WO1997032608A1 - Genetically engineered primary oligodendrocytes for transplantation-mediated gene delivery in the central nervous system - Google Patents

Abstract

The present invention pertains to a method for transplanting 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 with mitogens; (c) genetically engineering the cells by introducing transgenes through an eukaryotic expression vector for DNA-mediated gene transfer into the cells; and (d) transplanting the genetically engineered primary cells into the brain of the patient.

Description

GENEΗCALLY ENGINEERED PRIMARY OLIGODENDROCYTES l o FOR TRANSPLANT AΉON-MIΦIATED GENE DELIVERY

IN THE CENTRAL NERVOUS SYSTEM

BACKGROUND OF THE INVENTION

15 Field of the Invention

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.

Description of the Background

30

The disclosures referred to herein to illustrate the background of the invention and to provide additional detail with respect to its practice are incorporated herein by reference and, for convenience, are numerically referenced in the following text and respectively grouped in the appended bibliography.

35

Our understanding of the basis for many human neurological and neurodegenerative diseases is rapidly out pacing the development of therapeutic intervention strategies to treat such disorders. In numerous examples, effective treatments will require an effective system for gene delivery into the central nervous

40 system in order to provide a stable and continuous source of therapeutic gene products in the brain. Strategies currently under development include the direct injection of recombinant DNA or the use of viral vectors encoding within tiieir genomes specific sequences of recombinant DNA, which will be incorporated into endogenous cells within the central nervous system and therein be expressed as recombinant (therapeutic) proteins.

The Oligodendrocyte Lineage

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.

Studies on the oligodendrocyte lineage in vivo have demonstrated that 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). In vitro, 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). Lineage analysis using a panel of monoclonal antibodies has demonstrated that 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). Knowledge of growth factors involved in central nervous system myelin formation, as in neural development in general, comes largely from in vitro studies which suggest specific roles for a number of cytokines during myelin development, destruction, and repair

(6). Individually or in combination, these factors affect oligodendrocyte progenitor cell proliferation (bFGF, PDGF, IGF-1, NT-3), migration (PDGF), differentiation (IGF-1, TGF-β) and survival (PDGF, bFGF, IGF-1, CNTF). Several of these including PDGF act via transmembrane receptors with intrinsic ligand activated tyrosine kinase activity, or receptor tyrosine kinases (7). Oligodendrocyte progenitors express exclusively the α-chain form of PDGF receptors (PDGFRα) (8), and PDGFRα 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.

Somatic Transgenics

For many growth factor signaling systems, germ line mutations of either ligand or receptor genes results in early embryonic lethal phenotypes, as seen in Pdgfra^Pn (Patch) (10-12) and Fgfrl^ml1 (13,14) mutant mice. To date only two examples of altered signal transduction pathways are known to affect central nervous system myelin in vivo, the non-receptor tyrosine kinase yn (15) and IGF-1

(16,17). These issues point to the necessity of an alternative genetic approach to define the roles of specific molecules in vivo. In vitro mutagenesis of somatic cells, followed by transplantation in vivo, allows an analysis of specifically engineered somatic cells in a normal recipient animal.

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.

SUMMARY OF THE INVENTION

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;

(b) expanding the cells in vitro with mitogens;

(c) genetically engineering the cells by introducing transgenes through an eukaryotic expression vector for DNA-mediated gene transfer into the cells; and

(d) transplanting the genetically engineered primary cells into the brain of the patient.

BRIEF DESCRIPTION OF THE FIGURES

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

(panels v-vii), immunostained with monoclonal O4 (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 μg/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 μg RSVluc; graph B shows to 10 μg RSVluc plus increasing concentrations of carrier DNA; and graph C shows 10 μg 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) (bottom) shows pMo.PDGFRα.iresNeo, with the 3.4 kilobase human PDGFRα 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. 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 β-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

(30).

DETAILED DESCRIPTION OF THE INVENTION

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. Transplantation of either wild-type or genetically engineered primary cells into normal and myelin-deficient hosts allows an analysis of the effects of gene manipulations on this cell lineage in vivo. The application of these approaches for the analysis of growth factor receptor function during oligodendrocyte development is described.

