[go: up one dir, main page]

WO2004044157A2 - Vecteurs pseudotypes avec la gp64 et leurs utilisations - Google Patents

Vecteurs pseudotypes avec la gp64 et leurs utilisations Download PDF

Info

Publication number
WO2004044157A2
WO2004044157A2 PCT/US2003/035728 US0335728W WO2004044157A2 WO 2004044157 A2 WO2004044157 A2 WO 2004044157A2 US 0335728 W US0335728 W US 0335728W WO 2004044157 A2 WO2004044157 A2 WO 2004044157A2
Authority
WO
WIPO (PCT)
Prior art keywords
vector
cells
cell
retroviral
particle
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2003/035728
Other languages
English (en)
Other versions
WO2004044157A3 (fr
Inventor
Mukesh Kumar
Joshua Zimmerberg
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
National Institutes of Health NIH
US Department of Health and Human Services
Original Assignee
National Institutes of Health NIH
US Department of Health and Human Services
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 National Institutes of Health NIH, US Department of Health and Human Services filed Critical National Institutes of Health NIH
Priority to AU2003291426A priority Critical patent/AU2003291426A1/en
Publication of WO2004044157A2 publication Critical patent/WO2004044157A2/fr
Publication of WO2004044157A3 publication Critical patent/WO2004044157A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—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
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16045—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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/40—Systems of functionally co-operating 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
    • 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
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/60—Vector systems having a special element relevant for transcription from viruses
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • the present invention relates to pseudotyped lentiviral vectors that can be used for gene transfer as well as for large-scale production of high titer vims to be utilized in clinical and commercial applications.
  • Lentiviral vectors can insert large genes and can target both dividing and non- dividing cells. Therefore, they hold unique promise as gene transfer agents, the past, the native lentiviral envelope glycoprotein has been replaced with that of vesicular stomatitis vims G (VSV-G), and the genes of interest were packaged in non-replicating vectors using transient transfection with three plasmids.
  • VSV-G vesicular stomatitis vims G
  • the present invention provides a pseudotyped lentiviras vector using the baculovims gp64 envelope glycoprotein.
  • gp64 vectors When compared to VSV-G, gp64 vectors exhibit similar broad tropism and ⁇ 30% higher native titers. Gp64-pseudotyped vectors are also highly concentrated without losing titer. Since, unlike VSV-G, gp64 expression does not kill cells, 293T-based cell lines constitutively expressing gp64 were generated. The present invention demonstrates that the baculovims gp64 protein is an alternative to VSV-G for viral vectors for the large-scale production of high titer vims required for clinical and commercial applications.
  • the present invention provides a recombinant retroviral vector expression system comprising: a) a first vector comprising a retroviral nucleic acid sequence encoding retroviral gag and retroviral pol; b) a second vector comprising a retroviral nucleic acid sequence comprising cis-acting sequence elements for reverse transcription of the vector genome and a cloning site wherein a gene of interest may be inserted; and c) a third vector comprising a viral nucleic acid sequence, wherein the third vector expresses the GP64 envelope protein.
  • a method of producing a recombinant retroviral particle comprising transfecting a cell with: a) a first vector comprising a retroviral nucleic acid sequence encoding retroviral gag and retroviral pol; b) a second vector comprising an retroviral nucleic acid sequence comprising cis-acting sequence elements for reverse transcription of the vector genome and a gene of interest; and c) a third vector comprising a viral nucleic acid sequence, wherein the third vector expresses the GP64 envelope protein, wherein the cell produces a recombinant retroviral particle.
  • composition comprising: a) a first vector comprising an retroviral nucleic acid sequence encoding retroviral gag and retroviral pol; b) a second vector comprising an retroviral nucleic acid sequence comprising cis-acting sequence elements for reverse transcription of the vector genome and a cloning site wherein a gene of interest may be inserted; and c) a third vector comprising a viral nucleic acid sequence, wherein the third vector expresses the GP64 envelope protein.
  • the present invention further provides a method of producing a recombinant retroviral particle,comprising: a) transfecting a cell with a first vector comprising a viral nucleic acid sequence, wherein the first vector expresses the GP64 envelope protein; and b)transfecting the GP64 expressing cells of a) with a second vector comprising an retroviral nucleic acid sequence comprising cis-acting sequence elements for reverse transcription of the vector genome and a gene of interest and a third vector comprising an retroviral nucleic acid sequence encoding retroviral gag and retroviral pol wherein the cell produces a recombinant retroviral particle.
  • a retroviral particle comprising a genome, gag, pol, and a GP64 envelope protein, wherein the genome lacks at least one retroviral structural protein selected from the group consisting of : gag, pol, vif, vpu, vpr, nef and env.
  • retroviral particles comprising a genome, gag, pol, and a GP64 envelope protein, wherein the genome lacks at least one retroviral structural protein selected from the group consisting of : gag, pol, vif, vpu, vpr, nef and env.
  • Fig. 1 A shows the production of baculovims GP64-pseudotyped recombinant H ⁇ V-1.
  • the arrow indicates the GP64 band.
  • 293T cells were either transfected or not with plasmid pBsd-gp64, then grown for 48 hrs.
  • Samples of total cellular proteins were separated on SDS-PAGE, transferred to nitrocellulose membrane for immunob lotting using the anti-GP64 monoclonal antibodies ACV5.
  • Right two lanes Cells were transfected or not with three plasmids for 60 hrs.
  • FIG. 1 shows the production of baculovims GP64-pseudotyped recombinant
  • HIN-1 Purified recombinant vims was applied to formavar-coated copper grids, followed by negative staining and visualized by transmission electron microscopy.
  • Figure 1C shows the production of baculovims GP64-pseudotyped recombinant HIN-1.
  • 200 ⁇ l of purified GP64-pseudotyped vims suspension containing 10 mM sodium azide was overlaid over a monolayer of HOS cells in a 10 cm dish on ice for 30 min. The cells were then washed 4-5 times in excess chilled PBS containing 10 mM sodium azide to remove unbound vims.
  • Fig. 2 A shows transduction of a broad range of target cells by GP64- pseudotyped HIN- 1.
  • Pseudotyped vims was created by three-plasmid transfection methods in 293T cells. The same amount of unconcentrated vims-containing culture supernatant (lOO ⁇ l) was added to the medium on monolayers of HOS, MDCK, 293T and HeLa cells. Cells were observed for GFP (trans gene cloned into the transfer vector) expression using fluorescence microscopy 60h post-infection.
  • Figure 2B shows that the number of GFP-positive cells increases linearly with the amount of vims used.
  • HOS cells were infected with indicated amounts of GP64- pseudotyped HJV-GFP and imaged 60h post-infection.
  • FIG. 2C shows that HOS cell monolayers growing in 6-well plates were infected with indicated amounts of GP64-pseudotyped vims. At 60h post-infection cells were analyzed by FACS for percent GFP positive cells as described in methods.
  • D Genomic D ⁇ A was prepared from transduced cell lines and tested by Alu PCR for integration of the transfer vector, as described (Butler et al., 2001). The PCR products were separated on a 1% agarose gel and visualized by ethidium bromide staining. U, uninfected cells; I, Infected cells.
  • Figure 3A shows a permanent human cell line expressing GP64.
  • the level of expression of GP64 in various passages of the permanent cell line (KZ64) was measured using FACS analysis.
  • Cells from each passage were labeled with anti-GP64 monoclonal antibodies followed by cy5-labeled goat anti-mouse IgG as secondary and analyzed by FACS as described in the methods.
  • Figure 3B shows that the presence of gp64 gene was detected in each passage of
  • KZ64 by PCR of the genomic D ⁇ A followed by separation of the PCR product on agarose gel. Arrow indicates the gp64 gene ( ⁇ 1.5kb in size).
