WO1997032608A1 - Oligodendrocytes primaires obtenus par genie genetique et destines a l'administration de genes par transplantation dans le systeme nerveux central - Google Patents

Oligodendrocytes primaires obtenus par genie genetique et destines a l'administration de genes par transplantation dans le systeme nerveux central Download PDF

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WO1997032608A1
WO1997032608A1 PCT/US1997/003094 US9703094W WO9732608A1 WO 1997032608 A1 WO1997032608 A1 WO 1997032608A1 US 9703094 W US9703094 W US 9703094W WO 9732608 A1 WO9732608 A1 WO 9732608A1
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cells
oligodendrocyte
primary
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Randall D. Mckinnon
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University Of Medicine & Dentistry Of New Jersey
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2740/11011—Alpharetrovirus, e.g. avian leucosis virus
    • C12N2740/11041—Use of virus, viral particle or viral elements as a vector
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    • 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 pertains to a method for transplanting 20 genetically engineered primary cells into the central nervous system of a patient.
  • the method comprises the steps of (a) isolating primary oligodendrocyte progenitor cells from the brain of a patient; (b) expanding the cells in vitro witii mitogens; (c) genetically engineering the cells by introducing transgenes through an eukaryotic expression vector for DNA-mediated gene transfer into the cells; and (d) 25 transplanting the genetically engineered primary cells into tiie brain of the patient.
  • Myelin is the insulation of neuronal axons that is essential for rapid conduction of ax onal signals, allowing for a large number of fast conducting axons in a compact space. Myelination occurs relatively late during development, after the majority of the neuronal architecture is in place. It is formed by an elaboration of the cytoplasmic membrane of Schwann cells in the peripheral nervous system, and by oligodendrocytes (OL) in central nerve. Mature oligodendrocytes extend processes, each of which ensheathes a segment of an axon to form one myelin internode, and the combination of myelin internodes facilitate saltatory electrical conduction. The interests in the biology of myelin forming cells stem from the fact that the destruction of the myelin sheaths of oligodendrocytes is the principal pathophysiology of one of the most common neurological disorders in man, multiple sclerosis.
  • oligodendrocyte precursor cells originate in the ventral regions of the germinal neuroepithelium (1) then migrate into both grey and white matter areas before differentiating (2,3).
  • oligodendrocyte precursors were first characterized from neonatal rat optic nerve as bipolar, migratory cells which form either oligodendrocytes or type-2 astrocytes under different culture conditions, and were termed "O-2A progenitors" (4).
  • oligodendrocyte precursors progress from the bipotential O-2A stage (A2B5 + /O4 " ; bipolar, motile and proliferative), to pro- oligodendroblasts (O4 + /GC ⁇ ; stellate, non-motile and proliferative), to oligodendrocytes (GC + ; multipolar, non-motile and postmitotic) (5).
  • A2B5 + /O4 " bipolar, motile and proliferative
  • pro- oligodendroblasts O4 + /GC ⁇
  • stellate, non-motile and proliferative to oligodendrocytes
  • GC + multipolar, non-motile and postmitotic
  • oligodendrocyte progenitor cell proliferation bFGF, PDGF, IGF-1, NT-3
  • migration PDGF
  • differentiation IGF-1, TGF- ⁇
  • survival PDGF, bFGF, IGF-1, CNTF.
  • PDGF act via transmembrane receptors with intrinsic ligand activated tyrosine kinase activity, or receptor tyrosine kinases (7).
  • Oligodendrocyte progenitors express exclusively the ⁇ -chain form of PDGF receptors (PDGFR ⁇ ) (8), and PDGFR ⁇ expression in the developing central nervous system appears restricted to precursor cells found in a restricted subset of subventricular zone cells (9). While these studies imply a role for PDGF in oligodendrocyte development, to date there is no formal genetic proof that PDGF is involved in oligodendrocyte maturation in vivo.
  • the transplantation of precursor cells which have an inherent ability to mature upon injection into the brain represents an alternative strategy for production of specific gene products in vivo.
  • the present invention describes the application of this latter approach utilizing a unique precursor cell population as a vector for gene delivery into the central nervous system, the immediate progenitors of the myelm-forming oligodendrocytes in the central nervous system.
  • One important aspect of the precursor cell described herein which makes it especially attractive as a vector for cell delivery is its ability to migrate throughout the brain after transplantation. This dispersion facilitates the widespread distribution of cells capable of producing a therapeutic gene product in vivo.
  • the present invention pertains to a method for transplanting genetically engineered primary cells into the central nervous system of a patient which comprises the steps of: (a) isolating primary oligodendrocyte progenitor cells from the brain of a patient;
  • Figure 1 illustrates mitogen expanded primary oligodendrocyte progenitor cells.
