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WO1996009399A2 - Adenovirus chimerique permettant l'apport de genes - Google Patents

Adenovirus chimerique permettant l'apport de genes Download PDF

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Publication number
WO1996009399A2
WO1996009399A2 PCT/US1995/011537 US9511537W WO9609399A2 WO 1996009399 A2 WO1996009399 A2 WO 1996009399A2 US 9511537 W US9511537 W US 9511537W WO 9609399 A2 WO9609399 A2 WO 9609399A2
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dna
csf
adenovirus
chimeric adenovirus
interest
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PCT/US1995/011537
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English (en)
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WO1996009399A3 (fr
Inventor
Srinivas Shankara
Varavani Dwarki
Tarlochan Nijjar
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Somatix Therapy Corporation
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Priority to AU35511/95A priority Critical patent/AU3551195A/en
Publication of WO1996009399A2 publication Critical patent/WO1996009399A2/fr
Publication of WO1996009399A3 publication Critical patent/WO1996009399A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention is directed to novel adenovirus vectors useful for the delivery of cloned genetic material to target cells.
  • the chimeric adenovirus vectors comprise genetic material of interest which is flanked by adenoviral sequences, and may optionally comprise a suitable eucaryotic promoter to facilitate the expression of the genetic material of interest.
  • the chimeric adenovirus are produced by a process involving a recombinant adenovirus vector which is used in conjunction with replication deficient helper adenovirus genomes to generate recombinantly produced chimeric adenovirus particles comprising the genetic material of interest.
  • the resulting chimeric adenovirus may be used to infect target cells which subsequently express the cloned genetic material.
  • One class of novel chimeric adenovirus does not contain a selectable marker which obviates the need for a selection step after the genetic material of interest has been introduced into the target cells.
  • Mammalian cells may be transduced by any of a variety of well known processes. Techniques such as calcium phosphate precipitation and DEAE-dextran mediated transfection are widely used in the art. More recently, other techniques for delivery of exogenous DNA into cells such as electroporation or the use of liposomes have gained increased acceptance. Perhaps the most elegant methods of introducing recombinant nucleic acid into cells is viral mediated cell transduction. Recombinant retroviruses have been widely used in gene transfer experiments (see generally, Mulligan, R.C., Chapter 8, In: Experimental Manipulation of Gene Expression. Academic Press, pp. 155-173 (1983); Coffin, J. , In: RNA Tumor Viruses.
  • eucaryotic viruses which have been used as vectors to transduce mammalian cells include adenovirus, papilloma virus, herpes virus, adeno-associated virus, rabies virus, and the like (See generally, Sambrook et al . , Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, Vol. 3:16.1-16.89 (1989) .
  • Adenovirus have proved to be of particular interest because of several features of adenoviral biology (See generally, Berkner, K.L. (1992) Curr. Top. Microbiol . Immunol.
  • viral concentration may be an important factor in achieving high efficiency transduction of mammalian cells.
  • Adenovirus by virtue of their life-style, generally allow growth conditions which result in production of higher titer stocks then other mammalian virus.
  • adenovirus capsids are not enveloped. Because of this fact, adenovirus particles are quite stable, and may retain infectivity after any of a variety of laboratory procedures. Procedures of particular interest include methods of concentrating infective virus, e.g., CsCl centrifugation, or methods that allow virus to be stored for relatively long periods while retaining substantial infectivity.
  • adenoviral vectors are generally better suited than other viral vectors for the transduction of postmitotic, slowly proliferating, or nonreplicating cells.
  • replication deficient human, or murine adenovirus are available for the construction of recombinant virus particles that express a gene of interest.
  • recombinant adenovirus can be engineered to utilize viral coat proteins which normally facilitate the normal infection of human cells or cells of other species, rather then rely on the viral coats of a less specific, or amphotropic, nature. This species specificity appears to result in more efficient infection kinetics than can generally be obtained by virus with less specific infectivity.
  • An additional advantage of using adenovirus for gene delivery is that the genetic material transduced (to be expressed) into the host cell is DNA. Thus, expression of the transduced gene does not need to be preceded by reverse transcription. This is particularly advantageous where the intended recipient is undergoing treatment for the suppression of retroviral disease (i.e., AZT treatment to inhibit reverse transcriptase activity) , such as treatment for acquired immunodeficiency syndrome (AIDS) .