  • Genomic D ⁇ A from untransfected 293T cells was used as negative control, designated as "-ve”.
  • C Relative comparison of GP64 mR ⁇ A in different passages of KZ64 cells was carried out by qRT-PCR as described in methods. Small “p” represents passage number and the number following it denotes the passage number from which R ⁇ A was made. 293T cells transfected with pBsdGP64 and 48 hr later the transfected cells were plated into complete DMEM containing lO ⁇ g/ml Blasticidin. Resistant cells were harvested 8 days post-selection and used for total RNA purification. This sample was designated passage zero (pO).
  • a nucleic acid includes multiple copies of the nucleic acid and can also include more than one particular species of nucleic acid molecule.
  • the present invention provides a recombinant retroviral vector expression system comprising: a) a first vector comprising a retroviral nucleic acid sequence encoding retroviral gag and retroviral pol; b) a second vector comprising a retroviral nucleic acid sequence comprising cis-acting sequence elements for reverse transcription of the vector genome and a cloning site wherein a gene of interest may be inserted; and c) a third vector comprising a viral nucleic acid sequence, wherein the third vector expresses the GP64 envelope protein.
  • retroviral nucleic acid means that the sequence comprises at least part of a retroviral genome.
  • the retroviral nucleic acid can be a nucleic acid from an HJN-1 genome or from an HIN-2 genome. The entire human HIN-1 genome can be accessed via GenBank Accession
  • the human HIN-1 genome accessible via Accession No. NC_001802 includes nucleic acids encoding retroviral gag and pol, as well as nucleic acids encoding tat, vif, vpu, vpr, nef and env all of which are incorporated herein by this reference.
  • the entire human HIN- 2 genome can be accessed via GenBank Accession No. NC_001722 and is incorporated herein in its entirety by this reference.
  • NC_001722 includes nucleic acids encoding retroviral gag and pol, as well as nucleic acids encoding tat, vif, vpu, vpr, nef and env all of which are incorporated herein by this reference.
  • the vectors can also comprise a retroviral nucleic acid sequence from a self inactivating HIV sequence.
  • the retroviral nucleic acid sequences can also be sequences derived from bovine immunodeficiency vims, Jembrana disease vims, Equine infectious anemia vims, feline immunodeficiency vims, caprine arthritis-encephalitis vims, ovine lentivirus, Visna vims, simian immunodeficiency vims, simian-human immunodeficiency vims.
  • the first vector is a gag/pol retroviral expression vector which expresses proteins required for assembly and release of viral particles from cells, and includes the nucleic acids encoding retroviral gag and pol.
  • This vector can also comprise nucleic acids encoding vpr and vpu as well as a promoter sequence.
  • the promoter sequence may comprise a promoter of eukaryotic or prokaryotic origin . Suitable promoters include, but are not limited to, the cytomegalovims promoter, the Rous Sarcoma vims promoter, promoters derived from immunoglobulin genes, SV40, Adenovims and Bovine Papilloma Vims.
  • All of the vectors described herein can comprise a promoter, hi addition to the guidance provided in the Examples provided herein, standard techniques for the constraction of the vectors of the present invention are well-known to one of skill in the art and can be found in references such as Sambrook et al., Molecular Cloning: A Laboratory Manual 2 nd Ed. (Cold Spring Harbor, N.Y., 1989).
  • One of skill in the art can select from a variety of techniques available for the ligation of nucleic acids to construct the vectors of the present invention.
  • the first vector can also comprise a defective env gene. As used herein,
  • defective means that a nucleic acid sequence is not functional with regard to either encoding its gene product or serving as a signaling sequence.
  • a defective env nucleic acid sequence will not encode the env protein; in another example, a defective packaging signal will not facilitate the packaging of the nucleic acid molecule the defective packaging signal is located on.
  • Nucleic acid sequences may be rendered defective by means known in the art, including the deletion of some or all of the sequence, by mutating the nucleic acid sequence, by placing the sequence out of frame or by otherwise interfering with the sequence.
  • the second vector comprises a retroviral nucleic acid sequence comprising cis- acting sequence elements for reverse transcription of the vector genome and a cloning site wherein a gene of interest may be inserted.
  • the cis-acting sequences of the present invention include rev, tat and the long terminal repeats (LTRs) containing the packaging signals.
  • This second vector can also comprise a defective env gene.
  • the present invention also provides an expression system in which a defective env nucleic acid or gene is present in the first vector as well as in the second vector.
  • the second vector can also be deficient or defective for expression of at least one retroviral structural protein selected from the group consisting of: gag, pol, vif, vpu, vpr, nef and env.
  • the vectors of the present invention include such defective nucleic acids in order to exclude the possibility of generating replication-competent retrovims.
  • the second vector need only include the LTRs and the gene of interest.
  • the second vector contains a CMN promoter and one of the LTRs in order to minimize gene silencing.
  • the second vector can have active gag, pol, vif, vpu and vpr genes and inactive nef and env genes.
  • the present invention also contemplates the use of the self-inactivating (SL ⁇ ) construct of the HJN vims, hi this construct, a 400 base pair fragment from the 3' LTR is deleted such that the HIN genome can integrate only once and the chances of forming replication competent vims by random recombination is much lower, h these vectors, gag, pol, vif, vpr, vpu, nef and env are usually deleted.
  • SL ⁇ self-inactivating
  • the second vector of the expression system of the present invention is designed to serve as the vector for gene transfer and contains all of the cis-acting sequence elements required to support reverse transcription of the vector genome, as well as a multiple cloning site for insertion of a nucleic acid encoding a heterologous gene of interest that will be delivered to cells.
  • the vector encoding the gene of interest is a recombinant retroviral vector, for example, an HIN-1 or HIN-2 vector, that comprises a gene of interest to be transduced into a cell as well as cis-acting sequences necessary for the packaging and integration of the viral genome.
  • nucleic acid sequence encoding the heterologous gene of interest or “heterologous nucleic acid” is meant that any heterologous or exogenous nucleic acid, i.e. not normally found in the wildtype retroviral genome, can be inserted into the vector for transfer into a cell, tissue or organism.
  • any heterologous or exogenous nucleic acid i.e. not normally found in the wildtype retroviral genome, can be inserted into the vector for transfer into a cell, tissue or organism.
  • particles comprising a heterologous nucleic acid encoding a gene of interest can be isolated for delivery to cells.
  • the heterologous nucleic acid can be functionally linked to a promoter.
  • the promoter can promote expression of the heterologous nucleic acid, as is known in the art, and can include the appropriate orientation of the promoter relative to the heterologous nucleic acid.
  • the heterologous nucleic acid preferably has all appropriate sequences for expression of the nucleic acid.
  • the nucleic acid can include, for example, expression control sequences, such as an enhancer, a silencer and necessary information processing sites, such as ribosome binding sites, R ⁇ A splice sites, polyadenylation sites, and transcriptional terminator sequences.
  • the heterologous nucleic acid can encode beneficial proteins or polypeptides that replace missing or defective proteins required by the cell or subject into which the vector is transferred or can encode a cytotoxic polypeptide that can be directed, e.g., to cancer cells or other cells whose death would be beneficial to the subject.
  • the heterologous nucleic acid can also encode antisense R ⁇ As that can bind to, and thereby inactivate, mR ⁇ As made by the subject that encode harmful proteins.
  • the heterologous nucleic acid can also encode ribozymes that can effect the sequence-specific inhibition of gene expression by the cleavage of mR ⁇ As.