The present invention is further illustrated by the following examples which are not intended to limit the effective scope of the claims. All parts and percentages in the examples and throughout the specification and claims are by weight of the final composition unless otherwise specified. Examples

Animals include Sprague-Dawley rat pups (Taconic Farms, NY), and shiverer (MBP^sn*) mutant mice obtained from L. Rhodes Jr. (NINDS, NIH

Bethesda MD). 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). Recombinant growth factors bFGF (FGF-2) and PDGF (AA homodimer), obtained from Upstate Biotechnology Inc. (Lake Placid, NY), were stored in 10 μg/ml aliquots at -70°C in 0.04 N HCL plus 1 mg/ml bovine serum albumen. Geneticin (G418 sulphate, Gibco/BRL, Bethesda MD) was dissolved at 40 mg/ml in PBS, and used at 400 μg/ml for selection of G418^R transformed cell colonies and at 200 μg/ml for subculture of established cell lines. 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

MA), respectively. Plasmid vectors encoding firefly luciferase (Luc), chloramphenicol acetyltransferase (CAT), and growth factor receptors PDGFRα 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.

Primary Mixed Glial Cultures

Mixed 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

Hepes buffered media (MEM Hepes, Gibco/BRL). Using No. 5 forceps, 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. In a sterile culture hood, 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

21 gauge needles, then one pass through a 25 gauge needle. The suspension is then filtered through a cell strainer mesh (Falcon, No. 2350), centrifuged (1,000 rpm, 10 minutes), then resuspended in DMEM (Gibco/BRL, high glucose) containing 10% fetal bovine sera (FBS, Gibco/BRL) at a final volume of 10 ml DMEM/FBS per 2 original brains. In our experience, avoiding heat inactivation of FBS consistently gives the best rate of growth of these primary cultures. This cell suspension is plated in Falcon 75 cm² tissue culture flasks (10 ml per flask) and place in a 37°C incubator (10% CO2, 95% relative humidity). 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). On day three (first refeed) there is considerable 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.

Purification of 0-2 A Progenitor Cells

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. After a 10 minute incubation, 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 2xl0⁵ cells per ml in prewarmed (37°C) DMEM containing sodium pyruvate, penicillin-streptomycin, 0.5% FBS, 50 μg/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 μM selenium, 30 μM T3; Sigma Chemicals, St. Louis MO) are frozen in aliquots at -20°C, and bovine insulin is dissolved at 10 mg/ml in 0.01 N HCI and stored at 4°C. Resuspended cells are plated on polyornathine coated coverslips (Bellco Glass, Vineland NJ) or

Falcon tissue culture dishes, at a density of "100 cells/mm2 (1x10^ cells/12 mm coverslip, or 3xl0⁵ cells/60 mm dishes). Coated surfaces are prepared in advance by incubating culture dishes at room temperature for 30 minutes with 100 μg/ml polyormthine (Sigma Chemicals), diluted in H2O from a 1.0 mg/ml stock stored at 4°C in 0.15 M of Boric Acid, 0.15 M NaOH (pH 8.4), then removed by extensive washes in H2O. 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). The addition of 5 ng/ml bFGF (8), bFGF plus 10 ng/ml PDGF-AA (21), or 20% vol of B104 neuroblastoma conditioned media (B104-CM)

(22), will maintain these primary cultures as mitotic oligodendrocyte progenitor cells. For cell surface immunohistochemistry, cells are fixed in 4% paraformaldehyde and incubated with tissue culture supematants of monoclonal antibodies A2B5, Ol, O4, or RmAb, followed by fluorescent secondary conjugates (Pierce Chemical Co., Rockford, IL), and photographed on a Zeiss Axiovert lOOTV microscope using a 40X achrostigmat lens as described (8).

Secondary Oligodendrocyte Progenitor Cultures

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/mm², then filtered (0.45 μm) and aliquots stored at -20°C. Oligodendrocyte progenitor cells growing in OLΛB 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 . For cryoprotection, detached cells are resuspended at lxlO⁶ 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.

Karyotype Analysis

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 μg/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

Balanced Salt Solution, then at room temperature for 2.5 minutes in Harleco Giemsa Stain (EM Diagnostics) diluted in Gurr Buffer (Gibco/BRL) prepared fresh (0.4 ml Giemsa in 5 ml Gurr buffer), then rinsed in water and mounted for viewing.

Thymidine Incorporation

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 μCi/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.

