EP0735887A1 - THERAPIE GENIQUE $i(IN UTERO) DESTINEE AUX F TUS - Google Patents

THERAPIE GENIQUE $i(IN UTERO) DESTINEE AUX F TUS

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Publication number
EP0735887A1
EP0735887A1 EP95903185A EP95903185A EP0735887A1 EP 0735887 A1 EP0735887 A1 EP 0735887A1 EP 95903185 A EP95903185 A EP 95903185A EP 95903185 A EP95903185 A EP 95903185A EP 0735887 A1 EP0735887 A1 EP 0735887A1
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EP
European Patent Office
Prior art keywords
cells
gene
fetus
vector
viral
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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.)
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EP95903185A
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German (de)
English (en)
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EP0735887A4 (fr
Inventor
Robert C. Apartment N-104 MOEN
Lisa M. Morris
Esmail M. Zanjani
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Genetic Therapy Inc
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Genetic Therapy Inc
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Publication of EP0735887A1 publication Critical patent/EP0735887A1/fr
Publication of EP0735887A4 publication Critical patent/EP0735887A4/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • This invention relates to gene therapy, and in particular to in utero gene therapy. More particularly, this invention relates to in vivo gene therapy for fetuses by administering to a fetus at least one nucleic acid sequence encoding a therapeutic agent.
  • Certain inherited metabolic diseases such as Hurler's syndrome, Lesch-Nyhan disease, Tay Sachs disease, and alpha-thalassemia, may produce irreversible damage to the fetus before birth.
  • Infants born with other inherited diseases such as, for example, adenosine deaminase (ADA) deficiency, appear normal at birth; however, such diseases are manifested shortly thereafter.
  • ADA adenosine deaminase
  • Kantoff, et al., Blood, Vol. 73, No. 4, pgs. 1066-1073 discloses the retroviral-mediated transfer of a neomycin resistance (neo R ) gene into fetal sheep hematopoietic cells by exchange transfusion.
  • neo R neomycin resistance
  • blood was obtained from a sheep fetus, and mononuclear cells were harvested.
  • the cells then were transduced ex vivo with retroviral vectors including the neo R gene or a cDNA for human adenosine deaminase (ADA) .
  • the transduced cells then were reinfused into the sheep fetuses.
  • neo R gene After birth, the lambs were examined for presence of a functioning neo R gene. Out of ten lambs analyzed, six were positive for G418 resistant hematopoietic-progenitor cells. One sheep had blood cells which expressed the neo R gene for more than two years after birth.
  • Such an exchange transfusion procedure requires that one needs to wait until a period of time late in gestation in order for the fetus to be of a sufficient size in order to obtain sufficient blood cells to effect the gene transfer, and certain genetic diseases could be treated more successfully if gene transfer into fetal cells could be effected early in gestation.
  • such a procedure requires multiple manipulations of the fetus, which increases the risk of damage to the fetus.
  • transduction takes place only in cells removed from the fetus.
  • a process for effecting gene therapy in vivo in a fetus comprises transducing fetal cells in vivo with at least one nucleic acid (DNA or RNA) sequence encoding a therapeutic agent.
  • DNA or RNA nucleic acid
  • nucleic acid sequence means a DNA or RNA molecule, and includes complete and partial gene sequences, and includes polynucleotides as well. Such term also includes a linear series of deoxyribonucleotides or ribonucleotides connected one to the other by pho ⁇ phodiester bonds between the 3 ' and 5 ' carbons of the adjacent pentoses .
  • the term "therapeutic” as used herein is used in a generic sense and includes treating agents, prophylactic agents, and replacement agents .
  • the process of the present invention may be carried out during any stage of gestation, including the yolk sac stage. Such process may be applied to humans, wherein a human fetus may be injected intraperitoneally with a needle which is guided into the fetus by ultrasound, in a manner similar to that in which a fetus is given a blood transfusion for the treatment of alpha-thalessemia.
  • the nucleic acid sequence which encodes the therapeutic agent is contained in an appropriate expression vehicle which transduces the fetal cells.
  • expression vectors include, but are not limited to, eukaryotic vectors, prokaryotic vectors (such as, for example, bacterial vectors) , and viral vectors.
  • the expression vector is a viral vector.
  • Viral vectors which may be employed include, but are not limited to, retroviral vectors, adenovirus vectors, adeno-associated virus vectors, and Herpes virus vectors.
  • the viral vector is a retroviral vector.
  • a packaging cell line is transduced with a viral vector containing the nucleic acid sequence encoding the therapeutic agent to form a producer cell line which includes the viral vector.
  • the producer cells then are administered in vivo to the fetus, whereby the producer cells generate viral particles capable of transducing fetal cells .