  • Panel A top illustrates photomicrographs of primary O-2A progemtor cells (panels i-iv) (52) and mitogen-expanded oligodendrocyte cultures
  • Panel B is a graph illustrating stimulation of DNA synthesis in mitogen-expanded oligodendrocyte cultures, as measured by ⁇ H-thymidine incorporation in the absence (open) or presence (closed boxes) of 10 ⁇ g/ml insulin, in cultures treated for 24 hours with (1) DMEM, (2) oligodendrocyte media, (3) oligodendrocyte plus 5 ng/ml bFGF, (4) oligodendrocyte plus 10 ng/ml PDGF-AA, and (5) oligodendrocyte plus 5% B104- CM.
  • Figure 2 shows three graphs illustrating transient transfection of primary O-2A cells.
  • Graph A shows expression of firefly luciferase in oligodendrocyte progenitor cells exposed for 72 hours to a DNA precipitate containing 10 ⁇ g RSVluc;
  • graph B shows to 10 ⁇ g RSVluc plus increasing concentrations of carrier DNA;
  • graph C shows 10 ⁇ g each of RSVluc plus carrier DNA harvested at the indicated time points.
  • Figure 3 illustrates expression vectors for stable transfection.
  • Panel (A) top) shows pMo.iresNeo, including Moloney LTR promoter, the internal ribosome entry sequence (IRES), and neomycin (G418 R ) gene.
  • Panel (B) (middle) shows pMo.FGFRl.iresNeo, with cDNA insert encoding the murine FGFR1 receptor (53) (solid box). The arrow indicates the location of an in-frame stop codon introduced in construct FGFRx, encoding a dominant negative version of FGFR1 (32).
  • Panel (C) shows pMo.PDGFR ⁇ .iresNeo, with the 3.4 kilobase human PDGFR ⁇ cDNA (35) (solid box). The arrow indicates the location of an in-frame stop codon introduced in construct PDGFRx.
  • Figure 4 illustrates transplantation of O-2A progenitor cells in vivo.
  • Panel (A) shows PKH-26 dye labeled cells, 72 hours post transplant, in internal capsule.
  • Panel (B) shows ⁇ -galactosidase labeled oligodendrocyte progenitor cells, 12 days post transplant, scattered through brain parenchyma (indicated by asterisks). Arrow indicates site of transplant.
  • Figure 5 is a graph illustrating motor function of normal and shiverer mutant mice in terms of cumulative falls from a rotarod of homozygous mutant shiverer (shi) mice, shi mice that are heterozygous (shi/mbpl) or homozygous (shi/mbp2) for an MBP transgene (54), shi mice that received grafts of wild type oligodendrocyte progenitor cells at birth (shi/tspt), and heterozygous (shi/+) mice
  • the present invention pertains to the transplantation of genetically engineered primary cells into the central nervous system to allow an analysis of gene function that is often not otherwise possible, such as with germ line mutations that result in embryonic lethality or that have pleiotropic effects.
  • Primary oligodendrocyte progenitor cells, the myelin-forming cells of the central nervous system, were isolated from the neonatal rat brain, expanded in vitro with mitogens, and genetically altered by the introduction of transgenes. The development and use of an efficient eukaryotic expression vector for optimal DNA-mediated gene transfer in these progenitor cells is detailed.
  • mice include Sprague-Dawley rat pups (Taconic Farms, NY), and shiverer (MBP sn *) mutant mice obtained from L. Rhodes Jr. (NINDS, NIH
  • B104 neuroblastoma cells were obtained from D. Schubert (Salk Inst. , San Diego CA), and the oligodendrocyte progenitor line CG-4 from J.-C. Louis (UC San Diego).
  • G418 sulphate Gibco/BRL, Bethesda MD
  • PBS PBS
  • Retroviral vectors including LZ1 and DAP were obtained as producer cell lines from J. Sanes (Wash. U., St. Louis MO) and C. Cepko (Harvard Med., Boston
  • Plasmid vectors encoding firefly luciferase (Luc), chloramphenicol acetyltransferase (CAT), and growth factor receptors PDGFR ⁇ and FGFR1, were obtained from S. Nordeen (U. Colorado, Bolder CO), L. Hudson (NINDS, NIH Bethesda), T. Matsui and S. Aaronson (NCI, NTH Bethesda) and D. Ornitz (Wash U. St. Louis), respectively.