Retroviral-Mediated Gene Transfer

Viral producer cells LZ1 (23) and DAP (24) are expanded in DMEM/FBS in monolayer cultures. For viral infection of oligodendrocyte progenitor cultures, producer cells are plated in Falcon 12 mm cell culture inserts (1.0 μm pore size) maintained in Falcon 12 well plates in DMEM/FBS. When confluent, 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. For DAP, 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 μg/ml G418. For detection of β- galactosidase (LZl) or alkaline phosphatase (DAP), cells are transferred to coverslips for staining. For tissue sections from animals transplanted with retroviral infected cells, sections are processed on siliconized glass slides, β- galactosidase staining is performed at 4°C for 2-12 hours with X-gal (5-Bromo-4- chloro-3-indolyl-β-D-galactopyranoside, Fisher Biotech) in 5 mM K3Fe(CN)₍>, 5 mM K4Fe(CN)6, 2 mM MgCl2 as described (23). For 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

(room temperature, 2-24 hours) with 0.1 mg/ml 5-bromo-4-chloro-3-indolyl phosphate, 1 mg/ml nitroblue tetrazolium, with 0.1 mg/ml levamisol (Kirdegaard and Perry Labs Inc., Gaithersburg, MD) included to inhibit endogenous alkaline phosphatase, as described (24). Slides are mounted in Permount (Fisher Scientific) and examined by bright field microscopy.

DNA-mediated Gene Transfer

Gene transfer into mitogen-expanded oligodendrocyte progenitor cultures is performed using the calcium phosphate DNA co-precipitation technique

(the "calcium technique") (25,26) using plasmid DNA samples purified by CsCl gradient centrifugation (27). Transfections are performed with lxl 0⁵ cells/60 mm dish containing 5 ml of OIJB media plus 10% FBS, using 1-2 μg/dish plasmid DNA (empirically dependant on the transfection efficiency of individual constructs) plus 10 μg per dish of carrier DNA prepared from rat tissue or rat cell lines (28).

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). 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. For transient transfection studies, the cells are harvested after 48-72 hours exposure to the precipitate. For selection of drug resistant colonies such as transposon Tn5-derived neo vectors (29), the cultures are refed after 48 hours with OL/B medium containing 400 μg/ml G418 and refed with fresh medium every 3 days, and G418 concentrations reduced to 200 μg/ml after colonies are visible (7-10 days).

Cell Transplantation

For transplantation, 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. For neo cultures, cells are washed with PBS and cultured for at least 24 hours in medium lacking G418 prior to transplant. Cells lacking heritable markers (DAP, β-gal) are labeled for 5 minutes in vitro, immediately before injection, with the red fluorescent marker PKH26 (Sigma Immunochemicals) using conditions described by the manufacturer. Generally 50,000 cells are transplanted per pup, in a 1 μl 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%. Where graft rejection is a concern, immunosuppression with cyclosporin A (10 mg/kg/day for 7 days, 8 mg/kg/day thereafter) is used to enhance survival.

For analysis, 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. 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

(pH 7.6), then frozen in embedding compound (OCT, Miles Laboratories) and 20 μm frozen sections obtained on a cryostat. For BrdU analysis, animals are injected at 2 hour intervals (5 injections ip) prior to harvest with 100 μl of 2 mg/ml BrdU (Sigma), and incorporation is determined by identification of PKH26⁺/BrdU⁺ cells by immunohistochemistry (anti-BrdU, Becton Dickenson). For immunohistochemical studies, sections are stained overnight at 4°C in a humid chamber with primary antibodies, and for 2 hours with fluorescent-conjugated secondary antibodies, then mounted and visualized by fluorescence microscopy. Motor Function Analysis

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).

Biochemical Techniques

For determining 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 μl aliquots in 100 μl 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

Units (RLU) represents the mean +/- s.d. above background (RLU=200). CAT activity is measured using [*⁴C]-cMoramphenicol (Amersham) by liquid scintillation counting, as described (Promega Protocols).

Recombinant DN Constructs

The construct 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. This was then digested with BamHI (unique site 3' of Neo) and the recessed ends filled in with Klenow DNA polymerase plus dNTPs, then ligated to give a construct lacking a BamHI site. An EcoRI polylinker from plasmid pUC475B (R.McKinnon, unpublished) was then ligated into the unique EcoRI site 5' of IRES sequence, generating pMo.iresNeo (Figure 2, graph A). A 3.4 kb BamHI cDNA including the entire coding region of hPDGFRα (35) was inserted into pMo.iresNeo, generating pMo.hPDGFRα.iresNeo (Figure 2, graph C). A truncated (dominant-negative) form of hPDGFRα was produced in pGEM3Z. hPDGFRα 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 hPDGFRα was then inserted into pMo.iresNeo, generating pMo.hPDGFRx.iresNeo.

Results and Discussion

Mitogen Expansion of Oligodendrocyte Progenitor Cells

Primary oligodendrocyte progenitor cells purified from mixed glial cell cultures of the neonatal rat forebrain are immunoreactive with monoclonal antibodies A2B5 (18,19). 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). Using B104-CM, 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). As with bFGF-expanded oligodendrocyte progenitors, 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).

After extended passages in vitro, 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.

DNA Mediated Gene Transfer in Oligodendrocyte Progenitors

A variety of approaches were investigated for gene transfer into primary oligodendrocyte progenitor cells, including the use of viral vectors, electroporation, lipofectin, and calcium-mediated approaches. Oligodendrocyte progenitors 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 β-galactosidase and alkaline phosphatase, respectively. When oligodendrocyte precursors were co-cultured with DAP producer cells, G418- resistant OL*^^ precursors were obtained within one week and clonal G418 transformants were successfully isolated and expanded.

The use of DNA-mediated gene transfer approaches was examined for oligodendrocyte transfection using DNA-CaPO4 co-precipitation (the "calcium technique"), originally developed for oncogenic transformation with adenovirus DNA (25), and proven optimal for transfection of many mammalian cell types (26). Using firefly luciferase reporter constructs (37,38) in a transient transfection assay, we determined that the calcium technique was comparable to lipofectin and at least

100-fold more efficient than electroporation. 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). Using this approach, high level expression of chloramphenicol acetyl transferase (CAT) reporter genes was obtained under transcriptional regulation of several oligodendrocyte-lineage promoter elements, including pdgfra (39), the myelin genes MBP and PLP (40), and JC5' (data not shown).

Stable Expression of Transgenes in Oligodendrocyte Cultures

Stable transfection requires the integration of transgenes in a chromatin conformation allowing transcriptional activation. In order to facilitate high levels of transgene expression, we constructed the vector pMo.iresNeo (Figure 3, panel A) encoding a retroviral LTR promoter, a multicloning polylinker site, an internal ribosome entry sequence (IRES), and the neo (G418^R) drug resistance gene for selection of transformants (29). The LTR drives transcription of a polycistronic mRNA encoding the cDNA, inserted in at the polylinker site, and the neo gene. Translation initiation at the first open reading frame produces the cDNA product, and ribosome reassembly at the IRES allows reinitiation of translation of neomycin phosphotransferase (G418 ) (41). Thus G418 resistant cells must, barring intramolecular recombination events, express an RNA transcript encoding the upstream cDNA transgene. Using the calcium technique, the efficiency of stable gene transfer into oligodendrocyte progenitors using the plasmid vectors shown in Figure 3 is approximately 50 colonies per μg per IO⁵ cells (0.05%). Dose response studies in oligodendrocyte progenitor cultures yield a non-linear relationship, with few to no colonies obtained at DNA concentrations predicted to generate < 10-15 colonies per dish, suggesting paracrine effects of oligodendrocyte progenitor cells required for survival of transfectant colonies.

The potential of this vector for expression of transgenes was examined using cDNAs encoding growth factor receptor genes, including wild type and mutant versions of the murine FGF receptor FGFR1 (32), and the human PDGFRα (35) (Figure 2, graphs B and C). Mutant versions of these receptors encode truncated molecules lacking the cytoplasmic signaling domain, and act in a dominant-negative fashion (33) to interfere with signaling through endogenous receptors when introduced into cultured cells (32,34,42). Northern blot analysis of RNA isolated from clonally selected oligodendrocyte progenitor cells transfected with pMo.FGFRx.iresNeo (Figure 2, graph B) demonstrated that the FGFRx.neo transcript is expressed at a level at least 10-fold higher than the endogenous FGFR transcripts, and [¹²^I]-FGF ligand binding analysis indicated a transgene encoded protein present at a level at least 5-fold more abundant than the endogenous FGFR (43). 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). Thus 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.

Transplantation of Oligodendrocyte Progenitors in Syngeneic Rat Brain

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.

The potential of this approach was examined by transplanting rat oligodendrocyte progenitor cells into syngeneic neonatal rat thalamus, after labeling cells in vitro with the fluorescent dye PKH26 and after labeling cells by infection with the retroviral vector LZl. After 3 days, PKH26 labeled wild type (O2A²) 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. With LZl labeled cells, β-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).

In contrast to wild type cells, oligodendrocyte progenitor cells expressing the dominant negative FGFRx transgene (Figure 2, graph B) 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.

Transplantation of Oligodendrocyte Progenitors in Shiverer Mice - Motor Function Analysis

Using a second transplantation paradigm, we have transplanted wild type oligodendrocyte progenitors into neonatal shiverer (MBP^sm) mice central nervous system (30). 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). 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). This test is also able to discriminate shi/shi mice carrying either one or two copies of an MBP transgene (51) from non-transgenic mutant and unaffected shi/+ littermate controls (Figure 5). These observations indicate that the rotarod is a sensitive measure of motor function in animals with compromised central nervous system myelin, and is useful for assessing the results of experimental manipulations including cell transplantation.

These studies demonstrate the utility of cell transplant studies for in vivo analysis of oligodendrocyte development. The ability to introduce oligodendrocyte precursor cells with specific and defined mutations in growth factor receptor genes, and follow their fate after transplantation into the neonatal brain, allows a type of analysis not possible with germ line mutations. This approach therefore has the potential to reveal important insights into the role of specific gene products during oligodendrocyte development in vivo. The application of these approaches to other cell types in the central nervous system for which mitotic precursors can be isolated and manipulated in vitro, including macroglia, microglia and neural stem cells, should lead to further insights into the role of specific gene products in brain development, defense and repair. Table 1

Karyotype Analysis of Mitogen-Expanded Oligodendrocyte Progenitor Cell

Cultures

Cell type Passage n Mean ± sd Mode

rat brain 0 13 43 ± 7 43

O2A2 5 34 53 ± 8 50

O2A.2-1 4 23 40 ± 3 42

O2A3 13 21 57 ± 5 54

O2A3 DAP 14 67 63 ± 11 60

CG-4 23 43 52 ± 9 52

Karyotype analysis of primary brain endothelial cells, CG4 cells (22), mitogen expanded primary oligodendrocyte progenitor cultures O2 A² and O2 A^ (independent isolates), and derivatives of these cultures including 2-1 (clonal derivatives of 02 A² expressing hPDGFRα), and DAP (non-clonal derivatives of O2A^ expressing alkaline phosphatase).

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 μg/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 μg RSVluc; graph B shows to 10 μg RSVluc plus increasing concentrations of carrier DNA; and graph C shows 10 μg 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 ) 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) (bottom) shows pMo. PDGFRα. iresNeo, with the 3.4 kilobase human PDGFRα 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 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 β-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).

Throughout this application, various publications have been referenced. The disclosures in these publications are incorporated herein by reference in order to more fully describe the state of the art.

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While the invention has been particularly described in terms of specific embodiments, those skilled in the art will understand in view of the present disclosure that numerous variations and modifications upon the invention are now enabled, which variations and modifications are not to be regarded as a departure from the spirit and scope of the invention. Accordingly, the invention is to be broadly construed and limited only by the scope and spirit of the following claims.

Claims

We claim:

1. 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;

(b) expanding the cells in vitro with mitogens;

(c) genetically engineering the cells by introducing transgenes through an eukaryotic expression vector for DNA-mediated gene transfer into the cells; and (d) transplanting the genetically engineered primary cells into the brain of the patient.

2. The method according to claim 1, wherein the primary oligodendrocyte progenitor cells in step (a) are O-2A progemtor cells.

3. The method according to claim 1, wherein the mitogens in step (b) are bFGF, PDGF, or conditioned medium from neuroblastoma B104 cells (B104-CM).

4. The method according to claim 1, wherein the eukaryotic expression vector in step (c) is pMo.iresNeo.

5. The method according to claim 1, wherein DNA-mediated gene transfer into the cells in step (c) is carried out using DNA-CaPO4 co-precipitation, electroporation, or lipofectin.

6. 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;

(b) expanding the cells in vitro with mitogens;

(c) genetically engineering the cells by introducing transgenes through a retro viral-mediated gene transfer into the cells; and

(d) transplanting the genetically engineered primary cells into the brain of the patient.

7. The method according to claim 6, wherein the primary oligodendrocyte progenitor cells in step (a) are O-2A progenitor cells.

8. The method according to claim 6, wherein the mitogens in step (b) are bFGF, PDGF, or conditioned medium from neuroblastoma B104 cells (B104-CM).

9. The method according to claim 6, wherein the retroviral-mediated gene transfer is carried out with herpes simplex.