  • fetal cells may be located throughout the body and include, but are not limited to, somatic and germinal cells, including bone marrow cells, including hematopoietic stem cells; peripheral blood cells; cells of the central nervous system, including brain cells; lung cells; kidney cells; testicular cells; ovarian cells; and liver cells.
  • the viral vector is a retroviral vector.
  • retroviral vectors which may be employed include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus.
  • the retroviral vector is an infectious but non-replication competent retrovirus.
  • replication competent retroviruses may also be used.
  • Retroviral vectors are useful as agents to mediate retroviral-mediated gene transfer into eukaryotic cells.
  • Retroviral vectors are generally constructed such that the majority of sequences coding for the structural genes of the virus are deleted and replaced by the gene(s) of interest. Most often, the structural genes (i.e., gag, pol, and env), are removed from the retroviral backbone using genetic engineering techniques known in the art. This may include digestion with the appropriate restriction endonuclease or, in some instances, with Bal 31 exonuclease to generate fragments containing appropriate portions of the packaging signal.
  • Retroviral vectors have also been constructed which can introduce more than one gene into target cells. Usually, in such vectors one gene is under the regulatory control of the viral LTR, while the second gene is expressed either off a spliced message or is under the regulation of its own, internal promoter.
  • a packaging-defective helper virus is necessary to provide the structural genes of a retrovirus, which have been deleted from the vector itself.
  • the retroviral vector may be one of a series of vectors described in Bender, et al., J. Virol. 61:1639-1649 (1987), based on the N2 vector (Armentano, et al. , J. Virol. , 61:1647-1650) containing a series of deletions and substitutions to reduce to an absolute minimum the homology between the vector and packaging systems. These changes have also reduced the likelihood that viral proteins would be expressed. In the first of these vectors, LNL-XHC, there was altered, by site-directed mutagenesis, the natural ATG start codon of gag to TAG, thereby eliminating unintended protein synthesis from that point.
  • MoMuLV Moloney murine leukemia virus
  • pPr80 8ag another glycosylated protein
  • MoMuSV Moloney murine sarcoma virus
  • the vector LNL6 was made, which incorporated both the altered ATG of LNL-XHC and the 5' portion of MoMuSV.
  • the 5' structure of the LN vector series thus eliminates the possibility of expression of retroviral reading frames, with the subsequent production of viral antigens in genetically transduced target cells.
  • Miller has eliminated extra env sequences immediately preceding the 3' LTR in the LN vector (Miller, et al., Biotechni ⁇ ues. 7:980-990, 1989).
  • Safety is derived from the combination of vector genome structure together with the packaging system that is utilized for production of the infectious vector.
  • Miller, et al. have developed the combination of the pPAM3 plasmid (the packaging-defective helper genome) for expression of retroviral structural proteins together with the LN vector series to make a vector packaging system where the generation of recombinant wild-type retrovirus is reduced to a minimum through the elimination of nearly all sites of recombination between the vector genome and the packaging- defective helper genome (i.e. LN with pPAM3) .
  • the retroviral vector may be a Moloney Murine Leukemia Virus of the LN series of vectors, such as those hereinabove mentioned, and described further in Bender, et al. (1987) and Miller, et al. (1989).
  • Such vectors have a portion of the packaging signal derived from a mouse sarcoma virus, and a mutated gag initiation codon.
  • the term "mutated” as used herein means that the gag initiation codon has been deleted or altered such that the gag protein or fragments or truncations thereof, are not expressed.
  • the retroviral vector may include at least four cloning, or restriction enzyme recognition sites, wherein at least two of the sites have an average frequency of appearance in eukaryotic genes of less than once in 10,000 base pairs; i.e., the restriction product has an average DNA size of at least 10,000 base pairs .
  • Preferred cloning sites are selected from the group consisting of NotI, SnaBI, Sail, and Xhol.
  • the retroviral vector includes each of these cloning sites. Such vectors are further described in U.S. Patent Application Serial No. 919,062, filed July 23, 1992, and incorporated herein by reference.
  • a shuttle cloning vector which includes at least two cloning sites which are compatible with at least two cloning sites selected from the group consisting of NotI, SnaBI, Sail, and Xhol located on the retroviral vector.
  • the shuttle cloning vector also includes at least one desired gene which is capable of being transferred from the shuttle cloning vector to the retroviral vector.
  • the shuttle cloning vector may be constructed from a basic "backbone" vector or fragment to which are ligated one or more linkers which include cloning or restriction enzyme recognition sites. Included in the cloning sites are the compatible, or complementary cloning sites hereinabove described. Genes and/or promoters having ends corresponding to the restriction sites of the shuttle vector may be ligated into the shuttle vector through techniques known in the art.
  • the shuttle cloning vector can be employed to amplify DNA sequences in prokaryotic systems .