  • antisense polynucleotides can be produced from a heterologous expression cassette in a retroviral vector construct where the expression cassette contains a sequence that promotes cell-type specific expression (Wirak et al, EMBO 10:289 (1991)).
  • the expression cassette contains a sequence that promotes cell-type specific expression (Wirak et al, EMBO 10:289 (1991)).
  • Antisense RNA and DNA D. A. Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1988).
  • heterologous nucleic acids that can be utilized in the expression systems and methods of the present invention, include but are not limited to the following: nucleic acids encoding secretory and nonsecretory proteins, nucleic acids encoding therapeutic agents, such as tumor necrosis factors (TNF) and TNF- ⁇ ; interferons, such as interferon- ⁇ , interferon- ⁇ , and interferon- ⁇ ; interleukins, such as LL-1, IL-l ⁇ , and ILs -2 through -14; GM-CSF; adenosine deaminase; cellular growth factors, such as lymphokines; soluble CD4; Factor NIII; Factor IX; T-cell receptors; LDL receptor; ApoE; ApoC; alpha- 1 antitrypsin; ornithine transcarbamylase (OTC); cystic fibrosis fransmembrane receptor (CFTR); insulin; anti-apoptotic gene products; proteins promoting neuronal survival, such as
  • particles comprising a heterologous nucleic acid encoding a gene of interest can be made and used to target various cells.
  • the nucleic acid is chosen considering several factors, including the cell to be transfected.
  • the target cell is a blood cell
  • particularly useful nucleic acids to use are those which allow the blood cells to exert a therapeutic effect, such as a gene encoding a clotting factor for use in treatment of hemophilia.
  • Another target cell is the lung airway cell, which can be used to administer nucleic acids, such as those coding for the cystic fibrosis fransmembrane receptor, which could provide a gene therapeutic treatment for cystic fibrosis..
  • target cells include muscle cells where useful nucleic acids, such as those encoding cytokines and growth factors, can be transduced and the protein the nucleic acid encodes can be expressed and secreted to exert its effects on other cells, tissues and organs, such as the liver. Neurons and retinal cells can also serve as targets for delivery of heterologous nucleic acids.
  • the nucleic acid can encode more than one gene product, limited only, if the nucleic acid is to be packaged, by the size of nucleic acid that can be packaged.
  • suitable nucleic acids can include those that, when transferred into a primary cell, such as a blood cell, cause the transferred cell to target a site in the body where that cell's presence would be beneficial.
  • blood cells such as TIL cells can be modified, such as by transfer into the cell of a Fab portion of a monoclonal antibody, to recognize a selected antigen.
  • Another example would be to introduce a nucleic acid that would target a therapeutic blood cell to tumor cells.
  • Nucleic acids useful in treating cancer cells include those encoding chemotactic factors which cause an inflammatory response at a specific site, thereby having a therapeutic effect.
  • nucleic acids particularly blood cells, muscle cells, airway epithelial cells, brain cells and endothelial cells having such nucleic acids transferred into them can be useful in a variety of diseases, syndromes and conditions.
  • suitable nucleic acids include nucleic acids encoding soluble CD4, used in the treatment of AIDS and ⁇ - antitrypsin, used in the treatment of emphysema caused by ⁇ -antitrypsin deficiency.
  • diseases, syndromes and conditions in which such cells can be useful include, for example, adenosine deaminase deficiency, sickle cell deficiency, brain disorders such as Alzheimer's disease, Huntington's disease, lysosomal storage diseases, Gaucher's disease, Hurler's disease, Rrabbe's disease, motor neuron diseases such as amylotrophic lateral sclerosis and dominant spinal cerebellar ataxias (examples include SCA1, SCA2, and SCA3), thalassemia, hemophilia, diabetes, phenylketonuria, growth disorders and heart diseases, such as those caused by alterations in cholesterol metabolism, and defects of the immune system.
  • adenosine deaminase deficiency such as Alzheimer's disease, Huntington's disease, lysosomal storage diseases, Gaucher's disease, Hurler's disease, Rrabbe's disease, motor neuron diseases such as amylotrophic lateral sclerosis and dominant
  • the heterologous nucleic acid can also encode a reporter gene sequence or a selectable marker gene sequence.
  • a reporter gene sequence is any gene sequence which, when expressed, results in the production of a protein whose presence or activity can be monitored. Thus, the reporter protein can be specifically detected when expressed.
  • Many reporter proteins are known to one of skill in the art. These include, but are not limited to, beta-galactosidase, chloramphenicol acetyltransferase, beta- lactamase, luciferase, and alkaline phosphatase that produce specific detectable products. Fluorescent reporter proteins can also be used, such as green fluorescent protein (GFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP) and yellow fluorescent protein (YFP).
  • GFP green fluorescent protein
  • CFP cyan fluorescent protein
  • RFP red fluorescent protein
  • YFP yellow fluorescent protein
  • a selectable marker sequence is any gene sequence capable of expressing a protein whose presence permits one to selectively propagate a cell which contains it.
  • selectable marker genes include gene sequences capable of conferring host resistance to antibiotics, for example, arnpicillin, tetracycline, kanamycin etc., or of conferring host resistance to amino acid analogues, or of permitting the growth of bacteria on additional carbon sources.
  • the heterologous nucleic acid may also provide an immunogenic or antigenic protein or polypeptide that can be used to achieve an antibody response. These antibodies can then be collected from an animal in a body fluid such as blood, serum or ascites.
  • the third vector comprises a viral nucleic acid sequence, wherein the third vector expresses the GP64 envelope protein. Therefore, this vector comprises a nucleic acid sequence encoding GP64.
  • GP64 is a class I membrane glycoprotein, wliich constitutes the major envelope protein of the AcMNPN budded baculovims virion (Nolkman and Goldsmith, 1984; Nolkman et al, 1984).
  • the third vector is pseudotyped, which means that the viral particles made utilizing the expression system described herein contains nucleic acid from one vims and the envelope protein of another, different vims. Therefore, based on the teachings of the present invention, any recombinant vector system can utilize a psuedotyped GP64 vector for the production of recombinant viral particles.
  • Nucleic acids encoding GP-64 may be identical in sequence to the sequences which are naturally occurring for the GP-64 protein or may include alternative codons which encode the same amino acid as that which is found in the naturally occurring sequence. These nucleic acids can also be modified from their typical structure.
  • Such modifications include, but are not limited to, methylated nucleic acids, the substitution of a non-bridging oxygen on the phosphate residue with either a sulfur (yielding phosphorothioate deoxynucleotides), selenium (yielding phosphorselenoate deoxynucleotides), or methyl groups (yielding methylphosphonate deoxynucleotides).
  • methylated nucleic acids the substitution of a non-bridging oxygen on the phosphate residue with either a sulfur (yielding phosphorothioate deoxynucleotides), selenium (yielding phosphorselenoate deoxynucleotides), or methyl groups (yielding methylphosphonate deoxynucleotides).
  • Any of the nucleic acid sequences described herein, such as the GP-64 nucleic acid sequences and the heterologous nucleic acid sequences provided for by the present invention may be
  • One example of a method of obtaining a DNA molecule encoding a specific GP64 protein is to synthesize a recombinant DNA molecule which encodes the GP64 protein.
  • oligonucleotide synthesis procedures are routine in the art and oligonucleotides coding for a particular protein region are readily obtainable through automated DNA synthesis.
  • a nucleic acid for one strand of a double-stranded molecule can be synthesized and hybridized to its complementary strand.
  • One can design these oligonucleotides such that the resulting double-stranded molecule has either internal restriction sites or appropriate 5' or 3' overhangs at the termini for cloning into an appropriate vector.
  • Double-stranded molecules coding for relatively large proteins can readily be synthesized by first constmcting several different double-stranded molecules that code for particular regions of the protein, followed by ligating these DNA molecules together.
