベクター
US6165782A (en) Method and means for producing high titer, safe, recombinant lentivirus vectors
JP4436024B2 (ja) レンチウイルスの三重鎖ｄｎａ、並びにレンチウイルスの三重鎖ｄｎａを含有するベクター及び組換え細胞
WO1999051754A9 (fr) Vecteurs lentiviraux infectieux pseudotypes depourvus de porine et utilisation desdits vecteurs
AU749059B2 (en) Therapeutic use of lentiviral vectors
US7803582B2 (en) Recombinant vector and use in gene therapy
EP0970201A1 (fr) Vecteurs retroviraux pouvant transduire des cellules ne se divisant pas
US5580766A (en) Retroviral vector particles for transducing non-proliferating cells
CA2233867C (fr) Vecteur et procede permettant d'alimenter en acide nucleique des cellules ne subissant pas de division
AU758600B2 (en) Vector and method of use for nucleic acid delivery to non-dividing cells
DE69829174T2 (de) Exprimierung eines modifiziertem "foamy virus envelope protein" (hüllenprotein)
US20120034693A1 (en) Recombinant vector and use in gene therapy
Baranick Characterization and utilization of simple replication-competent retroviral vectors

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW AM AZ BY KG KZ MD RU TJ TM

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV GH

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH KE LS MW SD SZ UG ZW AT BE CH DE DK ES FI FR GB GR IE IT LU MC

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
ENP Entry into the national phase

Ref document number: 2265522

Country of ref document: CA

Ref country code: CA

Ref document number: 2265522

Kind code of ref document: A

Format of ref document f/p: F

ENP Entry into the national phase

Ref country code: JP

Ref document number: 1998 514725

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 1997940952

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWP Wipo information: published in national office

Ref document number: 1997940952

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1997940952

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载