  • the shuttle cloning vector may be prepared from plasmids generally used in prokaryotic systems and in particular in bacteria.
  • the shuttle cloning vector may be derived from plasmids such as pBR322; pUC 18; etc.
  • the vector includes one or more promoters.
  • Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechni ⁇ ues, Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and ⁇ -actin promoters).
  • Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, TK promoters, and B19 parvovirus promoters . The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
  • the vector then is employed to transduce a packaging cell line to form a producer cell line.
  • packaging cells which may be transfected include, but are not limited to, the PE501, PA317, -2, -AM, PA12, T19- 14X, VT-19-17-H2, CRE, CRIP, GP+E-86, GP+envAml2, PAT 2.4, and DAN cell lines. Representative examples of packaging cell lines also are described in Miller, Human Gene Therapy, Vol. 1, pgs. 5-14 (1990).
  • the vector containing the nucleic acid sequence encoding the therapeutic agent may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaP0 4 precipitation.
  • a first packaging cell line such as PE501
  • a second packaging cell line such as PA317
  • the producer cells are then administered in vivo to the fetus in an amount effective to produce a therapeutic effect in the fetus.
  • the producer cells are administered to the fetus in an amount of from about 1 x 10 4 cells to about 1 x 10'° cells, preferably from about 1 x 10 7 cells to about 1 x 10 9 cells, more preferably in an amount of from about 5 x 10 7 cells to about 1 x 10 8 cells.
  • the producer cells may be administered to the fetus systemically, such as by intraperitoneal administration, intravenous administration, or by direct injection into an organ or muscle.
  • the amount of producer cells to be administered is dependent upon various factors, including the disease to be treated and the extent and severity thereof.
  • the producer cells are administered in combination with a pharmaceutically acceptable carrier suitable for administration to a patient.
  • the carrier may be a liquid carrier such as, for example, a saline solution or a buffer solution or other isomolar aqueous solution.
  • the producer cells Upon administration of the producer cells to the fetus, the producer cells generate viral particles.
  • the viral particles then transduce fetal cells, such as, for example, hematopoietic stem cells, cells of the central nervous system, and cells of other tissues and organs, whereby the transduced cells express the therapeutic agent.
  • a viral supernatant containing viral particles may be administered to the fetus, whereby such viral particles transduce fetal cells as hereinabove mentioned.
  • the viral supernatant may be administered in an amount of from about 1 x 10 3 CFU to about 1 x 10" CFU, preferably from about 1 x 10' CFU to about 1 x 10' CFU.
  • Therapeutic agents which may be encoded by the at least one nucleic acid sequence include, but are not limited to, those which treat hematopoietic system deficiencies, immune deficiencies, lysosomal storage disorders, Lesch-Nyhan disease, and leukocyte adhesion deficiency.
  • therapeutic agents include, but are not limited to, Factor VIII, for treating hemophilia A; Factor IX, for treating hemophilia B; FACC, for treating Fanconi anemia; ⁇ -globin, for treating ⁇ - thalassemia; ⁇ -globin, for treating ⁇ -thalassemia and sickle cell anemia; adenosine deaminase (ADA), and PNP, for treating severe combined immunodeficiency; the T-cell receptor ⁇ -chain, for treating X-linked immunodeficiency; glucocerebrosidase, for treating Gaucher's disease; iduronate sulfatase, for treating Hunter's syndrome; -L- iduronidase, for treating Hurler's syndrome, ⁇ - galactosidase, for treating Fabry disease; the ⁇ -subunit of hexosaminidase A, for treating Tay-Sachs disease; HPRT, for treating Lesch-Nyhan disease
  • the process of the present invention may be applied to fetal animals in order to suplly to the fetal animal therapeutic agents such as growth hormones, and agents which confer resistance to disease.
  • the process of the present invention may be employed to provide a fetal animal with a gene encoding a human protein or therapeutic agent. For example, a fetal animal may be given a gene for human hemoglobin, which then may be harvested from the animal after birth to provide a human blood substitute.
  • process of the present invention may be employed to provide animal models for gene expression and gene therapy.
  • Plasmid pGlNaSvAd was derived from plasmid PG1 ( Figure 3). Plasmid pGl was constructed from pLNSX (Palmer, et al., Blood, Vol. 73, pgs . 438-445. The construction strategy for plasmid pGl is shown in Figure 1. The 1.6kb EcoRI fragment, containing the 5' Moloney Murine Sarcoma Virus (MoMuSV) LTR, and the 3.Okb EcoRl/Clal fragment, containing the 3' LTR, the bacterial origin of replication and the ampicillin resistance gene, were isolated separately.
  • MoMuSV Moloney Murine Sarcoma Virus
  • pGl Figure 3
  • pGl Figure 3
  • MCS 54 base pair multiple cloning site
  • the MCS was designed to generate a maximum number of unique insertion sites, based on a screen of non-cutting restriction enzymes of the pGl plasmid, the neo 1" gene, the ⁇ -galactosidase gene, the hygromycin 1" gene, and the SV40 promoter.
  • the "backbone" vector pGlNa was constructed from pGl and pN2 (Armentano, et al. , J. Virology. Vol. 61, pgs. 1647-1650 (1987)).
  • pGlNa was constructed by cutting pN2 ( Figure 4) with EcoRI and AsuII, filling in the ends of EcoRI/AsuII fragment containing the neo® gene, and ligating the fragment into SnaBI digested pGl to form pGlNa ( Figure 5).
  • pGlNa was linearized 3' to the neo R gene by a double enzyme cut with Sail and Hind III, and blunt ended with Klenow. The SvAd fragment then was ligated into pGlNa to form pGlNaSvAd ( Figure 7). A schematic of the construction of pGlNaSvAd is shown in Figure 6.
  • a producer cell line was made from vector plasmid and packaging cells.
  • the PA317/GlNaSvAd producer cell was made by the same technigues used to make previous clinically relevant retroviral vector producer cell lines .
  • the vector plasmid pGlNaSvAd DNA was transfected into a ecotropic packaging cell line, PE501. Supernatant from the PE501 transfected cells was then used to transinfect the amphotropic, hTK containing, packaging cell line (PA317).
  • Clones of transinfected producer cells were then grown in G418 containing medium to select clones that contain the Neo R gene. The clones were then titered for retroviral vector production. Several clones were then selected for further testing and finally a clone was selected for clinical use.
  • 5 x 10 5 PE501 cells (Miller, et al. , Biotechniques, Vol. 7, pgs. 980-990 (1989), incorporated herein by reference) were plated in 100 mm dishes with 10 ml high glucose Dulbecco's Modified Essential Medium (DMEM) growth medium supplemented with 10% fetal bovine serum (HGD10) per dish (3-100 mm dishes are required per transfection) . The cells were incubated at 37°C, in a 5% C0 3 atmosphere overnight.
  • DMEM Dulbecco's Modified Essential Medium
  • the plasmid pGlNaSvAd then was transfected into PE501 cells by CaP0 4 precipitation using 50 ⁇ g of DNA by the following procedure.
  • 50 ⁇ g of DNA, 50 ⁇ l 10 x CaCl 2 , and 450 ⁇ l of sterile H 2 0 was mixed in a 15 ml polypropylene tube to yield a 0.25M Ca Cl 2 solution containing 50 ⁇ g DNA, 0.5 ml 2x PBS (containing 50 mM N-N-bis- (2-hydroxyethyl)- 2-aminoethane- sulfonic acid, 280 mM NaCl, 1.5 mM Na 2 HP0 4 , and 50 mM Hepes, pH6.95), then was added to the tube and the contents of the tube were mixed by pipetting.
  • 0.5 ml 2x PBS containing 50 mM N-N-bis- (2-hydroxyethyl)- 2-aminoethane- sulfonic acid, 280 mM NaCl, 1.5 mM Na 2 HP0 4 , and 50 mM Hepes, pH6.95
  • the DNA solution then was left at room temperature for about 20 minutes to 1 hour, lml of DNA solution then was added to each culture dish, and each dish was swirled to ensure even distribution of the DNA.
  • the dishes then were incubated at 35°C in a 3% C0 2 atmosphere overnight.
  • a culture dish(es) with optimum precipitate following the overnight incubation then was selected.
  • the medium/DNA precipitate was aspirated from the dish(es), and 5 mL PBS was added to each dish.
  • the dish(es) was allowed to sit for 2 to 3 minutes to allow salts to dissolve.
  • the dish(es) then was washed again with PBS to remove the salt and the salt solution. 10 ml of HGD10 medium then was added to the dish(es), and the dish(es) incubated at 37°C in a 5% C0 2 atmosphere for about 48 hrs.
  • a 48 hour transient supernatant then was collected from the transfected cells by removing the supernatant from the cells and placing it in a 15 ml polypropylene tube.
  • the dish(es) then was rinsed with 5 ml PBS.
  • the PBS then was removed, and 1 ml trypsin-EDTA was added to each dish.
  • Three 15 ml polypropylene tubes then were labeled undiluted, 1:10, and 1:100, respectively. 9 ml of HGD10 plus 0.8 mg/ml of G418 were added to the 1:10 and the 1:100 tubes.
  • Serial dilutions of the cells then were made by adding 1 ml of undiluted cells to the 1:10 tube, and then by adding 1 ml of the 1:10 cells to the 1:100 tube. The cells then were mixed.