  • Cunningham, et al "Receptor and Antibody Epitopes in Human Growth Hormone Identified by Homolog-Scanning Mutagenesis," Science, 243:1330-1336 (1989), have constructed a synthetic gene encoding the human growth hormone gene by first constructing overlapping and complementary synthetic oligonucleotides and ligating these fragments together. See also, Ferretti, et al, Proc. Nat. Acad. Sci.
  • nucleic acid sequence of the desired GP64 protein is obtained, the sequence encoding specific amino acids can be modified or changed at any particular amino acid position by techniques well known in the art. For example, PCR primers can be designed which span the amino acid position or positions and which can substitute any amino acid for another amino acid. Then a nucleic acid can be amplified and inserted into the wild-type GP64 protein coding sequence in order to obtain any of a number of possible combinations of amino acids at any position of the GP64 protein.
  • nucleic acid encoding GP64 any nucleic acid encoding a heterologous nucleic acid of the present invention or any nucleic acid encoding retroviral nucleic acid sequences.
  • the present invention also provides an expression system in wliich the first vector is pHIN-PV, the second vector is pHIV-IRES-G " P ⁇ T " V " and the third vector is pcD ⁇ A-gp64. Also provided by this invention is an expression system in which the first vector is pHFN-PN, the second vector is pHIN- G ' P ' ETN " IRES-GFP and the third vector is pcD ⁇ A-gp64. These vectors and the methods of constructing these vectors are described in the Examples herein. Examples of other vectors that can be utilized as a third vector in this system include plasmids containing other envelope genes, e.g.
  • pME-VSVG also contains VSVG
  • pCB5HA contains influenza HA
  • pLTRMVG contains Makola Vims envelope
  • pHIT456 contains murine leukemia virus amphotrophic envelope
  • pCG6-Ebo-GP contains ebola virus glycoprotein
  • the present invention also provides a method of producing a recombinant retroviral particle,comprising transfecting a cell with: a) a first vector comprising a retroviral nucleic acid sequence encoding retroviral gag and retroviral pol; b) a second vector comprising an retroviral nucleic acid sequence comprising cis-acting sequence elements for reverse transcription of the vector genome and a gene of interest; and c) a third vector comprising a viral nucleic acid sequence, wherein the third vector expresses the GP64 envelope protein, wherein the cell produces a recombinant retroviral particle.
  • the present invention further provides a method of producing a recombinant retroviral particle,comprising transfecting a cell with: a) a first vector comprising a retroviral nucleic acid sequence encoding retroviral gag and retroviral pol; b) a second vector comprising an retroviral nucleic acid sequence comprising cis-acting sequence elements for reverse transcription of the vector genome and a gene of interest; c) a third vector comprising a viral nucleic acid sequence, wherein the third vector expresses the GP64 envelope protein, wherein the cell produces a recombinant retroviral particle; d) growing the cell to allow production of recombinant retroviral particles in the cell; and e) obtaining the recombinant retroviral particles from the cell.
  • the cells that can be utilized to make the particles of the present invention include, but are not limited to, 293 cells, COS7 cells, COS cells and CEM cells.
  • the methods of the present invention allow the preparation of infectious, replication-defective retroviral particles. These methods can be utilized with those available in the art to prepare viral particles.
  • Viral particles can be made by contacting lentivims-permissive cells or producer cells with the vector expression system or the compositions comprising the vectors of the present invention, producing the retro viral- derived particles in the transfected cells and collecting the vims particles from the cell. Transfection of cells can be accomplished utilized any standard method in the art such as electorporation, LEPTOFECTAMiNE mediated transfection, DEAE-dextran transfection etc.
  • Production of the infectious viral particles in the cells can be carried out using conventional techniques, such as standard cell culture techniques.
  • collection can be carried by techniques known in the art.
  • infectious particles can be collected by cell lysis, or collection of the supernatant of the cell culture, as described in the Examples and as is known in the art.
  • the vims particles can be further purified. Such purification techniques are known in the art.
  • the GP64-pseudotyped viral particles of the present invention can be concentrated without significant loss of titer.
  • the present invention also provides recombinant retroviral particles produced by the methods of the present invention as well as cells comprising the recombinant particles produced by the methods of the present invention.
  • a composition comprising: a) a first vector comprising an retroviral nucleic acid sequence encoding retroviral gag and retroviral pol; b) a second vector comprising an retroviral nucleic acid sequence comprising cis-acting sequence elements for reverse transcription of the vector genome and a cloning site wherein a gene of interest may be inserted; and c) a third vector comprising a viral nucleic acid sequence, wherein the third vector expresses the GP64 envelope protein. All of the compositions described herein can further comprise a carrier.
  • cells comprising the compositions of the present invention.
  • the present invention also provides a method of producing a recombinant retroviral particle,comprising: a) transfecting a cell with a first vector comprising a viral nucleic acid sequence, wherein the first vector expresses the GP64 envelope protein; and b) transfecting the GP64 expressing cells of a) with a second vector comprising an retroviral nucleic acid sequence comprising cis-acting sequence elements for reverse transcription of the vector genome and a gene of interest and a third vector comprising an retroviral nucleic acid sequence encoding retroviral gag and retroviral pol wherein the cell produces a recombinant retroviral particle.
  • Also provided by the present invention is a method of producing a recombinant retroviral particle, comprising: a) transfecting a cell with a first vector comprising a viral nucleic acid sequence, wherein the first vector expresses the GP64 envelope protein; b) transfecting the GP64 expressing cells of a) with a second vector comprising an retroviral nucleic acid sequence comprising cis-acting sequence elements for reverse transcription of the vector genome and a gene of interest and a third vector comprising an retroviral nucleic acid sequence encoding retroviral gag and retroviral pol wherein the cell produces a recombinant retroviral particle; c) growing the cell to allow production of recombinant retroviral particles in the cell; and d) obtaining the recombinant retroviral particles from the cell.
  • the present invention also provides a retroviral particle comprising a genome, gag, pol, and a GP64 envelope protein, wherein the genome lacks at least one retroviral structural protein selected from the group consisting of : gag, pol, vif, vpu, vpr, nef and env.
  • the genome can lack any one of these structural proteins or any combination thereof. Therefore, the genome can lack all of the structural proteins (gag, pol, vif, vpu, vpr, nef and env).
  • the retroviral particle of the present invention can further comprise rev and/or tat.
  • the retroviral particle of the present invention can be a particle wherein the genome is an HIN-1 genome and vpu, vpr, vif, tat and nef are absent or are dismpted. Any of the above-described particles of the present invention can further comprise a nucleic acid sequence encoding a gene of interest.
  • the present invention also provides an isolated cell or cells comprising the particles of the present invention.
  • the present invention also provides a method of expressing a gene of interest comprising contacting a cell with a retroviral particle of the present invention comprising a nucleic acid encoding a gene of interest.
  • the cells in which a gene of interest can be expressed include, but are not limited to, fibroblasts, neurons, retinal cells, kidney cells, lung cells, bone marrow stem cells, hematopoietic stem cells, retinal cells and neurons.
  • the cells in which the gene of interest can be expressed can be dividing cells such as MDCK cells, BHK cells, HeLa cells, 3T3 cells, CN1 cells, COS7 cells, HOS cells and 293 cells.