  • glial cultures are established by dissociation of neonatal rat forebrains (18), and O-2A progenitor cells purified from these cultures using antibody panning (19). Dissections are done at room temperature under aseptic conditions, and up to 10 pups can be harvested concurrently on a laboratory bench in an isolated room. Postnatal day 2 pups are decapitated and the heads immobilized on a styrofoam board with pins, rinsed with 70% ethanol, then the cranium is opened and the entire brain removed with curved forceps and placed in
  • MEM Hepes buffered media Gibco/BRL
  • the brains are split into two halves, the olfactory bulb and hindbrain/cerebellum are removed, then meningeal membranes are peeled away under a dissecting microscope.
  • the cleaned brains are placed in 10 ml of fresh MEM Hepes and the tissue is dissociated using a 20 cc syringe by six passes each through 19 and
  • the cultures are refed fresh DMEM/FBS every 3 days, and grow aggressively during the first week to form a confluent monolayer of astrocytes (type-1) with overlying microglia (loosely attached phase bright cells) and O-2A progenitor cells (small, round cells attached to the astrocyte monolayer).
  • day three first refeed
  • debris and unattached cells in the media from which live cells can be recovered by centrifugation, resuspended in fresh DMEM/FBS, and plated on new flasks to generate secondary mixed glial cultures with growth characteristics comparable to the original flasks.
  • O-2A progenitors are harvested from primary mixed glial cultures once they reach confluence, generally seven to eight days after the initial plating.
  • the microglial cells are depleted from the cultures by placing flasks with closed caps on a horizontal plane rotary shaker ( " 180 rpm, 4 hours, 37°C), then refeeding to remove unattached cells.
  • O-2A progenitors are then purified from these cultures by mitotic shake-off followed by antibody panning (19). The flasks are placed on the rotary shaker overnight, then the media containing unattached cells (microglia and O-2A progenitors) is collected and the primary cultures are refed fresh DMEM/FBS and returned to the CO2 incubator.
  • Progenitors can be harvested 2-3 times from these cultures.
  • the detached cells are recovered from the harvested media by centrifugation (1,000 rpm, 10 minutes), then resuspended in 10 ml of MEM Hepes containing 0.5% FBS and monoclonal antibody A2B5 (American Type Culture Collection, Rockville MD) (20) (1:100 dilution of ascites fluid, or 1:10 dilution of A2B5 tissue culture media) sterilized by centrifugation through Spin-X filter units (Costar, Cambridge MA) immediately prior to use.
  • A2B5 American Type Culture Collection, Rockville MD
  • the cells are plated on a 100 mm Falcon culture dish and left undisturbed for 7 minutes, then unattached (antibody-bound) O-2A progenitor cells are manually resuspended by gentle swirling (seven rotations) and collected from the culture fluid by centrifugation (19).
  • Recovered cells (generally 5x10" 0-2A progenitor cells/10 flasks per 20 animals) are resuspended at 2xl0 5 cells per ml in prewarmed (37°C) DMEM containing sodium pyruvate, penicillin-streptomycin, 0.5% FBS, 50 ⁇ g/ml transferrin, 30 nM selenium, 30 nM tri-idothyronine, and 50 ng/ml bovine insulin (OL media). Oligodendrocyte medium is prepared in advance and stored at 4°C until use. Stock solutions (50 mg/ml transferrin, 30 ⁇ M selenium, 30 ⁇ M T3; Sigma Chemicals, St.
  • O-2A progenitor cells cultured in oligodendrocyte media differentiate into mature galactocerebroside positive oligodendrocytes within 3 days under these conditions, and into type-2 astrocytes in oligodendrocyte media the presence of 20% FBS (4).
  • Primary O-2A cultures can be expanded as secondary cultures in oligodendrocyte media supplemented with bFGF plus PDGF-AA (OIJFP media), or with B104-CM (OL/B media) as used in this study.
  • B104 neuroblastoma cells are expanded in monolayers with DMEM/FBS, and CM is collected after 48 hours from cells plated in oligodendrocyte media at a density of 150 cells/mm 2 , then filtered (0.45 ⁇ m) and aliquots stored at -20°C.
  • Oligodendrocyte progenitor cells growing in OL ⁇ B media are given fresh mitogen every 48 hours, refed with OIJB media every 4-6 days, and passaged when confluent by rinsing plates once with PBS then once with ATV trypsin solution (Irvine Scientific, Irvine CA). The trypsin solution is immediately removed, and after 1-2 minutes the plates are gently tapped, the detached cells are suspended in OL/B media, and cells are seeded on polyornithine-coated dishes at a density of 100 cells/mm .
  • detached cells are resuspended at lxlO 6 cells/ml in a fresh prepared solution of 90% FBS and 10% DMSO (Sigma, stored in 1 ml aliquots at -70°C), placed in 2 ml polypropylene freezer vials (VWR Scientific), frozen for 24 hours at -70°C, then transferred to a liquid nitrogen container for long term storage.