  • retroviral disease i.e., AZT treatment to inhibit reverse transcriptase activity
  • Ad.RSV replication deficient adenovirus vector
  • Ad.RSV beta gal which expresses the bacterial ⁇ - galactosidase gene
  • An ideal replication deficient adenovirus for the delivery of genetic material of interest would comprise a variety of structural and functional elements. It would readily infect target cells of interest; it would place the gene of interest under the control of a well-characterized eucaryotic promoter element; it would create a gene structure flanking the gene of interest which would provide properly spaced and oriented genetic elements to allow optimum translational efficiency and mRNA stability; and it would produce high titer and substantially helper-free stocks of the recombinant adenovirus . 3.
  • the present invention relates to replication deficient chimeric adenovirus that allow for the rapid insertion and expression of deoxyribonucleic acid (DNA) of interest into mammalian cells, either .in vitro or in vivo.
  • the DNA of interest can optionally comprise a gene, or fraction thereof, oriented to express either a polypeptide or protein of interest, or a "sense” or “antisense” nucleic acid of structural or regulatory importance.
  • the DNA of interest will be placed in an expression cassette that contains a eucaryotic promoter and/or enhancer region; nucleotide sequence corresponding to a retroviral Psi- packaging site; and a substantially noncoding 3' DNA which facilitates the stability, polyadenlyation, or splicing of the transcript.
  • the chimeric adenovirus are thus useful for both the transduction of mammalian cells, and the expression of DNA of interest to produce regulatory factors or proteins.
  • the regulatory factors or proteins may optionally be produced in culture or otherwise such that they can be subsequently purified and used for therapeutic, medicinal or diagnostic purposes.
  • the chimeric adenovirus are particularly useful for gene therapy, replacement, or insertion because of the high infectivity inherent in adenovirus biology; the high viral concentrations which may be produced during the culture and subsequent concentration of the chimeric adenovirus; and the relatively long storage life of the chimeric particles.
  • Either murine, or human adenovirus of serotypes A, B, or C may be used in the present invention.
  • type C adenovirus used in the present invention which retain infectivity while generally being considered nononcogenic.
  • Figure 1 is a schematic representation of the method of producing chimeric adenovirus via the recombination of cotransfected plasmids.
  • One plasmid, pXCJL-GMCSF contains a "cassette” comprising the gene encoding the cytokine granulocyte/macrophage colony stimulating factor (GMCSF) situated such that it is transcribed, processed, and translated under the regulatory control of flanking viral sequences.
  • the second plasmid, pJM17 comprises a replication and packaging deficient adenovirus "helper" genome.
  • the two plasmids must recombine to produce a packagable genome, and thus substantially all of the resulting virus comprise the chimeric adenovirus desired (Recombinant El-deleted GM-CSF adenovirus) .
  • Figure 2 presents a schematic diagram and partial restriction map of pJM17.
  • Figures 3A-E disclose the DNA sequence of pXJCL-hGM-CSF (SEQ. I.D. NO. 1) , the plasmid used to construct the human GM- CSF expression cassette, and in the recombinatory insertion of the GM-CSF expression cassette into the replication deficient genome contained in pJM17.
  • the sequence of the murine GM-CSF is disclosed in foreign patent EP177568B1, herein incorporated by reference.
  • Figures 4A and 4B show the transient expression of human GM-CSF after one month old Balb/c mice were intramuscularly injected with either 10 9 or 10 8 pfu of Ad.hGM-CSF respectively. Serum samples were taken up to twenty one days after infection and GM-CSF levels were assayed by ELISA. Individual mice are represented by number and correspond to the indicated bars on the graphs.
  • Figure 5 shows the expression of human GM-CSF (as quantified by ELISA) after Ad.hGM-CSF injection and reinjection into adult Balb/C mice.
  • Ad.hGM-CSF as quantified by ELISA
  • mice 103 and 105 or I.M. mice 201, 203, and 205) . All mice were reinjected (I.M.) with 10 9 pfu of Ad.hGM-CSF at day 31.
  • Figure 6 shows the expression of human GM-CSF (as quantified by ELISA) after Ad.hGM-CSF injection and reinjection into adult SCID mice. SCID mice were injected (I.V.) with 10 ⁇ pfu of Ad.hGM-CSF, and GM-CSF blood serum levels were subsequently monitored. All mice were reinjected (I.M.) with 10 9 pfu of Ad.hGM-CSF at day 31, and monitored for GM-CSF expression through day 71.