  • the cells can also be embryonic stem cells of mouse, rhesus, human, bovine or sheep origin, as well as stem cells of neural, hematopoietic, muscle, cardiac, immune or other origin.
  • the embryonic stem cells of the present invention such as stem cells of neural, hematopoietic, muscle, cardiac, immune or other origin can be dividing or nondividing embryonic stem cells.
  • ⁇ ondividing cells can also be contacted with a particle of the present invention to express a gene of interest.
  • Such cells include, but are not limited to hematopoietic stem cells and embryonic stem cells that have been rendered non-dividing by specific media.
  • Stem cells can be rendered non-dividing by growing them in media lacking growth factors required by stem cells, namely interleukins, LL-3, IL-6, GM-CSF and stem cell factor.
  • cells can be contacted ex vivo, in vitro or in vivo with the particles of the present invention.
  • Administration can be an ex vivo administration directly to a cell removed from a subject, such as any of the cells listed above, followed by replacement of the cell back into the subject, or administration can be in vivo administration to a cell in the subject.
  • cells are isolated from a subject by standard means according to the cell type and placed in appropriate culture medium, again according to cell type (see, e.g., ATCC catalog). Viral particles are then contacted with the cells as described above, and the vims is allowed to transfect the cells. Cells can then be transplanted back into the subject's body, again by means standard for the cell type and tissue (e. g., for neural cells, Dunnett, S.B. and Bj ⁇ rklund, A., eds., Transplantation: Neural Transplantation-A Practical Approach,
  • the cells can be studied for degree of transfection by the vims, by known detection means and as described herein.
  • a particle of the present invention comprising a reporter protein.
  • the reporter protein By measuring the expression of the reporter protein in the cell or tissue, one of skill in the art can determine whether the cell is amenable for transduction with the particles of the present invention. The skilled artisan can also determine dosages utilizing such in vitro studies.
  • Vims particles can be administered orally, parenterally (e.g., intravenously), by intramuscular inj ection, by direct tissue or organ inj ection, by intraperitoneal inj ection, topically, transdermally, via aerosol delivery, via the mucosa or the like.
  • the present compositions can include various amounts of the selected viral particle in combination with a pharmaceutically acceptable carrier and, in addition, if desired, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc.
  • Parental administration, if used, is generally characterized by injection.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Dosages will depend upon the mode of administration, the disease or condition to be treated, and the individual subject's condition, but will be that dosage typical for and used in administration of viral vectors. For example, a typical dosage can be from about 10 to about 10 7 viral particles, depending on the age and the species of the subject. Often a single dose can be sufficient; however, the dose can be repeated if desirable.
  • Administration methods can be used to treat brain disorders such as Parkinson's disease, Alzheimer's disease, and demyelination disease.
  • Other diseases that can be treated by these methods include metabolic disorders such as , muscoloskeletal diseases, cardiovascular disease, cancer, and autoimmune disorders.
  • Administration of recombinant particles to the cell can be accomplished by any means, including simply contacting the particle, optionally contained in a desired liquid such as tissue culture medium, or a buffered saline solution, with the cells.
  • the particle can be allowed to remain in contact with the cells for any desired length of time, and typically the particle is administered and allowed to remain indefinitely.
  • the particle can be administered to the cell by standard viral fransduction methods, as known in the art and as exemplified herein. Titers of vims to administer can vary, particularly depending upon the cell type, but will be typical of that used for retroviral fransduction in general which is well known in the art. Additionally the titers used to transduce the particular cells in the present examples can be utilized.
  • the cells that can be transduced by the present particles of the present invention particle can include any desired cell, such as the following cells and cells derived from the ⁇ following tissues, human as well as other mammalian tissues, such as primate, horse, sheep, goat, pig, dog, rat, and mouse: Adipocytes, Adenocyte, Adrenal cortex, Airway epithelial cells, Alveolar cells, Amnion, Aorta, Ascites, Astrocyte, Bladder, Bone, Bone marrow, Brain, Breast, Bronchus, Cardiac muscle, Cecum, Cerebellar, Cervix, Chorion, Colon, Conjunctiva, Connective tissue, Cornea, Dennis, Duodenum, Endometrium, Endothelium, Endothelial cells, Ependymal cells, Epithelial tissue, Epithelial cells, Epidermis, Esophagus, Eye, Fascia, Fibroblasts, Foreskin, Gastric, Glial cells
  • the particles of the present invention can be used to deliver a nucleic acid to these cells.
  • cells comprising the particles of the present invention such as, but not limited to, fibroblasts, kidney cells, neurons, retinal cells, lung cells, bone manow stem cells, hematopoietic stem cells, MDCK cells, BHK cells, HeLa cells, 3T3 cells, CV1 cells, COS7 cells, HOS cells and 293 cells.
  • Other cells that can comprise the particles of the present invention include embryonic stem cells of mouse, rhesus, human, bovine or sheep origin, as well as stem cells of neural, hematopoietic, muscle, cardiac, immune or other origin.
  • Nondividing cells can also comprise the particles of the present invention. These cells include, but are not limited to hematopoietic stem cells and embryonic stem cells that have been rendered non- dividing by specific media.
  • the present invention also provides a population of retroviral particles, e.g., more than one particle, comprising a genome, gag, pol, and a GP64 envelope protein, wherein the genome lacks at least one retroviral structural protein selected from the group consisting of : gag, pol, vif, vpu, vpr, nef and env. Also provided by the present invention is a composition comprising a population of retroviral particles of the present invention.
  • the present invention is a method of expressing a gene of interest comprising contacting a population of cells with a population of retroviral particles of the present invention.
  • the cells can be, but are not limited to, fibroblasts, neurons, retinal cells, kidney cells, lung cells, bone marrow stem cells and hematopoietic stem cells.
  • the population of cells can be a population of dividing cells.
  • the population of cells can also be a population of non-dividing cells.
  • populations of cells comprising a population of retroviral particles comprising a genome, gag, pol, and a GP64 envelope protein, wherein the genome lacks at least one retroviral structural protein selected from the group consisting of : gag, pol, vif, vpu, vpr, nef and env.
  • a composition comprising a population of retroviral particles of the present invention.
  • the cells can be, but are not limited to, fibroblasts, neurons, retinal cells, kidney cells, lung cells, bone marrow stem cells and hematopoietic stem cells.
  • the population of cells can be a population of dividing cells.
  • the population of cells can also be a population of non-dividing cells.
  • pseudotyping 1 Since many native envelope glycoproteins have restricted target cell ranges, investigators have developed novel vectors by replacing the native envelope glycoprotein with ones from other vimses, a process called pseudotyping 1 .
  • the most prevalent pseudotyped lentiviral vector is HJN-1 with the vesicular stomatitis viras G (NSN-G) glycoprotein substituting for the env glycoprotein 2"8 .
  • NSN-G vesicular stomatitis viras G glycoprotein substituting for the env glycoprotein 2"8 .
  • These recombinant gene transfer vectors are produced by transient co-transfection of human embryonic kidney 293T cells using three different plasmids encoding the helper (packaging), gene transfer and envelope functions 2 ' 9"11 .
  • NSNG the cunent pseudotype of choice, is cytotoxic to the cells used to produce the recombinant HIN- based gene therapy vectors 1 . This prevents isolation and propagation of stable producer clones in which it is constitutively expressed, an essential step in the production of large quantities of standardized HIN-based vectors repetitively.