  • the six plates of cells were examined daily. The medium was changed if there was a great amount of cell death. Such medium changes were repeated until few dead cells were observed. At this point, live cells or colonies were allowed to grow to a size such that the colonies are large enough to clone out (i.e., the colonies are visible to the naked eye when looking up through the bottom of the plate). Viral supernatants from such colonies of PE501 cells were collected in amounts of from about 5 ml to about 10 ml, placed in cryotubes, and frozen in liquid nitrogen at about -70°C.
  • PA317 cells (ATCC Accession No. CRL 9078) (Miller et al., Mol. Cell. Biol.. 6:2895-2902 (1986)) and described in U.S. Patent No. 4,861,719, then were plated at a density of 5 x 10 4 cells per 100 mm plate on Dulbecco's Modified Essential Medium (DMEM) including 4.5 g/1 glucose, glutamine supplement, and 10% fetal bovine serum (FBS).
  • DMEM Dulbecco's Modified Essential Medium
  • FBS fetal bovine serum
  • the viral supernatant then was thawed, and 8 ⁇ g/ml of polybrene was added to viral supernatant from PE 501 cells, and the supernatant and polybrene were mixed and loaded into a syringe with a 0.22 ⁇ m filter unit.
  • the DMEM was suctioned off the plate of cells, and 7 to 8 ml of viral supernatant was added for overnight infection.
  • the viral supernatant then was removed and replaced with fresh 10% FBS.
  • the medium was changed to 10% FBS and G418 (800 ⁇ g/ml).
  • the plate then was monitored, and the medium was changed to fresh 10% FBS and G418 to eliminate dying or dead cells whenever necessary.
  • the plate also was monitored for at least 10 to 14 days for the appearance of G418 resistant colonies by scanning the bottom of the dish without a microscope. When colonies are large enough to see, they then were selected as clones.
  • the medium then was aspirated from the dish and replaced with 5 ml PBS.
  • the cells then were rinsed and most of the PBS was aspirated. About 0.5 to 1.0 ml of the PBS was left on the plate to keep it moist. Cloning rings then are placed on all selected colonies . Two drops of trypsin-EDTA then 2 were placed on each cloning ring.
  • the dish then was placed in an incubator, and tapped periodically until the cells are released from the dish. 5 ml of HGD10 plus 0.8 mg/ml was added to as many wells as needed in six well dishes.
  • the clones then were observed for confluent growth. When a clone was confluent or almost confluent, the clone was trypsinized and expanded in a 100 ml dish.
  • the old medium was removed and replaced with 1O ml of fresh HGD10 medium.
  • the dish was returned to the incubator for 20 to 24 hours.
  • the supernatant was removed from the dish, and placed in a 15 ml polypropylene tube. The tube was centrifuged at 1,200 to 1,500 rpm for 5 minutes to pellet out any cells which may have been in the supernatant. The supernatant then was aliquoted into six cryovials (1 ml/vial). The aliquots were stored in liquid nitrogen. 5 ml of PBS were added to the dish and the cells were rinsed.
  • the medium was aspirated off the cell pellet.
  • the pellet then was resuspended in 1ml HGD 10 and 1 ml of 2xDMS0 freezing medium, and 1 ml of cells was aliquoted into each of two cryovials.
  • the cryovials were placed on dry ice, and, when frozen, were transferred to liquid nitrogen.
  • PA317/GlNaSvAd.24 cells are thawed at 37°C and recultured as rapidly as possible to avoid damage to the cells by the cryopreservative dimethylsulfoxide (DMSO) .
  • DMSO dimethylsulfoxide
  • the contents of one working cell bank cryovial are placed in a T75 flask containing 25 ml of medium containing high glucose (4.5g/l) DMEM with 10% FBS and 2mM glutamine. The contents of this flask then are split into 4 to 10 75 cm 2 flasks. The contents of these flasks then can be split into additional 75 cm 2 flasks. When the cells are about 90% confluent, the medium in all the flasks is changed.
  • Supernatant then is collected from the flasks, and pooled in a large spinner flask. A sample is taken for Mvcoplasma screening. The remaining supernatant is filtered through a 0.22 micron filter, pooled (samples taken for further lot release testing) , and aliquoted into the final containers. More medium is added to each flask.
  • This vector contains the Thymidine Kinase (hTK) gene from Herpes Simplex Virus I regulated by the retroviral promoter and the bacterial gene, neomycin phosphotransferase (Neo R ) driven by an SV40 promoter.
  • hTK Thymidine Kinase
  • the hTK gene confers sensitivity to the DNA analogs acyclovir and ganciclovir, while the Neo R gene product confer resistance to the neomycin analogue, G418.
  • pGlTkSvNa a three step cloning strategy was used. First, the herpes simplex thymidine kinase gene (Tk) was cloned into the Gl plasmid backbone to produce pGlTk. Second, the Neo R gene (Na) was cloned into the plasmid pSvBg to make pSvNa. Finally, SvNa was excised from pSvNa and ligated into pGlTk to produce pGlTkSvNa. pGl was constructed as described in Example 1.
  • the restriction sites in the linkers were chosen because they are not present in the neomycin resistance gene, the ⁇ -galactosidase gene, the hygromycin resistance gene, or the SV40 promoter.
  • the 27 bp ribosomal binding signal was included in the 5 ' linker because it is believed to enhance mRNA stability (Hagenbuchle, et al. , Cell 13:551-563, 1978 and Lawrence and Jackson, J. Mol. Biol. 162:317-334, 1982).
  • the Kozak consensus sequence (5 '-GCCGCCACCATGG-3 ' ) has been shown to signal initiation of mRNA translation (Kozak, Nucl.Acids Res. 12:857-872, 1984).
  • the Kozak consensus seguence includes the Ncol site that marks the ATG translation initiation codon.
  • pBR322 (Bolivar et al. Gene 2:95, 1977) was digested with Ndel and EcoRI and the 2.1 kb fragment that contains the ampicillin resistance gene and the bacterial origin of replication was isolated.
  • the ligated 5' linker - lacZ - 3 ' linker DNA described above was ligated to the pBR322 Ndel/EcoRI vector to generate pBg.
  • pBg has utility as a shuttle plasmid because the lacZ gene can be excised and another gene inserted into any of the restriction sites that are present at the 5 ' and 3 ' ends of the lacZ gene. Because these restriction sites are reiterated in the pGl plasmid, the lacZ gene or genes that replace it in the shuttle plasmid construct can easily be moved into pGl.
  • a 1.74 kB Bglll/PvuII fragment containing the Herpes Simplex Virus Type I thymidise kinase gene (GenBank accession no. V00467, incorporated herein by reference) was excised from the pXl plasmid (Huberman, et al. , Exptl . Cell Res. Vol. 153, pgs 347-362 (1984) incorporated herein by reference), blunted with the large (Klenow) fragment of DNA polymerase I, and inserted into the unique SnaBI site in the pGl multiple cloning site, to form plasmid pGlTK. ( Figure 9) .
  • Producer cell line PAT 2.4/GlTkSvNa.90 was prepared according to the method disclosed in Example 1 for the preparation of producer cell line PA317/GlNaSvAd.24 except that packaging cell line PAT 2.4 was used instead of packaging cell line PA317.
  • PAT 2.4 was made according to the method for the preparation of PA317 cells disclosed in Miller et al. , Mol. Cell. Biol. , 6:2895-2902 (1986) and in U.S. Patent No.
  • the method is summarized as follows.
  • a population of the NIH 3T3 TK-minus cells was co-transfected with two plasmid DNAs at a ratio of 20:1 using standard CaP0 4 transection methodology.
  • the first plasmid was pPAM3 (ATCC accession number 40234), which contains the promoter, gag, pol, and env sequences of amphotropic murine leukemia virus. Seventy micrograms of DNA were used.
  • the second plasmid was pY3, which contained the hygromycin resistance gene.
  • the hygromycin resistance gene also is found in other plasmids which are available to those skilled in the art.
  • the population of cells was selected in hygromycin, and hygromycin resistant cells were frozen as primary and secondary seed lots.
  • the population was analyzed for viral envelope expression, and the packaging function was tested in TK vector producer cell clones to look for high titer vectors .
  • Oligo-clonal populations were created by seeding one 24-well plate with 5-10 cells per well and another plate with 10-20 cells per well. A total of 46 populations were created. Each oligoclone was expanded and frozen.
  • Each population was tested individually for packaging efficiency by generating a producer population with supernatant from the packaging cell PE501, which had been transfected with the plasmid pGlTkSvNa, containing the neomycin resistance gene according to the procedure of Example 1.
  • a subpopulation capable of producing high titer producer cell lines was identified according to the procedure of Example 1 and designated PAT 2.4/GlTkSvNa.90.
  • Viral supernatant containing GITkSvNa was prepared from this producer cell in accordance with Example 1.
  • Ten ewes with confirmed dates of pregnancy were prepared for surgery by withholding food for 48 hours and water for 18 to 24 hours. Each ewe was sedated with intramuscular keta ine (lOmg/kg), and given a 0.5 to 10% halothane-oxygen inhalation mixture via an endotracheal tube. Each animal received intravenous fluids and antibiotics during surgery. The uterus was exposed by a lower midline incision, and each fetus was accessed through a small hysterotomy and transverse incision of the myometrium and chorion. The amnion was left intact. The fetus then was visualized, and gently manipulated into an amniotic bubble.
  • the fetus then was immobilized for injection within the bubble under gentle applied pressure. At this time, the fetus is within view fully, which insures that the injected cells or viral supernatant remain within the peritoneal cavity.
  • the sheep were injected with one of the following: (i) 2 ml of viral supernatant from GlNaSvAd.24, having a titer of 1 x 10" CFU/ml; (ii) 1 ml of 5 x 10 7 PA317/ GlNaSvAd.24 producer cells; (iii) 1 ml of 5 x 10 7 PA317/GlNaSvAd.24 producer cells that were irradiated with 3,000 rads in a cesium irradiator; (iv) 2 ml containing 1 x 10 8 PAT2.4/GlTkSvNa.90 producer cells; (v) 1 nil of 5 x 10' PAT2.4/Gl
  • PA317/GlNaSvAd.24 1 571 ml producer cells
  • PA317/GlNaSvAd.24 2 571 ml producer cells
  • each fetus was returned to the primary amniotic space, and the myometrium was closed in a double layer. Following the closing of all incisions, each ewe was observed for 48 hours and, unless scheduled for further experimentation, was returned to a large animal facility for the remainder of the gestation period. Each ewe that survived was examined one week before the expected date of delivery.
  • CFU-E, BFU-E, CFU- GM, or CFU-Mix bone marrow cells were taken from various sheep and analyzed for the expression of the neo R gene by culturing the cells in the presence of G418, and/or for expression of the Herpes Simplex thymidine kinase (TK) gene by culturing the cells in the presence of ganciclovir.
  • TK Herpes Simplex thymidine kinase
  • CFU-E cells and BFU-E cells were tested in a plasma clot culture assay as follows:
  • G418 in amounts of 0 mg/ml, 0.5 mg/ml, 1.0 mg/ml, 1.5 mg/ml, 2.0 mg/ml, 2.5 mg/ml, or 3.0 mg/ml, and/or 0 ⁇ M, 3 ⁇ M or 6 ⁇ M of ganciclovir was added to each tube.
  • Ganciclovir interacts with Herpes Simplex thymidine kinase in order to kill cells which express the Herpes Simplex thymidine kinase gene.
  • the amount of Iscove's solution for each tube was calculated to bring the final volume of the tubes, including the cells, to 1 ml.
  • the Iscove's solution was added to the tube, followed by the G418 and/or the ganciclovir, followed by the cells.
  • 100 ⁇ l of citrated plasma, which aids in clotting, is added to the first tube.
  • the contents of the tube are mixed by pipetting the contents up and down with a 1 ml pipette, and 200 ⁇ l of the contents is dispensed to each of eight plasma clot wells . Four wells are in one 6 well plate, and four wells are in another 6 well plate.
  • the plates are removed from the incubator, and allowed to sit under a hood for 10 minutes. During this time, P0 3 buffer is placed into a petri dish.
  • Coated slides then are set up on a cafeteria tray and labeled. Four clots are lifted out for each group onto the same slide, and are arranged in an offset pattern so that the clots do not come in contact with each other.
  • a filter paper then is dipped into the P0 3 buffer, and a piece is laid over each slide.
  • the filter papers and the slides are allowed to sit for 1 to 2 minutes.
  • a large piece of Baxter filter paper then is obtained and laid over all the slides.
  • the paper is pressed firmly to blot out all of the excess buffer.
  • the large filter paper is discarded, and 3% glutaraldehyde is sguirted onto each slide's P0 3 buffer coated filter paper.
  • the slide and filter paper are allowed to sit for 1 minute.
  • the slide's filter papers are removed and discarded.
  • the slides are set in a drying rack, and are allowed to dry for several hours before staining.
  • Each slide then was treated with methanol for one minute, and then treated with 1% benzid.ne in methanol for 5 minutes.
  • Each slide then was treated with peroxidase for 3 minutes, and then washed with distilled water for 1 minute.
  • Each slide then was stained with hematoxylin for 8 minutes, and then placed under cool running tap water for 10 minutes.
  • CFU-GM and CFU-Mix colonies were evaluated for neomycin resistance or expression of the Herpes Simplex thymidine kinase gene through a methylcellulose culture assay as follows:
  • Bone marrow aspirates were drawn from sheep and mononuclear cells were isolated by layering 10 ml of bone marrow (diluted 1:3 in IMDM) onto a cushion of 5 ml of Ficoll-Hypaque buffer. The tubes were subjected to centrifugation at 1,500 rpm for 30 minutes. The mononuclear fraction was removed with a sterile transfer pipette and washed twice with IMDM. The cells were pelleted and cultured at 2 x 10 s cells/ml. Each plate consisted of 3 wells, each containing one-third of the following mixture:
  • PHA-LCM phytohemagglutinin-stimulated leukocyte conditioned medium
  • G418 was added to the plates at concentrations of 0 mg/ml, 0.5 mg/ml, 1.0 mg/ml, 1.5 mg/ml, 2.0 mg/ml, 2.5 mg/ml, and 3.0 mg/ml, and/or ganciclovir was added at 0 ⁇ M, 3 ⁇ M, or 6 ⁇ M. These plates then were cultured for 7 days (CFU-GM), and 12 days (CFU-Mix), and colonies were counted under a dissecting scope.