  • cell karyotypes The analysis of cell karyotypes is performed on cells growing on cell culture dishes. Proliferating cells are incubated at 37°C for 75 minutes with 200 ⁇ g/ml colcemid (KaryoMAX, Gibco/BRL), rinsed with 0.85% sodium citrate, then incubated in 0.85% sodium citrate for 25 minutes at room temperature. When the cells have swelled in the hypotonic solution, they are fixed (room temperature, 20 minutes) by adding 1 volume of 3:1 methanol: acetic acid, then with fresh fixative every 20 minutes for 60 minutes. The cells are dried with a stream of warm air on the bottom of the dish, while inverted over a boiling water steam bath to maintain humidity, then on a 60°C hot plate for 1 hour. For staining, the cells are incubated (room temperature, 80 seconds) in 0.025% trypsin solution (Gibco/BRL) in Hank's
  • Proliferation assays are performed on cells which have been removed from mitogens for 24 hours prior to exposure to growth factors. Oligodendrocyte progenitors plated in 96 well plates (2,000 cells/well) are incubated for 24 hours in oligodendrocyte medium, then for 24 hours in oligodendrocyte media with a range of concentrations of mitogens, with 0.5 ⁇ Ci/ml ⁇ H-thymidine (Amersham Life Sciences, Arlington Heights IL; specific activity 40 Ci/mmole) present for the final 4 hours. Assays are run in triplicate (three wells for each growth factor concentration), and include a positive responding cell type for comparison between experiments. The cells are trypsonized then recovered on Whatman GF/C filters using an automatic harvester (Brandel, Gaithersburg MD), and the incorporated radioactivity is measured by liquid scintillation counting.
  • Viral producer cells LZ1 (23) and DAP (24) are expanded in DMEM/FBS in monolayer cultures.
  • producer cells are plated in Falcon 12 mm cell culture inserts (1.0 ⁇ m pore size) maintained in Falcon 12 well plates in DMEM/FBS.
  • the inserts are refed with OIJB media and transferred to 24 well plates containing oligodendrocyte progenitor cells growing in OL/B, and after 24-48 hours co-culture the inserts are removed and the oligodendrocyte cultures refed OIJB.
  • G418 R producer cells are maintained in media lacking G418 after plating in the inserts, and after co-culture the retroviral infected oligodendrocyte progenitors are selected in OIJB plus 400 ⁇ g/ml G418.
  • LZl ⁇ - galactosidase
  • DAP alkaline phosphatase
  • ⁇ - galactosidase staining is performed at 4°C for 2-12 hours with X-gal (5-Bromo-4- chloro-3-indolyl- ⁇ -D-galactopyranoside, Fisher Biotech) in 5 mM K3Fe(CN) ( >, 5 mM K4Fe(CN)6, 2 mM MgCl2 as described (23).
  • X-gal 5-Bromo-4- chloro-3-indolyl- ⁇ -D-galactopyranoside
  • AP staining sections are incubated in PBS (30 minutes, 65°C), permeablized with 0.3% Triton X-100 in PBS (10 minutes, room temperature), rinsed in 0.1 M Tris (pH 7.4) and incubated
  • DNA co-precipitates are prepared in 15 ml polyethylene tubes by mixing plasmid and carrier DNA at room temperature in 10 mM Tris (pH 7.5), slowly adding 10% vol. of 2.5 M CaCl2 with swirling to mix, then adding this mixture dropwise to an equal volume of 2X Hepes-buffered saline (10 g/L HEPES acid, 16 g/L NaCl, 0.74 g/L KC1, 0.25 g/L Na2HPO4, 2 g/L D-glucose, final pH 7.1) while vigorously mixing the solution with air bubbles (26).
  • 2X Hepes-buffered saline 10 g/L HEPES acid, 16 g/L NaCl, 0.74 g/L KC1, 0.25 g/L Na2HPO4, 2 g/L D-glucose, final pH 7.1
  • a flocculent white precipitate is visible immediately upon mixing the two solutions, and the cells are exposed to the DNA precipitates by adding 1:10 vol DNA-CaCl2 co-precipitate suspension directly to the media in the culture dish.
  • the cells are harvested after 48-72 hours exposure to the precipitate.
  • the cultures are refed after 48 hours with OL/B medium containing 400 ⁇ g/ml G418 and refed with fresh medium every 3 days, and G418 concentrations reduced to 200 ⁇ g/ml after colonies are visible (7-10 days).
  • neonatal pups are anesthetized with isoflurane (Anaquest Inc., Liberty Corner NJ) and a hole is introduced into the cranium 1 mm lateral to midline and 3 mm caudal to Bregma using a 24 gauge needle.
  • isoflurane Asaquest Inc., Liberty Corner NJ
  • a hole is introduced into the cranium 1 mm lateral to midline and 3 mm caudal to Bregma using a 24 gauge needle.
  • cells are washed with PBS and cultured for at least 24 hours in medium lacking G418 prior to transplant.
  • Cells lacking heritable markers DAP, ⁇ -gal
  • PKH26 red fluorescent marker
  • 50,000 cells are transplanted per pup, in a 1 ⁇ l volume delivered into one hemisphere, with decolorizing charcoal (Mallinkrodt) included to mark the graft site.
  • the cells are suspended in DMEM and delivered manually either through a Hamilton syringe or a drawn glass capillary pipette, to a depth of 3 mm into the thalamus.
  • the cell suspension is introduced slowly over a period of 2 minutes, the needle withdrawn, and the pups maintained at 37°C until revived then returned to their mother. Survival for this procedure is generally 100%.
  • immunosuppression with cyclosporin A (10 mg/kg/day for 7 days, 8 mg/kg/day thereafter) is used to enhance survival.
  • animals are anesthetized (65 mg/kg sodium pentobarbital) and perfused intracardially first with saline plus 2 units/ml heparin (Elkins-Sinn, Inc., Cherry Hill NJ) then with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer, pH 7.4.
  • PFA paraformaldehyde
  • the central nervous system is dissected into coronal sections and post-fixed in 4% PFA for 6 hours at 4°C, washed for 24 hours in PBS
  • the motor function of normal, transgenic, and transplanted shiverer mice (30) is determined using the rotarod test (31).
  • Transplanted litters receive a single cell type and are coded at the time of injection, and animals are weaned and separated by gender at 3 to 4 weeks.
  • the rotarod device consists of a 1 " diameter wooden rod powered by a variable speed motor, suspended 18" over a drop box, assembled such that the animals walk toward the experimenter in treadmill fashion. Each animal is tested for four consecutive days at between 6 and 7 weeks of age, selected in random order by lottery each test day, with the experimenter single blind to the transplant condition. The cumulative number of falls during one minute at each of two test speeds (12, 18 rpm) is totaled, with the timing stopped when an animal is off the rod (30).
  • reporter gene (CAT, Luc) enzymatic activity the cell monolayers are washed with PBS then removed from the culture dish by scraping into 0.1 ml of Reporter Lysis Buffer (Promega Biotech, Madison WI) as described (Promega Protocols). The cells are then lysed by three cycles of freeze- thaw (-70°C, 37°C) and centrifuged to clear cellular debris. Luciferase activity is measured in triplicate on 10 ⁇ l aliquots in 100 ⁇ l of 0.1 M KPO4 (pH 7.8), 15 mM MgSO 5 mM ATP, using a Monolight 2010 Luminometer (Analytical Luminescence Lab. , San Diego) with a 10 second window and 1 mM D-luciferin in 0.1 M KPO4 (pH 7.8) as substrate. Luciferase activity measured in Relative Light
  • CAT activity is measured using [* 4 C]-cMoramphenicol (Amersham) by liquid scintillation counting, as described (Promega Protocols).
  • pMo.FGFRl.iresNeo ( Figure 2, graph B) contains the murine FGFR1 cDNA (32), while pMo.FGFRx contains a stop codon introduced by insertion of an Xbal linker and encodes a truncated FGFR1 (32) and acts in a dominant-negative fashion (33) to interfere with signaling through multiple types of endogenous FGF receptors (34) when introduced into cultured cells (32).
  • the expression vector pMo.iresNeo ( Figure 2, graph A) was constructed by excising an EcoRI fragment from pMo.FGFRl.iresNeo, generating a plasmid lacking mFGFRl.
  • hPDGFR ⁇ A truncated (dominant-negative) form of hPDGFR ⁇ was produced in pGEM3Z.
  • hPDGFR ⁇ by Klenow polymerase fill-in of a unique Spel site (nucleotide position 1929), producing a frame-shift mutation at codon 594 immediately downstream of the transmembrane coding domain.
  • a 3.4 kb BamHI fragment encoding the mutant version of hPDGFR ⁇ was then inserted into pMo.iresNeo, generating pMo.hPDGFRx.iresNeo.
  • oligodendrocyte progenitor cells purified from mixed glial cell cultures of the neonatal rat forebrain are immunoreactive with monoclonal antibodies A2B5 (18,19).
  • monoclonal antibodies A2B5 When cultured in the presence of bFGF they acquire immunoreactivity to monoclonal O4 antibody ( Figure 1, panel A, panels i-iii), and in the absence of mitogens they differentiate into postmitotic, galactocerebroside- positive oligodendrocytes ( Figure 1, panel A, panel iv).
  • These precursor cells can be stimulated to divide in the presence of mitogens including bFGF, PDGF, and conditioned medium from neuroblastoma B104 cells (B104-CM) ( Figure 1, panel B).
  • the primary rat oligodendrocyte progenitor cultures were expanded for over 20 passages, including repeated freezing then retrieval from liquid nitrogen storage, as originally described to generate the oligodendrocyte progenitor cell line CG-4 (22).
  • these cells are stellate and non motile ( Figure 1, panel A, panel v), and they differentiate into OLs (50% Ol + after 72 hours) upon removal of mitogens ( Figure 1, panel A, panels vi, vii).
  • These mitogen-expanded progenitors also form myelinating OLs after transplantation in vivo (30,36).
  • mitogen expanded oligodendrocyte precursors can lose both their dependence on mitogens for expansion, and their ability to differentiate upon withdrawal of mitogens (R. McKinnon, unpublished). This loss of mitogen dependence for proliferation correlates with abnormal karyotypes (Table 1), and may represent a selection for mitogen-independent cells to expand within these cultures upon extended subculturing. It is presently not clear whether such mitogen-independent cultures are able to differentiate after transplantation in vivo.
  • the use of mitogens to expand primary oligodendrocyte precursors for in vitro analysis therefore requires careful monitoring, to ensure that the cells under study retain the ability to differentiate upon mitogen withdrawal, as a criteria for further analysis.
  • Oligodendrocyte progenitor cells can be infected with both papova virus (SV40) and ecotrophic murine retroviral vectors, and the retroviral vectors LZl and DAP (23,24) were used to introduce ⁇ -galactosidase and alkaline phosphatase, respectively.
  • SV40 papova virus
  • LZl and DAP LZl and DAP
  • Critical parameters for transient transfection of oligodendrocyte progenitors include the concentration of carrier DNA and the time of exposure to the DNA co-precipitate ( Figure 2).
  • CAT chloramphenicol acetyl transferase
  • Stable transfection requires the integration of transgenes in a chromatin conformation allowing transcriptional activation.
  • vector pMo.iresNeo Figure 3, panel A
  • the LTR drives transcription of a polycistronic mRNA encoding the cDNA, inserted in at the polylinker site, and the neo gene.
  • G418 resistant cells must, barring intramolecular recombination events, express an RNA transcript encoding the upstream cDNA transgene.
  • the efficiency of stable gene transfer into oligodendrocyte progenitors using the plasmid vectors shown in Figure 3 is approximately 50 colonies per ⁇ g per IO 5 cells (0.05%).
  • Oligodendrocyte progenitors expressing the FGFRx transgene were specifically non-responsive to bFGF stimulation, as revealed by Fura-2 measurements of intracellular free Ca + + and by ⁇ H-thymidine incorporation studies (data not shown).
  • the vectors shown in Figure 2 are competent for high level expression of cDNA encoding dominant-negative transgenes when introduced into primary oligodendrocyte progenitor cell cultures.
  • oligodendrocyte progenitor cells The ability to isolate primary oligodendrocyte progenitor cells, manipulate them in culture, then reintroduce these cells back into an animal allows an analysis of progenitor cell fate in vivo under a variety of experimental conditions.
  • Transplanting oligodendrocyte precursors into the newborn rodent central nervous system allows an analysis of their fate during normal myelin development.
  • Transplanting cells into an adult brain, after induction of a demyelinating lesion allows an examination of the participation of grafted cells in brain repair.
  • Transplanting genetically engineered cells into wild type host allows a direct examination of the consequences of specific gene manipulations on the grafted cells, since the effects of the mutations should be independent of other cells in the transplant environment. Mutagenesis of cells in vitro, or "somatic transgenics", thus can facilitate a type of analysis that may not otherwise be possible using either classical genetics or targeted disruption of germ line genes.
  • PKH26 labeled wild type (O2A 2 ) cells were observed distributed in brain parenchyma, including regions of axonal tracts such as the internal capsule ( Figure 4, panel A) as well as hippocampal fimbria and the corpus callosum.
  • LZl labeled cells ⁇ -galactosidase positive cells were found two weeks post transplant that had apparently migrated through the internal capsule as far as 5 mm rostral to the site of injection ( Figure 4, panel B).