  • NSN-G pseudotyped vims is inactivated by the human complement system in the blood 17 .
  • NSN-G ' " Several attempts have been made to construct packaging cell lines with inducible expression of NSN-G ' " .
  • packaging cell lines have been shown to initially produce reasonable titers of lentiviral vectors, they do not retain their expression properties long-term, presumably due to negative selection of NSN-G expressing cells.
  • An alternative method is to use other non-toxic heterologous envelope gene.
  • envelope genes have been tried for pseudotyping human immunodeficiency vims type-1 (H ⁇ N-l)-based vectors 13 ' 21 ' 22 .
  • Gp64 is the major baculoviral envelope protein. It is a bonafide fusion protein as the expression of gp64 in insect cells is necessary and sufficient for a cell-cell fusion phenotype 23 ' 24 . Gp64 has been extensively studied to understand the mechanism of membrane fusion 23"28 . The present invention provides the use of gp64 for pseudotyping
  • HIN-1 based vectors By transient transfection of three plasmids (one containing gp64) into 293T cells, retroviral vectors that efficiently transduced various cell types were produced. More notably, gp64 expression was not cytotoxic to host cells. Thus, derivation of a permanent cell line constitutively expressing gp64 at high levels over many passages was successful. This novel cell line produced gp64 pseudotyped lentiviral vectors when transfected with helper and vector plasmids.
  • the present invention demonstrates that baculovims gp64 meets the requirements of an envelope glycoprotein for pseudotyping lentiviruses without the associated cytotoxic effects of NSN-G. Packaging cell lines based on gp64-pseudotyping can thus be easily created, making the production of large amounts of standardized vector necessary for efficient gene transfer in in vivo and ex vivo studies practical.
  • the plasmid for GP64 expression (pBsd-gp64) was transfected into 293T cells and expression of GP64 confirmed by Western blotting using anti-GP64 monoclonal antibodies (ACV5) (Fig. 1 A).
  • GP64-pseudotyped vims containing the gene for green fluorescence protein (GFP) was created using three-plasrnid transient transfection followed by purification. Electron microscopic analysis of the purified viral particles showed uniform virions ⁇ 100 nm in diameter (Fig. IB).
  • GP64-pseudotyped virus could transduce a variety of cell types (293T, HOS, HeLa, MDCK, BHK, NTH3T3) indicating a wide cell tropism (Fig 2A).
  • Fig. 2A Using the same amount of vims different numbers of GFP-positive cells were obtained in different cell types (Fig. 2A). Titers of the two vector preparations were assayed on several cell lines in parallel, using the expression of GFP in the target cell to detect successful fransduction.
  • GP64- and VSVG-pseudotyped vimses were prepared in parallel as described in below and titers for GP64-pseudotyped vimses on different cell lines were compared to that for VSVG pseudotyped vimses by two methods (Table 1). Titers obtained from GP64-pseudotyped vims are 15-50% higher than the VSVG- pseudotyped vims, depending on the cell type used and this difference is statistically significant, as determined by two-way ANOVA (with replication) (Table IB). The titers depend on the type of cells used with interaction between the cell type and the vector used (Table IB).
  • GP64 could also efficiently pseudotype HIV-2-based gene transfer vectors.
  • VSVG expression is cytotoxic to 293T cells (Yang et al. 1995, Ory et al, 1996).
  • GP64 expression was cytotoxic to 293T cells.
  • cells were transfected with a GP64 expression plasmid containing the Blasticidin resistance gene. Two days after transfection, the cells were split into selection medium and Blasticidin-resistant clones of cells were isolated.
  • KZ64 The Blasticidin-resistant cell clones were tested for GP64 expression by immunofluorescence and Western blotting, and one clone (called KZ64) was selected based on a high level constitutive expression of GP64.
  • KZ64 cells exhibit a steady high-level constitutive expression of GP64 up to 12 passages as tested by FACS analysis (Fig. 3A).
  • the GP64 gene could also be detected by genomic PCR (Fig. 3B). Relative comparison of GP64 mRNA in different passages of KZ64 cells by qRT-PCR exhibited a stable expression of GP64 gene (Fig. 3C).
  • GP64-pseudotyped vectors efficiently transduced various mammalian cell types. Vector particles were usually concentrated without apparent loss of titer. For any given target cell type, GP64-pseudotyped vector titers were modestly higher than
  • VSVG pseudotyped HIN vectors Lack of toxicity was shown by the generation of a cell line from 293T cells constitutively expressing high levels of GP64 up to 16 passages. High-titers of GP64-pseudotyped recombinant virus were simply and efficiently produced from this new cell line by transient transfection of the packaging and gene transfer plasmids alone. Incorporation of GP64 did not influence the size, shape or maturation of the HIN vector as far as we could detect. Thus the marked reduction of cytopathic effects of cells expressing GP64, compared to cells expressing NSNG, is a distinct advantage for the large-scale production of vectors.
  • Baculovims GP64 protein has been studied extensively as the protein essential for the entry of the baculovims nucleocapsid into insect cells. It is known that native baculovims produced in insect cells, and insect cells expressing GP64 fuse with mammalian cells (Hofmann et al., 1995, Boyce and Bucher, 1996, Plonsky et al.,
  • lentiviral vectors have several advantages over conventional oncoretro viruses: 1) Transduction of cells without requiring passage through the M phase of the cell cycle to traverse the nuclear membrane ( ⁇ aldini et al., 1996, Miyoshi et al., 1999). 2) A higher efficiency of transduction (Burns et al., 1993, Kafri et al., 1997, Kafri et al., 1999, Kumar et al., 2001) now enhanced further by using a central D ⁇ A flap, (Zennou et al., 2001).
  • lentiviral vectors can accommodate large, strong and complex genetic regulatory- elements conferring high-level expression and lineage-specificity (e.g. Kumar et al, 2001, Cui et al., 2002), critical to the success of many experimental and therapeutic gene-transfer strategies.
  • Recent modifications to the HIN sequences in the helper and vector genomes like the creation of self-inactivating (SLN) transfer vectors (Zufferey et al., 1998) and the removal of most of the HIV genes from the vector plasmid (Dull et al., 1998), are thought to get rid of the possibility of recombination and productive HIN infection (Dunbar, 2002).
  • SSN self-inactivating
  • GP64 When GP64 was expressed in human cell lines, it trafficked to the plasma membrane to incorporate into the budding envelope of a recombinant mammalian viras (HIN-1). This budded vims was able to fuse to native target cells of human origin, and integrate its engineered gene into that target cell for subsequent expression.
  • Moloney murine leukemia virus-based retroviral vector has been successfully pseudotyped by an insect retroviral envelope to infect insect cells (Teysset et al, 1998).
  • the present invention providse the first study where a protein from an insect viras has been used to pseudotype a HINl-based vector for infecting mammalian cells.
  • GP64 confers all the advantages of a heterologous envelope for pseudotyping gene therapy vectors without any associated cytotoxicity.
  • attempts to construct packaging cell lines constitutively expressing NSNG were of limited use (Oil et al., 1996, Kafri et al, 1999).
  • the production of a permanent cell line for GP64 demonstrates the ease of constructing packaging cell lines for HIN-based vectors, something that until now has been a major hurdle in their use for clinical gene therapy.
  • DMEM Dulbecco's modified Eagle's medium
  • Plasmid vector construction pHIN-rRES eGFP GT ⁇ V " (Agag, Apol, Aenv, Avi Avpr) (IRES, internal ribosome entry site from encephalomyocarditis viras), pHJN-PN (PN, packaging vector) and pME NSNG was provided by Dr. Richard E. Sutton, Baylor College of Medicine, Houston, TX (Sutton et al., 1998, Kumar et al., 2001).
  • the fransfer vector is based upon T-tropic isolate NL4-3, has large deletions in Gag, Pol, Env, Vif and Vpr; and carries LRES followed by the enhanced green fluorescence protein (GFP) gene (Clontech) in place of nef.
  • GFP enhanced green fluorescence protein
  • Baculovims AcMNPV gp64 gene was cut out from the insect expression vector ATH-11 (Kingsley et al., 1999) and sub-cloned into mammalian expression vector pCDNA 3.1 (Invitrogen, CA).
  • the blasticidin resistance gene from pCMV-Bsd (Invitrogen, CA) was subsequently sub-cloned into the above plasmid.