  • the present invention provides for chimeric adenovirus which are useful for transducing mammalian cells with DNA of interest, as well as methods of producing and using the chimeric adenovirus.
  • Previous recombinant adenovirus expression vectors have specifically taught the expression of the genetic material of interest under the control of endogenous adenoviral promoters, or have suggested that the DNA of interest be inserted into recombinant adenovirus under the control of an RSV promoter already present in the vector Ad.RSV.
  • the particular DNA of interest is first constructed as an expression cassette which comprises a gene, or portion thereof, of interest that is flanked by sequences of viral origin which are spatially organized to optimize the expression of the DNA of interest.
  • expression refers to the transcription of the DNA of interest, and the splicing, processing, stability, and, optionally, translation of the corresponding mRNA transcript.
  • the recombinant DNA cassette is subsequently recombined into a replication deficient helper adenovirus to produce the infective chimeric adenovirus of interest.
  • This method best ensures the maximal expression of the DNA of interest and additionally provides a method that is generally applicable to the relatively facile production of chimeric adenovirus which express a wide variety of DNAs.
  • the particular advantage of using an expression cassette stems from the fact that the recombinant Ad.RSV vector is rather large (over 36kb) . This large size makes plasmids which contain the Ad.RSV genome somewhat difficult to engineer as the number of unique (and hence useful) restriction sites tends to diminish as the amount of DNA sequence increases.
  • the utilization of a smaller plasmid to construct the expression cassette better enables a wide variety of genetic engineering techniques which may allow the fine tuning of the expression of the DNA of interest (see generally, Sambrook et al. (1989) Molecular Cloning Vols.
  • replication defective adenovirus refers to a adenovirus that are incapable of self replication within host cells that, absent infection or transfection, do not ' express at least one adenovirus gene or gene product .
  • transcriptional promoters and enhancers may be used in the expression cassette, including, but not limited to, the herpes simplex thymidine kinase promoter, cytomegalovirus promoter/enhancer, SV40 promoters, and retroviral long terminal repeat (LTR) promoter/enhancers.
  • LTR retroviral long terminal repeat
  • retroviral promoters particularly the Moloney murine leukemia virus (MLV) LTR promoter and the human immunodeficiency virus (HIV) LTR.
  • recombinant DNA techniques have been used to construct expression cassettes in plasmid pXCJl.1 which comprise genes coding for the murine or human forms of granulocyte macrophage colony stimulating factor (GM-CSF) , which have been placed under the transcriptional control of the Moloney murine leukemia virus (MLV) long terminal repeat (LTR) .
  • GM-CSF granulocyte macrophage colony stimulating factor
  • MLV Moloney murine leukemia virus
  • LTR long terminal repeat
  • an SV40 poly-adenylation sequence flanks the 3' end of the GM-CSF gene.
  • the transcript produced by either GM-CSF expression cassette is transcribed using the MLV LTR promoter and enhancer sequences, poly-adenylated using an SV40 poly-adenylation sequence, spliced using the MLV splice donor and splice acceptor sequences, and the mRNA is presumably translated using the endogenous MLV translation initiation sequence of the MLV gag gene.
  • the DNA expression cassette By engineering the DNA expression cassette such that the resulting transcript surrounds the coding region with naturally occurring viral control sequences, near optimum mRNA stability is obtained.
  • DNA expression cassette or simply “expression cassette” both refer to a DNA molecule comprising a eucaryotic promoter and/or enhancer region, a DNA of interest to be transcribed by the promoter, and a substantially noncoding 3' region of DNA that facilitates the stability, polyadenlyation, or splicing of the transcript.
  • the GM-CSF expression cassette is inserted into a replication defective helper adenovirus via homologous recombination after two circular plasmids (one containing the GM-CSF expression cassette and the other containing the replication defective adenovirus genome) are co-transfected into the appropriate cell line (see Fig. 1) .
  • the resulting chimeric adenovirus expresses a mammalian gene (human or murine GM-CSF) that is expressed under the transcriptional and translational control of MLV and SV40 control sequences.
  • the chimeric adenovirus can subsequently be purified by any of a number of well established techniques including, but not limited to, plaque purification, purification by limiting dilution, or the like. Purified chimeric adenovirus can then be propagated to relatively high titers by infection of appropriate host cells, for example 293 cells (human kidney epithelial cells which 5 constitutively produce adenovirus E1A) .
  • chimeric adenovirus infections will generally produce highly concentrated viral preparations, one may elect to further concentrate and purify the chimeric adenovirus to achieve titers of about l-5xl0 u plaque forming units (pfu) /ml) by
  • Ad.mGM-CSF murine GM-CSF
  • Ad.hGM-CSF human GM-CSF
  • Ad.hGM-CSF and Ad.mGM-CSF make both ideally suited for applications where GM-CSF expression by any of a broad range of target cells may be desired.
  • Ad.hGM-CSF or Ad.mGM-CSF to transduce primary tumor cells. It has previously been established that vaccinations with tumor cells engineered to secrete GM-CSF can stimulate anti-tumor immunity in mice (Dranoff et al . (1993) Proc. Natl. Acad. Sci. U.S.A.
  • Ad.hGM-CSF has been used to transduce primary human melanoma, renal cell carcinoma, and colon carcinoma cells which subsequently produced microgram quantities (about l-5 ⁇ g/10 s cells) of human GM-CSF (see Tables 2a-d) . Additionally, Ad.mGM-CSF has been used to infect and transduce
  • Ad.hGM-CSF was also injected into Balb/c or SCID mice at various anatomical locations, and in vivo expression of GM-CSF
  • Ad.hGM-CSF has been deposited (received at the ATCC on September 23, 1994) at the American Type Culture Collection, Rockville, MD, under the accession number under the terms of the Budapest Treaty. Applicants further agree to make this deposit available, without restriction to responsible third parties upon the granting of a patent from this application in the United States, and comply with existing laws and regulations pertaining thereto, without limitation, except as to third parties adherence to applicant rights as prescribed by the claims of a patent issuing from this application.
  • the present invention provides a method of producing chimeric adenovirus comprising the recombinatory insertion of a DNA expression cassette contained in a circular plasmid into a replication deficient helper adenovirus genome contained in a circular plasmid to produce a chimeric adenovirus capable of transducing mammalian cells.
  • the use of two circular plasmid is an important feature of the method of the present invention, since there is no need to linearize the adenoviral helper genome prior to cotransfection.
  • the chimeric adenovirus of the present invention exhibit very high infectivity and thus high levels of cellular transduction and expression of a DNA of interest.
  • modified forms of the GM-CSF genes may be utilized which have been altered by deletion or insertion, or to optimize codon usage for the specific target cells intended.
  • DNA expression cassettes may also be constructed which allow the subsequent production of chimeric adenovirus which are capable of transducing any of a number of heterologous mammalian genes (i.e., DNAs of interest, subject to the restriction that the net size of the insert is less the about 9 kb in length) .
  • heterologous genes of particular interest include, but are not limited to, nerve growth factor (NGF) , tyrosine hydroxylase (TH) , ciliary neurotropic factor (CNTF) , brain-derived neurotropic factor (BDNF) , factors VIII and IX, tissue plasminogen activator (tPA) , interleukins 1-2 and 4-6, tumor necrosis factor- ⁇ (TNF- ⁇ ) , or ⁇ interferons, and erythropoietin.
  • NNF nerve growth factor
  • TH tyrosine hydroxylase
  • CNTF ciliary neurotropic factor
  • BDNF brain-derived neurotropic factor
  • tPA tissue plasminogen activator
  • interleukins 1-2 and 4-6 interleukins 1-2 and 4-6
  • TNF- ⁇ tumor necrosis factor- ⁇
  • ⁇ interferons erythropoietin.
  • Chimeric adenovirus that express any of the above genes, or portions thereof, may be particularly useful for
  • chimeric adenovirus containing the genes for these factors may also be used to generate transient expression of the factors in vivo as required to therapeutically treat medical crisis.
  • an infusion of chimeric adenovirus containing a tPA expression cassette would provide transient expression of tPA during the critical period following a heart-attack or stroke.
  • chimeric adenovirus may be used to deliver genes into a variety of cell types to correct genetic defects associated with diseases including but not limited to 5-thalassemia, phenylketonuria, sickle-cell anemia, cystic fibrosis, or adenosine deaminase deficiency.