  • oligodendrocyte progenitor cells expressing the dominant negative FGFRx transgene did not migrate into brain parenchyma, and were found either at the site of injection or within the ventricles three days post transplant (43). Mutant cells thus behave similar to oligodendrocyte progenitors that have been allowed to mature into the 04 + stage of maturation prior to transplantation (44), suggesting that FGF-signaling may be essential for O4 + cells to revert to the migratory A2B5 + state for migration in vivo. This analysis thus indicates the potential of cell transplantation to address issues of gene function during central nervous system development.
  • MBP sm neonatal shiverer mice central nervous system
  • Shiverer have a deletion of the gene encoding myelin basic protein (MBP) resulting in uncompacted central nervous system myelin lacking major dense line, and has been used extensively for oligodendrocyte transplantation studies (45,46).
  • MBP myelin basic protein
  • the presence of MBP in transplant recipients confirms the survival and differentiation of grafted cells, and our analysis indicated that both late passage oligodendrocyte progenitors (02 A , passage 6-14) and CG4 cells (passage 25) are able to survive and differentiate into MBP+ OLs (30).
  • Transplanted oligodendrocyte progenitor cells have been shown to produce extensive myelin in neonatal and adult recipients (36,44,47), and glial grafts can enhance action potential conduction in myelin-deficient rats (48). Since shiverers have characteristic motor dysfunction (49), we adapted a classical motor function test to our transplant analysis in order to quantitatively assess the effects of oligodendrocyte progenitor grafts (30). These studies have demonstrated that the rotarod (31), a forced activity which tests for balance and coordination (50), could discriminate between the motor function of shi/shi mutants and shi shi mice which received transplants of wild type oligodendrocyte progenitors cells (Figure 5).
  • Figure 1 illustrates mitogen expanded primary oligodendrocyte progenitor cells.
  • Panel A top illustrates photomicrographs of primary O-2A progenitor cells (panels i-iv) (52) and mitogen-expanded oligodendrocyte cultures (panels v-vii), immunostained with monoclonal 04 (panels i-iii, vii), RmAb (panel iv), or in phase contrast (panels v, vi).
  • Panel B (bottom) is a graph illustrating stimulation of DNA synthesis in mitogen-expanded oligodendrocyte cultures, as measured by ⁇ H-thymidine incorporation in the absence (open) or presence (closed boxes) of 10 ⁇ g/ml insulin, in cultures treated for 24 hours with (1) DMEM, (2) oligodendrocyte media, (3) oligodendrocyte plus 5 ng/ml bFGF, (4) oligodendrocyte plus 10 ng/ml PDGF-AA, and (5) oligodendrocyte plus 5% B104- CM.
  • Figure 2 shows three graphs illustrating transient transfection of primary O-2A cells.
  • Graph A shows expression of firefly luciferase in oligodendrocyte progenitor cells exposed for 72 hours to a DNA precipitate containing 10 ⁇ g RSVluc;
  • graph B shows to 10 ⁇ g RSVluc plus increasing concentrations of carrier DNA;
  • graph C shows 10 ⁇ g each of RSVluc plus carrier DNA harvested at the indicated time points.
  • FIG. 3 illustrates expression vectors for stable transfection.
  • FIG. 1 shows pMo.iresNeo, including Moloney LTR promoter, the internal ribosome entry sequence (IRES), and neomycin (G418 ) gene.
  • Panel (B) shows pMo.FGFRl.iresNeo, with cDNA insert encoding the murine FGFR1 receptor (53) (solid box). The arrow indicates the location of an in-frame stop codon introduced in construct FGFRx, encoding a dominant negative version of FGFR1 (32).
  • Figure 4 illustrates transplantation of O-2A progenitor cells in vivo and the location of rat oligodendrocyte progenitor cells transplanted into neonatal rat brain.
  • Panel (A) shows PKH-26 dye labeled cells, 72 hours post transplant, in internal capsule.
  • Panel (B) shows ⁇ -galactosidase labeled oligodendrocyte progenitor cells, 12 days post transplant, scattered through brain parenchyma (indicated by asterisks). The arrow indicates the site of transplant.
  • Figure 5 is a graph illustrating motor function of normal and shiverer mutant mice in terms of cumulative falls from a rotarod of homozygous mutant shiverer (shi) mice, shi mice that are heterozygous (shi/mbpl) or homozygous (shi/mbp2) for an MBP transgene (54), shi mice tiiat received grafts of wild type oligodendrocyte progenitor cells at birth (shi/tspt), and heterozygous (shi +) mice (30).

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Abstract

Cette invention concerne un procédé permettant de transplanter dans le système nerveux central d'un patient, des cellules primaires obtenues par génie génétique. Ce procédé comprend les étapes suivantes: (a) isoler des cellules progénitrices d'oligodendrocytes primaires à partir du cerveau du patient; (b) développer ces cellules in vitro avec des mitogènes; (c) modifier les cellules par génie génétique en introduisant des transgènes par l'intermédiaire d'un vecteur d'expression eucaryote pour le transfert de gènes par ADN dans les cellules; et (d) transplanter les cellules primaires obtenues par génie génétique dans le cerveau du patient.
PCT/US1997/003094 1996-03-04 1997-02-28 Oligodendrocytes primaires obtenus par genie genetique et destines a l'administration de genes par transplantation dans le systeme nerveux central WO1997032608A1 (fr)

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Cited By (7)

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US7285415B2 (en) 2002-07-11 2007-10-23 The Regents Of The University Of California Oligodendrocytes derived from human embryonic stem cells for remyelination and treatment of spinal cord injury
US7579188B2 (en) 2002-07-11 2009-08-25 The Regents Of The University Of California Oligodendrocytes derived from human embryonic stem cells for remyelination and treatment of spinal cord injury
US8367615B2 (en) 2006-03-30 2013-02-05 Research Foundation Of City University Of New York Stimulation of neuron regeneration by secretory leukocyte protease inhibitor
US8513009B2 (en) 2008-01-30 2013-08-20 Geron Corporation Synthetic surfaces for culturing stem cell derived oligodendrocyte progenitor cells
US8647874B2 (en) 2005-06-16 2014-02-11 Ramot At Tel-Aviv University Ltd. Isolated cells and populations comprising same for the treatment of CNS diseases
US8663987B2 (en) 2008-05-28 2014-03-04 Ramot At Tel-Aviv University Ltd. Mesenchymal stem cells for the treatment of CNS diseases
WO2017153982A1 (fr) 2016-03-06 2017-09-14 Yeda Research And Development Co. Ltd. Procédé de modulation de la myélinisation

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Cited By (16)

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US7579188B2 (en) 2002-07-11 2009-08-25 The Regents Of The University Of California Oligodendrocytes derived from human embryonic stem cells for remyelination and treatment of spinal cord injury
EP2273268A2 (fr) 2002-07-11 2011-01-12 The Regents of The University of California Oligodendrocytes dérivés de cellules souches embryonnaires humaines pour remyélinisation et traitement de lésion de la moelle épinière
US10611998B2 (en) 2002-07-11 2020-04-07 The Regents Of The University Of California Oligodendrocytes derived from human embryonic stem cells for remyelination and treatment of spinal cord injury
US7285415B2 (en) 2002-07-11 2007-10-23 The Regents Of The University Of California Oligodendrocytes derived from human embryonic stem cells for remyelination and treatment of spinal cord injury
US9879225B2 (en) 2005-06-16 2018-01-30 Ramot At Tel-Aviv University Ltd. Isolated cells and populations comprising same for the treatment of CNS diseases
US10869899B2 (en) 2005-06-16 2020-12-22 Ramot At Tel-Aviv University Ltd. Isolated cells and populations comprising same for the treatment of CNS diseases
US8647874B2 (en) 2005-06-16 2014-02-11 Ramot At Tel-Aviv University Ltd. Isolated cells and populations comprising same for the treatment of CNS diseases
US8367615B2 (en) 2006-03-30 2013-02-05 Research Foundation Of City University Of New York Stimulation of neuron regeneration by secretory leukocyte protease inhibitor
US10221390B2 (en) 2008-01-30 2019-03-05 Asterias Biotherapeutics, Inc. Synthetic surfaces for culturing stem cell derived oligodendrocyte progenitor cells
US8513009B2 (en) 2008-01-30 2013-08-20 Geron Corporation Synthetic surfaces for culturing stem cell derived oligodendrocyte progenitor cells
US9474787B2 (en) 2008-05-28 2016-10-25 Ramot At Tel-Aviv University Ltd. Mesenchymal stem cells for the treatment of CNS diseases
US10052363B2 (en) 2008-05-28 2018-08-21 Ramot At Tel-Aviv University Ltd. Mesenchymal stem cells for the treatment of CNS diseases
US8900574B2 (en) 2008-05-28 2014-12-02 Ramot At Tel-Aviv University Ltd. Mesenchymal stem cells for the treatment of CNS diseases
US8663987B2 (en) 2008-05-28 2014-03-04 Ramot At Tel-Aviv University Ltd. Mesenchymal stem cells for the treatment of CNS diseases
US11185572B2 (en) 2008-05-28 2021-11-30 Ramot At Tel-Aviv University Ltd. Mesenchymal stem cells for the treatment of CNS diseases
WO2017153982A1 (fr) 2016-03-06 2017-09-14 Yeda Research And Development Co. Ltd. Procédé de modulation de la myélinisation

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