  • the resulting plasmid, pBsd-gp64 had both the gp64 and Blasticidin resistance genes with separate CMV promoters.
  • Pseudotyped HIV supematants were prepared as described earlier, without the addition of pcRev or butyrate (Sutton and Littman, 1996).
  • 293T cells maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin (complete DMEM), were passaged into 25 cm 2 culture flasks and then transiently transfected at 50-60% confluency with equal mass amounts of pHIN-PV, vector plasmid and either pME-VSVG or pBsd-gp64 using TransIT-LTl transfection reagent (Minis, WI).
  • Vims-containing supematants were collected approximately 60h later, centrifuged at 3000 x g for 10 min. at 25°C to remove cell debris, and used for infections.
  • Both the GP64- and VSV G-pseudotyped vimses were titered on various target cells (HOS, 293T, HeLa, COS7, CV1 , ⁇ TH3T3, MDCK or BHK) in two different ways.
  • Target cells grown in large flasks were lifted and seeded into 6-well plates ( ⁇ 5 x 10 5 cells per well). After 24 hrs, wells were infected with increasing dilutions of both viras preparations in complete DMEM.
  • the first method for titer determination was based upon the counting of GFP-positive foci using fluorescence microscopy as described earlier (Cashion et al, 1999), with modification. After 48-60h, foci of GFP-positive cells (defined as small clusters (2-8 cells) of GFP-positive cells apparently due to division of a transduced cell occurring between the times of infection and counting) were counted using a calibrated field of a digital cooled CCD camera (ORCA-ER, Hamamatsu) on a fluorescent microscope. The total number of positive foci per well was estimated by counting green foci in 9 distinct calibrated areas of the well randomly selected and knowing the total surface area of a well.
  • the viral titer was determined by multiplying the number of positive foci per well by the viral dilution.
  • the biological end-point titer was estimated from the three highest dilution wells that contained GFP positive foci.
  • viral titers were calculated from the percent GFP-positive cells in a well, obtained by FACS analysis, multiplied by the mean number of cells present at the time of infection in a parallel set of wells, to give the number of cells infected followed by calculation of infectious titer per ml.
  • FACS analysis cells were lifted with trypsin-EDTA, made into single cell suspensions by pipetting, and fixed with 4% parafonnaldehyde.
  • GFP expressing cells were analyzed with FACScan (Beckton Dickinson). Cells were counted for scatter characteristics, and the data was analyzed with WinList software (Verity Software House, ME) using uninfected cells as negative controls.
  • FACS analysis a qualitative method for titer estimation, paired percentage method, was used to compare the titers of different vims preparations. For this method, cells plated in 6-well plates were infected with different dilution of the respective unconcentrated or concentrated viras preparations so as to give 20-80% GFP positive cells.
  • the percent GFP-positive cells were estimated visually using fluorescence microscopy and multiplied by the mean number of cells present at the time of infection for each dilution followed by calculation of infectious titer per ml of the viras preparation.
  • the culture supernatant was centrifuged at 100, 000 X g for 1 hr at 4°C to pellet the viras and the viras pellet was resuspended in 1/100 of the volume in DMEM and used for infection.
  • CD34+ HPCs For infection of CD34+ HPCs, the virus-containing culture supernatant was ultracentrifuged as described above and the pellet suspended in either 1/100 of the volume or the same volume of LMDM (to correspond for unconcentrated supernatant).
  • CD34+ cells were typically transduced overnight with the GFP-containing viras, using 3 X 10 5 cells in 500 ⁇ l volume then infected with 100 ⁇ l unconcentrated or concentrated vims suspension in the presence of 4 ⁇ g of polybrene per ml, either in the presence or absence of the cytokine cocktail as described above.
  • Initial marker analysis was carried out 48 to 96 hr later. GFP expressing cells were analyzed with FACScan (Beckton Dickinson). Cells were counted for scatter characteristics, and the data was analyzed with CELLQUEST software (Beckton Dickinson). Confrols were uninfected cells.
  • Recombinant viras was concentrated from the culture supernatant by ulfracentrifugation as described above and further purified by another cycle of ulfracentrifugation on a 50% sucrose bed. Such purified viras was tested for integrity and presence of GP64 by electron microscopy (EM), Western blotting and immunofluorescence. For EM purified virus was applied to formavar coated copper grids, negatively stained in 2% phosphotungstic acid (PTA) solution for 1 min, and then visualized using transmission electron microscopy (100CX, JEOL, Japan).
  • EM electron microscopy
  • PTA 2% phosphotungstic acid
  • HOS cells were infected with GP64 pseudotyped virus in cold and in the presence of lOmM sodium azide (a potent inhibitor of endocytosis, Schwartz, et al., 1982), followed by labeling with anti-GP64 monoclonal (ACV1, that binds to native conformation of GP64) as primary and Cy3 labeled goat anti-mouse IgG polyclonal antibodies (Amersham Pharmacia, NJ) as secondary.
  • the labeled cells were visualized using an inverted fluorescence microscope (Zeiss Axiovert 25) and ⁇ imaged with a 16-bit cooled CCD camera (ORCA-ER, Hamamatsu, Japan) using Metamorph imaging software (Universal Imaging, PA).
  • Western blot analysis was carried out by separating purified viras on SDS-PAGE, followed by Western blot analysis using anti-GP64 monoclonal antibodies.
  • Genomic DNA was prepared from transduced cells using Wizard genomic DNA purification kit (Promega, WI). The genomic DNA was subjected to Alu PCR as described earlier (Butler et al., 2001) to test for integration of the vector DNA.
  • 293T cells were transfected with pBsd-gp64 plasmid using TransIT LT1 transfection reagent. 48 hr post-transfection the cells were lifted and plated into selection medium (complete DMEM containing 1 O ⁇ g per ml Blasticidin (Life Tech., NY)). The resistant colonies were propagated till 10-14 days. Individual cell colonies were grown and tested for GP64 expression by immunofluorescence, FACS analysis using anti-GP64 monoclonal antibodies, Western blotting and PCR. Cell lines with high expression level were selected and passaged in complete medium containing Blasticidin.
  • GP64-expressing cell line Each passage of the selected GP64-expressing cell line (called KZ64) was tested for the level of GP64 expression by FACS analysis, immunofluorescence and quantitative RT-PCR (qRT-PCR) for comparing level of GP64 rnRNA in different passages of KZ64 cells.
  • qRT-PCR quantitative RT-PCR
  • total RNA from ⁇ 2 X 10 6 cells was extracted using Trizol reagent (Life Tech., MD) and 5 ⁇ g of total RNA from each preparation was used for each test using QuantiTech S YBR green RT-PCR kit (Qiagen, CA) and primers 5'-ATGCGGCCGCA-TGGTAAGCGCTATTGTT-3' and 5'-
  • GATCCTCGAGTTAATATTGTCTATTAC-GG-3' The qRT-PCR were carried on the ABI 7700 system (Applied Biosystems, CA) with conditions of 94°C, 58°C and 72°C each for 45 sec.
  • packaging and gene transfer plasmids were transiently transfected in the KZ64 cells and the virus- containing culture supernatant harvested 60h post-transfection. This supernatant was tested for viras titers as described above.
  • VSV-G pseudotyped lentiviral vector particles produced in human cells are inactivated by human serum. Mol Ther. 2, 218-222 (2000).
  • Table 1 A, Transduction of several cell types by GP64-pseudotyped HIN-1. Paired dishes of the appropriate cells were grown at the same time and then freated with equal volumes of either GP-64 vector or NSV-G vector at differing dilutions. After 48-60 hrs, titers were determined by three methods as discussed in the text. In each case, dilutions were tested in triplicate and the mean and SD is given.
  • B Statistical analysis of variance between infections of cell lines infected with either GP64- or VSVG- pseudotyped virus. Titers obtained by each method were analyzed by two-way ANOVA with replication and the results of the analysis for each tifration type are given.
  • Table 3 High titer GP64-pseudotyped viras could be created from KZ64 cells. Each passage of KZ64 cells was fransfected with transfer and packaging vectors alone, and supernatant harvested 60hr post-transfection. Unconcentrated viras from each passage indicated was used to fransduce MDCK cells, and titers per ml were calculated by the paired percentage method as described in the text. Each observation is average of two independent experiments. o