  • the chimeric adenovirus of the present invention may be used to transduce mammalian cells either in vitro or in vivo. Where transduction in vitro is contemplated, cells may be infected at multiplicities of infection (moi's) of between about 1:1 to about 5000:1, and generally in the range of about 100:1 to about 2,500:1. Moi's of up to about 1000:1 have produced good expression of the DNA of interest without evidence of serious cellular toxicity effects, and moi's of about 200:1 have resulted in no toxicity. Using similar methodologies, chimeric adenovirus may be used to infect resected primary tissue or cells which may subsequently be reintroduced into the body of an individual by established surgical or medical procedures.
  • moi's multiplicities of infection
  • chimeric adenovirus capable of transducing and expressing the DNA of interest may be introduced in vivo by any of a number of established methods.
  • chimeric adenovirus may be administered by inhalation.
  • chimeric adenovirus suspensions may also administered by intravenous (I.V.) , intraperitoneal (I.P.), or intramuscular (I.M.) injection.
  • the chimeric adenovirus may also be injected directly into tumors.
  • a chimeric adenovirus which encodes a bacterial lacZ gene was injected into B16 melanoma tumors in C57 mice.
  • adenovirus mediated transduction and in vivo expression of / S-galactosidase was observed in the tumors.
  • Other in vivo studies have established that a single bolus of as much as about 10 9 pfu (in lOO ⁇ l total volume) of Ad.hGM-CSF can be injected (I.V. or I.M.) into mice without apparent toxicity effects (see Fig. 4A) .
  • Possible cell types or tissues that may serve as targets for chimeric adenovirus gene delivery include, but are not limited to, hepatocytes, fibroblasts, endothelial cells, bone marrow stem cells, lymphocytes, neural tissue, astrocytes, alveolar tissue, and granulocytes.
  • An additional embodiment of the present invention is chimeric adenovirus containing expression cassettes which further comprise a specific retroviral Psi-packaging sequence. More particularly, a Psi-packaging sequence which corresponds to that recognized and used by any of a number of ecotropic and amphotropic Moloney murine leukemia virus packaging cell lines including, but not limited to, PA317 or PsiCRIP.
  • the chimeric adenovirus may be used to transiently infect MLV packaging cell lines and produce amphotropic or ecotropic retrovirus which package RNA genomes transcribed by the expression cassette of the chimeric adenovirus. Infection of the appropriate cells by the resulting retrovirally packaged chimeric adenovirus transcripts will result in the integration and stable expression of the DNA of interest contained in the expression cassette of the chimeric adenovirus.
  • the chimeric adenovirus described above provide the user with increased versatility relative to previously disclosed retroviral or adenoviral transduction vectors. This is because a single chimeric adenovirus allows the user to choose between the increased storage life, infectivity, and transient expression inherent in the high titer chimeric adenovirus system, or the stable integration and expression inherent in the MLV packaging system. Alternatively, an optimal mixture of the two delivery systems may be preferred.
  • the present invention also provides for replication defective chimeric adenovirus which contain an expression cassette which further comprises nucleotide sequence corresponding to a MLV Psi- packaging site.
  • An additional embodiment of the present invention is chimeric adenovirus which place the expression of genes whose products are toxic to the cell under the strict control of a trans-activated promoter, such as an HIV LTR promoter.
  • a trans-activated promoter such as an HIV LTR promoter.
  • Toxic genes which may be employed in these vectors include, but are not limited to, sequence coding for diphtheria toxin A chain, polio virus protein 2A, and the like (or modified forms thereof) . Since the HIV promoter generally requires virally encoded trans- activators, chimeric adenovirus will generally only express the toxic products (hence killing the cells) in HIV infected cells.
  • chimeric adenovirus since the expression of genes contained in chimeric adenovirus is not dependent on cell division or proliferation (unlike retrovirally expressed genes) , the above chimeric adenovirus may find utility in targeting and killing non-replicating or quiescent HIV- infected cells.
  • PXCJL The starting plasmid, designated PXCJL1, was constructed 5 from a modified Ad5 adenovirus genome cloned into pBR322. A deletion was made from the map units 1.3 to 9.3, and a multiple cloning site was inserted at the unique Xbal site. This construct was obtained from Dr. Frank Graham of McMaster University (McGrory, W.J. et al . , Virology 163: 614-617, o 1988) -
  • MFGs is an unpublished three nucleotide modification of the MFG vector, as
  • MFG-GM-CSF represented by MFG-GM-CSF (Dranoff, et al . , Proc. Natl. Acad. Sci. 90:3539-3543, 1993; the modification has no effect on expression levels or transduction efficiencies) .
  • MFGs-GM-CSF DNA was first digested to completion with Hindlll and BamHI and the ends were blunt-ended with the Klenow fragment. The
  • the purified GM- CSF cDNA (Fragment 1) was blunt-end ligated to the linearized PXCJL1 with T4 ligase to generate the intermediate plasmid PXCJL GM-CSF (I) .
  • Xbal and BamHI sites were regenerated in the intermediate plasmid only if the insert was in the correct orientation, as determined by restriction endonuclease (EcoRI and BamHI) analysis.
  • SV40 polyadenylation sequence was generated by polymerase chain reaction (PCR) using the pRC/CMV vector as the DNA template.
  • the PCR primers were designed as follows: the sense primer containing the BamHI site- GAG GAT CCT ATC GCC TTC TTG ACG and the antisense primer containing the Sail site-
  • PCR conditions were 95°C for 1 min., 55°C for 2 min. , and 72°for 3 minutes, for 35 cycles.
  • the PCR product was cloned into a TA plasmid and sequenced.
  • the product with the correct SV40 poly(A) sequence was digested with BamHI and Sail and the 216 bp SV40 poly(A) sequence was ligated to PXCJL GM-CSF(I) (Fragment 3) with T4 ligase.
  • the resulting cDNA expression plasmid, PXCJL, GM-CSF contains the entire GM-CSF cassette, including the 5' MLV LTR, Psi-packaging and splicing sequences, the GM-CSF cDNA, and the SV40 poly (A) sequences, flanked by adenovirus sequences. Both murine and human GM-CSF cDNA were subcloned into PXCJL1 following the same strategy.
  • PXCJL-GMCSF plasmid by the calcium phosphate method following the standard transfection protocol. 36 hours after transfection, cells were overlaid with 0.8% Noble agar containing DMEM with 10% heat inactivated fetal calf serum.
  • Plaques visible by 8 days after transfection were picked and resuspended in 1 ml of medium and freeze-thawed three times to release the virus. These supernatants were used as viral lysates in subsequent experiments. 0.2 ml of the viral supernatant from each individual plaque was added to the 1 ml of medium and used to infect confluent monolayers of 293 cell in a 6-well plate for four hours. After 24 hours, the cells began to show complete cytopathic effects.
  • the colonies were harvested, and the medium was analyzed for GM-CSF secretion.
  • the cells were lysed by three rounds of freeze-thaw, and the medium was used to infect NIH 3T3 cells in a 6-well plate. 80% confluent monolayers of NIH 3T3 cells in a 6-well plate were infected with 0.1 ml of crude virus stock in 1 ml of medium for four hours. 24 hours after infection fresh growth medium was added, and the GM-CSF secreted for the next 24 hours was analyzed by ELISA.
  • the values for GM-CSF produced by Ad/human GM-CSF and Ad/mouse GM- CSF-transduced NIH 3T3 cells ranged from 300-400ng in 24 hours.
  • Confluent monolayers of 293 cells in 100mm dishes plated on day 1 were infected in 5 ml of medium on day 2 with 0.1 ml of viral supernatant obtained by resuspending virus containing agar block, as described above. After 1 hour of infecting at 37°C, the virus-containing medium was removed and overlaid with the agar-containing medium that had been prepared earlier. The cells were incubated at 37°C for 4-5 days and well isolated plaques were picked and analyzed for the ability to transduce NIH 3T3 cells with GM-CSF, as described earlier. O 96/09399 PCMJS95/11537
  • the virus band was collected and repurified by CsCl banding.
  • the purified virus was then dialyzed against lOmM Tris/l mM MgCl 2 , pH 7.4, and stored in 10% glycerol at -70°C.
  • NIH 3T3 cells were infected with purified virus at different multiplicities of infection (moi) for four hours, supernatants from 24-48 hours post-infection were collected and GM-CSF secretion was measured by ELISA. Results are shown in Table 1.
  • Tables 2a-d Expression of GM-CSF ( ⁇ g/lxlO* cells/24 hour) in Ad.hGM-CSF transduced primary tumor cells.
  • Ad.hGM-CSF could also mediate transient expression of human GM-CSF in adult mice, and whether or not the route of injection substantially affected expression
  • four month old Balb/C mice were injected with 10 8 pfu of Ad.hGM-CSF either intravenously (I.V.) or intramuscularly (I.M.) .
  • Serum samples were drawn at 3, 7, 14, and 31 days after injection and assayed for GM-CSF levels by ELISA. Serum levels of GM- CSF were generally lower than those observed in one month old mice, peaked between three to seven days after injection, and were undetectable fourteen days after infection.
  • mice Thirty one days after the initial injection the mice were reinjected (I.M.) with 10 9 pfu of Ad.hGM-CSF and serum samples were drawn and analyzed for GM-CSF at 2, 4, and 9 days after reinjection. After reinjection, serum levels of GM-CSF peaked after two days and were undetectable after four days. The mode of primary injection apparently made little difference (see Fig. 5) . 6.9.
  • MOLECULE TYPE DNA (genomic)
  • CAGCCCTCAG CAGTTTCTAG AGAACCATCA GATGTTTCCA GGGTGCCCCA AGGACCTGAA 1200
  • TTCTGCTCCC CGAGCTCAAT AAAAGAGCCC ACAACCCCTC ACTCGGGGCG CCAGTCCTCC 1320
  • TTCTCTAGGC GCCCCCATAT GGCCATATGA GATCTTATAT GGGGCACCCC CGCCCCTTGT 2400
  • GATTGTGACT GACTTTGCTT TCCTGAGCCC GCTTGCAAGC AGTGCAGCTT CCCGTTCATC 4020 CGCCCGCGAT GACAAGTTGA CGGCTCTTTT GGCACAATTG GATTCTTTGA CCCGGGAACT 4080
  • CTCCCCTCCC AATGCGGTTT AAAACATAAA TAAAAAACCA GACTCTGTTT GGATTTGGAT 4200
  • CTGTCCGTGT CCCCGTATAC AGACTTGAGA GGCCTGTCCC TCGACCGATG CCCTTGAGAG 5940 CCTTCAACCC AGTCAGCTCC TTCCGGTGGG CGCGGGGCAT GACTATCGTC GCCGCACTTA 6000
  • CTTCGGGGCG AAAACTCTCA AGGATCTTAC CGCTGTTGAG ATCCAGTTCG ATGTAACCCA 9300

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Abstract

L'invention concerne un adénovirus chimérique pouvant effectuer la transduction de cellules mammaliennes avec un ADN d'intérêt. Ces adénovirus servent à introduire des gènes clonés dans l'organisme d'un individu et sont donc également utilisés pour traiter les maladies et les troubles génétiques chez les mammifères.
PCT/US1995/011537 1994-09-23 1995-09-12 Adenovirus chimerique permettant l'apport de genes WO1996009399A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU35511/95A AU3551195A (en) 1994-09-23 1995-09-12 Chimeric adenovirus for gene delivery

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US31148594A 1994-09-23 1994-09-23
US311,485 1994-09-23

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WO1996009399A2 true WO1996009399A2 (fr) 1996-03-28
WO1996009399A3 WO1996009399A3 (fr) 1996-07-18

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997015679A1 (fr) * 1995-10-27 1997-05-01 The Trustees Of The University Of Pennsylvania Virus recombines contenant des elements genetiques mobiles et procedes d'utilisation desdits virus en therapie genique
FR2741358A1 (fr) * 1995-11-17 1997-05-23 Centre Nat Rech Scient Production de vecteurs retroviraux par l'intermediaire de vecteurs viraux a base de virus a adn
WO1997012986A3 (fr) * 1995-10-02 1997-06-19 Cornell Res Foundation Inc Vecteurs adenoviraux n'appartenant pas au groupe c
US5849561A (en) * 1997-05-22 1998-12-15 Cornell Research Foundation, Inc. Method for the production of non-group C adenoviral vectors
EP1320621A4 (fr) * 2000-09-15 2005-11-23 Merck & Co Inc Vaccins adenoviraux de premiere generation evolues, exprimant les proteines gag, pol et nef du vih-1 a optimisation des codons et leurs modifications
WO2005121343A1 (fr) 2004-06-07 2005-12-22 Chengdu Kanghong Biotechnologies Co., Ltd. Construction d'une recombinaison d'adenovirus oncolytique exprimant de facon specifique un facteur immunomodulateur gm-csf dans des cellules tumorales et utilisations correspondantes
US7125706B2 (en) 1998-12-01 2006-10-24 Introgen Therapeutics, Inc. Method for the production and purification of adenoviral vectors
EP1707631A3 (fr) * 1996-11-20 2006-12-27 Introgen Therapeutics, Inc. Procédé amélioré pour production et purification de vecteurs d'adénovirus
US7235391B2 (en) 1998-11-16 2007-06-26 Introgen Therapeutics, Inc. Formulation of adenovirus for gene therapy
US9428768B2 (en) 1996-11-20 2016-08-30 Crucell Holland B.V. Method for the production and purification of adenoviral vectors

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK0596881T3 (da) * 1991-08-01 1997-10-13 Fond Nat Transfusion Sanguine Ekspression i ikke-tumurale humane lymofoblastoider med en integrativ vektor

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997012986A3 (fr) * 1995-10-02 1997-06-19 Cornell Res Foundation Inc Vecteurs adenoviraux n'appartenant pas au groupe c
WO1997015679A1 (fr) * 1995-10-27 1997-05-01 The Trustees Of The University Of Pennsylvania Virus recombines contenant des elements genetiques mobiles et procedes d'utilisation desdits virus en therapie genique
FR2741358A1 (fr) * 1995-11-17 1997-05-23 Centre Nat Rech Scient Production de vecteurs retroviraux par l'intermediaire de vecteurs viraux a base de virus a adn
WO1997019182A1 (fr) * 1995-11-17 1997-05-29 Centre National De La Recherche Scientifique Production de vecteurs retroviraux par l'intermediaire de vecteurs herpetiques
US7510875B2 (en) 1996-11-20 2009-03-31 Introgen Therapuetics, Inc. Methods for producing purified adenoviral vectors
US7445930B2 (en) 1996-11-20 2008-11-04 Introgen Therapeutics Inc. Method for the production and purification of adenoviral vectors
US9428768B2 (en) 1996-11-20 2016-08-30 Crucell Holland B.V. Method for the production and purification of adenoviral vectors
EP1760151A1 (fr) * 1996-11-20 2007-03-07 Introgen Therapeutics, Inc. Procédé amélioré pour production et purification de vecteurs d'adénovirus
EP1707631A3 (fr) * 1996-11-20 2006-12-27 Introgen Therapeutics, Inc. Procédé amélioré pour production et purification de vecteurs d'adénovirus
US5849561A (en) * 1997-05-22 1998-12-15 Cornell Research Foundation, Inc. Method for the production of non-group C adenoviral vectors
US7235391B2 (en) 1998-11-16 2007-06-26 Introgen Therapeutics, Inc. Formulation of adenovirus for gene therapy
US7888096B2 (en) 1998-11-16 2011-02-15 Crucell Holland B.V. Liquid adenovirus formulations
US7888097B2 (en) 1998-11-16 2011-02-15 Crucell Holland B.V. Formulation for adenovirus storage
US7125706B2 (en) 1998-12-01 2006-10-24 Introgen Therapeutics, Inc. Method for the production and purification of adenoviral vectors
US7732129B1 (en) 1998-12-01 2010-06-08 Crucell Holland B.V. Method for the production and purification of adenoviral vectors
EP1320621A4 (fr) * 2000-09-15 2005-11-23 Merck & Co Inc Vaccins adenoviraux de premiere generation evolues, exprimant les proteines gag, pol et nef du vih-1 a optimisation des codons et leurs modifications
US7951585B2 (en) 2004-06-07 2011-05-31 Chengdu Kanghong Biotechnologies Co., Ltd. Construction of oncolytic adenovirus recombinant specifically expressing immune modulatory factor GM-CSF in tumor cells and uses thereof
WO2005121343A1 (fr) 2004-06-07 2005-12-22 Chengdu Kanghong Biotechnologies Co., Ltd. Construction d'une recombinaison d'adenovirus oncolytique exprimant de facon specifique un facteur immunomodulateur gm-csf dans des cellules tumorales et utilisations correspondantes

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WO1996009399A3 (fr) 1996-07-18

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