  • Example 5 The effect of G418 on CFU-Mix, BFU-E, CFU-GM, and CFU-E cells of three of the sheep which were born alive, and now at 7 months of age (10 months after intraperitoneal administration of viral supernatant or producer cells), was evaluated and compared with the effect of G418 on the CFU- Mix, BFU-E, CFU-GM, and CFU-E bone marrow cells of six control sheep which were not given any producer cells or viral supernatant.
  • one of the three treated sheep received GlNaSvAd.24 viral supernatant, one sheep received 5 x 10 7 irradiated PA317/GlNaSvAd.24 producer cells, and 1 sheep received 5 x 10 7 PAT2.4/GlTkSvNa.90 producer cells.
  • the percentage of G418 resistant colonies of each cell type was determined according to the assay procedures of Example 3, and the average percentage of G418 resistant colonies was calculated for each sheep.
  • Example 6 Four sheep at age 6 months were evaluated for the presence of G418-resistant CFU-E, BFU-E, and CFU-GM cells in the presence of 2 mg/ml G418 according to the assay procedures of Example 3. Sheep 1 received 5 x 10' PAT2.4/GlTkSvNa.90 producer cells. Sheep 2 received 1 x 10 8 PAT2.4/GlTkSvNa.90 producer cells. Sheep 3 received 1 ml of 1 x 10' CFU/ml of GlNaSvAd.24 viral supernatant, and Sheep 4 received 5 x 10 7 irradiated PA317/GlNaSvAd.24 producer cells. The percent resistance of the cells from each sheep to 2 mg/ml G418 is given in Table II below:
  • Example 7 All the sheep that were born alive, which received producer cells, which produce viral particles expressing the neo" gene, and all sheep that were born alive which received viral supernatant, were monitored for a period of 20 months for G418-resistant colonies of bone marrow cells according to the assays hereinabove mentioned in Examples 3 and 5. The average percent of G418-resistant cells for all cell types, and then for all sheep which received viral supernatant or producer cells, was calculated. The results of such monitoring are shown in Figure 13.
  • PCR assays were conducted on DNA isolated from the lung, liver, kidney, and testes for presence of the neo R gene. The PCR assays were conducted a ⁇ follows:
  • PCR polymerase chain reaction
  • Primer 1 has the following sequence: 5-GGT GGA GAG GCT ATT CGG CTA TGA-3 '
  • Primer 2 has the following sequence: 5'-ATC CTG ATC GAC AAG ACC GGC TTC-3 '
  • These primers amplify a 440 base pair fragment of the neomycin resistance gene.
  • the samples were overlaid with 100 ⁇ l of mineral oil, heated to 95°C for 10 minutes to inactivate the UDG, and then were subjected to 40 cycles of PCR.
  • the reactions were run in an automated PCR temperature cycling block that allowed denaturation of the DNA at 95°C for 1 minute, annealing of the primers at 65°C for 1.5 minutes, and extension of the primers at 72°C for 1.5 minutes, and extension of the primers at 72°C for 1.5 minutes. After the 40th cycle, the reactions were held at 72°C to allow complete extension of the amplification products and to prevent damage due to residual UDG activity. Chloroform extractions were performed on the reactions, and 15 ⁇ l of each resultant aqueous phase was loaded onto a 2% agarose gel and electrophoresed in Tris- acetate-EDTA.
  • PCR analysis of DNA from the brain showed that brain cells contained the neo R gene.
  • direct injection of an engineered retrovirus into a fetus can deliver a gene to the brain of a developing fetus.

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Abstract

Procédé de thérapie génique s'adressant à un f÷tus in utero, selon lequel on transduit des cellules f÷tales in vivo avec au moins une séquence nucléotidique codant un agent thérapeutique. Les cellules f÷tales peuvent être transduites avec un vecteur viral (tel qu'un vecteur rétroviral) qui comprend la séquence nucléotidique codant l'agent thérapeutique. Le vecteur viral peut être contenu dans un surnageant viral qui est administré au f÷tus ou qui peut être généré par une lignée cellulaire productrice administrée au f÷tus.
EP95903185A 1993-12-03 1994-12-02 THERAPIE GENIQUE -i(IN UTERO) DESTINEE AUX F TUS Withdrawn EP0735887A4 (fr)

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