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)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention a trait à de vecteurs pseudotypés avec la GP-64 pouvant être utilisés pour le transfert génétique ainsi que pour la production à grande échelle de virus à titre élevé destinés à être utilisés dans des application cliniques et commerciales.
PCT/US2003/035728 2002-11-12 2003-11-10 Vecteurs pseudotypes avec la gp64 et leurs utilisations Ceased WO2004044157A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003291426A AU2003291426A1 (en) 2002-11-12 2003-11-10 Gp64-pseudotyped vectors and uses thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US42585302P 2002-11-12 2002-11-12
US60/425,853 2002-11-12

Publications (2)

Publication Number Publication Date
WO2004044157A2 true WO2004044157A2 (fr) 2004-05-27
WO2004044157A3 WO2004044157A3 (fr) 2004-09-30

Family

ID=32313059

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2003/035728 Ceased WO2004044157A2 (fr) 2002-11-12 2003-11-10 Vecteurs pseudotypes avec la gp64 et leurs utilisations

Country Status (2)

Country Link
AU (1) AU2003291426A1 (fr)
WO (1) WO2004044157A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1856250A4 (fr) * 2005-02-15 2009-03-11 Univ North Carolina Nouveaux vaccins à base de virus vivant
US7862829B2 (en) 2004-07-09 2011-01-04 University Of North Carolina At Chapel Hill Viral adjuvants
ES2411804A1 (es) * 2011-12-02 2013-07-08 Universidad De Zaragoza Compuestos inhibidores de la agregación del péptido beta amiloide.

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0432216A1 (fr) * 1988-09-01 1991-06-19 Whitehead Institute For Biomedical Research Retrovirus de recombinaison a gammes d'hotes amphotropiques et ecotropiques
US6013516A (en) * 1995-10-06 2000-01-11 The Salk Institute For Biological Studies Vector and method of use for nucleic acid delivery to non-dividing cells
KR100556864B1 (ko) * 1997-05-13 2006-03-10 더 유니버시티 오브 노쓰 캐롤라이나 엣 채플 힐 렌티바이러스-기원 유전자 전달 벡터
US6863884B2 (en) * 2002-05-01 2005-03-08 Cell Genesys, Inc. Pseudotyped retroviral vectors

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7862829B2 (en) 2004-07-09 2011-01-04 University Of North Carolina At Chapel Hill Viral adjuvants
US8821897B2 (en) 2004-07-09 2014-09-02 The University Of North Carolina At Chapel Hill Viral adjuvants
EP1856250A4 (fr) * 2005-02-15 2009-03-11 Univ North Carolina Nouveaux vaccins à base de virus vivant
US8486420B2 (en) 2005-02-15 2013-07-16 The University Of North Carolina At Chapel Hill Live virus vaccines
ES2411804A1 (es) * 2011-12-02 2013-07-08 Universidad De Zaragoza Compuestos inhibidores de la agregación del péptido beta amiloide.

Also Published As

Publication number Publication date
AU2003291426A8 (en) 2004-06-03
AU2003291426A1 (en) 2004-06-03
WO2004044157A3 (fr) 2004-09-30

Similar Documents

Publication Publication Date Title
CA2328404C (fr) Nouvelles cellules lentivirales d'encapsidation
Kumar et al. Large-scale production of pseudotyped lentiviral vectors using baculovirus GP64
CN100425702C (zh) 慢病毒三链体dna、及含有慢病毒三链体dna的载体和重组细胞
Olsen Gene transfer vectors derived from equine infectious anemia virus
JP4979851B2 (ja) 高いタイターで安全な組換えレンチウイルスベクターの作製方法
US6323031B1 (en) Lentiviral vectors derived from SIVagm, methods for their preparation and their use for gene transfer into mammalian cells
AU686181B2 (en) Generation, concentration and efficient transfer of VSV-G pseudotyped retroviral vectors
Kafri Gene delivery by lentivirus vectors: an overview
JP2021502799A (ja) レトロウイルスベクター
US6863884B2 (en) Pseudotyped retroviral vectors
US20080124308A1 (en) Gene Therapy of Solid Tumours by Means of Retroviral Vectors Pseudotyped With Arenavirus Glycoproteins
WO2000015819A9 (fr) Lignees de cellules d'encapsidation pour particules de vecteur retroviral derive du vih
CN112714790A (zh) 用于生产vsv-g假型逆转录病毒载体颗粒或其病毒颗粒的ldlr阴性包装细胞系
US9840720B2 (en) Materials and methods relating to packaging cell lines
WO2004044157A2 (fr) Vecteurs pseudotypes avec la gp64 et leurs utilisations
DE69829174T2 (de) Exprimierung eines modifiziertem "foamy virus envelope protein" (hüllenprotein)
Marusich et al. Spleen necrosis virus-based vector delivery of anti-HIV-1 genes potently protects human hematopoietic cells from HIV-1 infection
Sauter et al. A highly efficient gene delivery system derived from feline immunodeficiency virus (FIV)
JP2000502261A (ja) 毒性タンパク質発現ウイルスおよびプロデューサー細胞株
Kafri Gene Delivery by Lentivirus Vectors
Brady et al. High-Titer Human Immunodeficiency Virus
JP2008512110A (ja) Ｃａｅｖ系ベクターシステム
HK1056576A1 (zh) 含有血凝素活性膜蛋白质的假型逆转录病毒载体
HK1056576B (en) Pseudotype retroviral vectors containing membrane proteins having hemagglutinin activity

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP