A PLASMID DNA CONSTRUCT INCLUDING THE GENE ENCODING A MAMMALIAN BETA-NERVE GROWTH FACTOR

Abstract: A plasmid DNA construct comprising essentially DNA sequences endogenous to mammalian cells excluding DNA sequences of viral origin including the gene encoding a mammalian beta -nerve growth factor ( beta -NGF) or an active polypeptide fragment thereof, and DNA sequences necessary for replicating the plasmid in a bacterial cell; and a method for the treatment of neurodegenerative diseases.

Description: A plasmid DNA construct including the gene encoding a mammalian beta-nerve growth factor. The present invention relates to a plasmid DNA con¬ struct including a gene encoding a nerve growth factor. The invention also includes a method for the treatment of neuro- degenerative diseases in mammals. β-Nerve growth factor (NGF) is a target-derived prote¬ in that in the peripheral nervous system is required for the development and maintenance of sympathetic and sensory neu¬ rons (Levi-Montalcini, R. & Angeletti, P.U. (1968) Physiol. Rev. 8, 534-569; Thoenen, H. & Barde, Y-A. (1980) Physiol. Rev. 60, 1284-1335) . NGF mediates its neurotrophic effects b interaction with specific NGF receptors, and in the central nervous system basal forebrain cholinergic neurons have NGF receptors and retrogradely transport exogenous NGF (Schwab, M.E. , Otten, U., Agid, Y. & Thoenen, H. (1979) Brain Res. 168, 473-483; Seiler, M. & Schwab, M.E. (1984) Brain Res. 300, 33-39) . The target areas of these neurons, hippocampus and cortex, contain the highest levels of NGF mRNA in the brain ( hittemore, S.R., Ebendal, T., Larkfors, L. , Olson, L., Seiger, A., Stromberg, I & Persson, H. (1986) Proc . Natl. Acad.Sci. USA 83, 817-821) . The adult basal forebrain choli¬ nergic neurons respond to exogenous NGF by increasing the levels of choline acetyl transferaεe, the key enzyme in ace- tylcholine synthesis. Cholinergic neurons in the medial sep- turn can be prevented from degeneration after transection of the septohippocampal pathway by chronic infusion of exogenous NGF (Hefti, F. (1986) J. Neurosci. 6, 2155-2162; Kromer, L.F. (1987) Science 235, 214-216). The basal forebrain cholinergic system is associated with cognitive functions and in rodents a strong correlation exists between impairments in learning and memory and atroph of cholinergic neurons. A loss of forebrain cholinergic neu¬ rons occurs during normal aging (Bartus, T.T. , Dean, R.L. , Beer, B. & Lippa, A.S. (1982) Sience, 217, 408-417) and it i conceivable, although by no means proven, that the age-depen dent atrophy is caused by a deficient supply or utilization of one or several trophic factor(s). Interestingly, the le¬ vels of both NGF RNA and protein are signi icantly reduced in the forebrain of aged rats (Larkfors, L., Ebendal, T. , Whittemore, S.R., Persεon, H., Hoffer, B. & Olson, L. (1987) Mol .Brain.Res . 3, 55-60). In addition, intracerebral infusion of NGF in aged rats reduces basal forebrain cholinergic neu¬ ron atrophy and results in an improved spatial memory in be- haviourally impaired rats (Fischer, W., Wictorin, K. , Bjόrk- lund, A., Williams, L.R., Varon, S. & Gage, F.H. (1987) Natu- re (Lond.) 329, 65-68) . A pronounced loss of cholinergic neurons in the basal forebrain is seen in Alzheimer's senile dementia and the cor¬ responding degeneration of cholinergic terminals in cortical areas is thought to be causally linked to the progressive loss of memory and other cognitive disturbances characteris¬ tic of the disease. The main purpose of the present invention is to provi¬ de new techniques enabling successful treatment of neuro- degenerative diseases in mammals including man. Another object of the invention is to provide a novel plasmid DNA construct comprising DNA sequences endogenous to mammalian cells one of which is a mammalian gene encoding a β-nerve growth factor (NGF) , including the human NGF gene enabling expression of said nerve growth factor in eucaryotic cells. Yet another object of the invention is to provide a method for the treatment of neurodegenerative diseases in mammals including man. Still another object of the invention is to provide a method capable of stimulating central cholinergic neurons in a mammal, including man. For these and other purposes which will be clear from the following description the invention provides for a plas¬ mid DNA construct comprising essentially DNA sequences endo- genous to mammalian cells excluding DNA sequences of viral origin including the gene encoding a mammalian β-nerve growth factor (β-NGF) or an active polypeptide fragment thereof, as well as DNA sequences necessary for propagating the plasmid construct in a bacterial cell. According to a preferred embodiment of the invention such plasmid DNA construct contains a DNA sequence encoding the gene for a human β-nerve growth factor (β-NGF). The invention also provides a transfected mammalian host cell which is selected by resistance to an antibiotic marker and therefore constitutes a stable cell line harbou¬ ring a recombinant mammalian expression plasmid vector based on the plasmid DNA construct as defined above. The invention also covers a method for the treatment of neurodegenerativθ diseases in mammals including man. Said method is based on implantation into the brain of a mammal, including man, of an effective amount of the transfected recombinant NGF producing and selected stable mammalian cell as defined above. According to a preferred embodiment of the method of the invention the amount of biologically active recombinant β β-NGF used is effective to stimulate central cholinergic neu- rons in the mammal. In the method of the invention the cells are preferab¬ ly used either in the form of a cell suspension or imbedded in a collagen gel. It is preferred that the transfected mammalian host cell according to this invention is a cell of human origin. The present invention will now be further exemplified by non-limiting examples in conjunction with the appended drawing, wherein: Fig. 1 illustrates construction and analysis of a stable cell line producing biologically active NGF; Fig. 2 illustrates growth responses evoked after im¬ plantation of cells producing recombinant NGF into the rat brain; and Fig. 3 is an illustration of semiquantitative estima- tions of cholinergic fiber densities in cholinergically de- nervated cerabral cortex around grafts of cholinergic neurons and/or fibroblasts. Detailed description of the contents of the drawings. Figure 1 (a) Schematic representation of the OVEC-NGF plasmid. ATG and TGA refer to the initiation and termination codons of preproNGF. Sequences comprising the mature NGF are shown in the hatched box and proteolytic cleavage sites are indicated by arrows. Also the position of the rabbit β-globin TATA-box (TATA) and four tandemly repeated copies of an 18 oligonu- cleotide comprising a metal ion-inducible enhancer from the mouse metallothionein-I gene (MRE) are shown in the figure. Filled boxes indicate exons from the rabbit β-globin gene and the open box represents the second intron from the same gene. (AATAAA) indicates a polyadenylation signal from the β-globin gene . (b) Southern blot analysis of genomic DNA from the parental cell line (3T3) or from the OVEC-NGF transfected cell line (3E), restricted with Bam HI. (c) Northern blot analysis of total RNA prepared from 3T3 cells, 3E cells, or from adult rat brain. The left-hand side of figure lc is from a 1-h exposure of the autoradio- gram, wheras the three right-most lanes are from an 18-h ex¬ posure. The NGF mRNA in the parental 3T3 cells as well as in the rat brain is 1.3kb, whereas the transgene NGF transcript in the 3E cells is 2.2kb. (d) Conditioned medium from 3E cells, or from the pa¬ rental 3T3 cells, were assayed for the presence of NGF in a bioassay using chicken sympathetic ganglia (Ebendal, T., Olson, L., Seiger, A. & Hedlund, K.O. (1980) Nature 286, 25- 28) . Note the dense nerve fiber halo formed with medium from the 3E cell line. Figure 2 (a) AChE histochemistry of a coronal section from cho- linergically denervated cerebral cortex 4 weeks after graf¬ ting of a dissociated cell suspension of embryonic day 17 to 18 rat-basal forebrain cholinergic neurons mixed with 3E cells. Note marked hyperinnvervation by AChE positive fibers in graft area (dark and dense stained area at midright) as well as around grafted neurons (e.g. midleft) and in adjacent cortical neuropil. The inset shows two grafted cholinergic neurons with several nerve fibers formed in response to 3E cells . (b) AChE immunohistochemistry 6 weeks after implanting a 3E cell-rich gel into intact striatum of adult rats. The density of AChE immunoreactive terminals is markedly increa¬ sed around the implant. Higher magnifications of interface between gel implants (left) and adjacent striatal neuropil (right) 4 weeks after implanting gels containing (c) 3E cells, (d) 3T3 cells or (e) no cells. The three different graft types were processed together. Arrow in e indicates one AChE-immunoreactive nerve cell, typical of the type of in¬ trinsic cholinergic neurons found throughout the striatal neuropil. Scale bars, (a) and (b) lOOμm, inset and (c) 50μm. Figure 3 When cholinergic neurons are mixed with or placed ad¬ jacent to 3E cells the fiber density is significantly higher than when grafted neurons are mixed with or placed adjacent to parental 3T3 cells. Implantation of 3E cells in the absen¬ ce of fetal cholinergic grafts caused a local increase in density of intrinsic AChE-positive nerve fibers which is not seen after implantation of parental 3T3 cells. Density of AChE-positive nerve fibers was semiquan itatively estimated on coded slides using a 0-5 scale with 10 steps. Means + s.e.m. Numbers of grafts are given below each bar. Statisti¬ cal evaluations were made within experiments using Student's t-test. ** p<0.01; ***p<0.001. EXAMPLE 1 Isolation of a genetically modified cell line that produces excess levels of recombinant NGF. The 3'-exon of the rat NGF gene (Whittemore , S.R. , Persson, H. , Ebendal , T., Larkfors, L. , Larhammar, D. & Erik- sson, A. (1987) Nato Advanced Study Institutes Series: Life Sciences ( Springer-Verlag, Berlin) 22, 245-256) was restric¬ ted with BstEII and Pstl and a 771bp fragment was isolated and blunt-ended using T4 DNA polymerase. Pstl-linkers were ligated to the fragment followed by insertion into the Pstl site of the plasmid MRE (4x)-0VEC (Westin, G. , Gerster, T. , Muller, M.M., Schaffner, G. & Schaffner, W. (1988) Nucl.Acids Res. 15, 6787-6798). The ligation mixture was transformed into E. coli MC1061. The plasmid containing the rat NGF gene in the correct orientation for translation was purified and cotransfected with a plasmid containing a neomycin resistance gene (neo r ) (molar ratio, 10 OVEC-NGF to 1 neomycin plasmid) into mouse fibroblaεt 3T3 cells using the Ca3(PO4)2/glycerol technique (Graham, F.L. & Van der Eb, A.J. (1973) Virology 52, 456-467). Transfected cells resistant to the neomycin analogue G-418 (1.5mg/ml) were isolated by serial dilution and conditioned medium from the resulting cell clones were assayed for NGF using chicken sympathetic ganglia (Ebendal, T., Olson, L., Seiger, A. & Hedlund, .O. (1980) Nature 286, 25-28) . The transfected cell line 3T3/3E, as well as the pa¬ rental 3T3 cells, grown in DMEM containing 10% fetal calf serum and 2.5 x 10-*-- M ZnSθ4 were disrupted in 4 M guanidine isothiocyanate, 0.025 M sodium citrate pH 7.0, and 0.14 M β- mercaptoethanol, followed by centrifugation on a cushion of 5.7 M CsCl in 0.025 M sodium acetate pH 5.0. The cellular DNA and RNA was separately collected from the gradient and etha- nol precipitated. The DNA was restricted with BamHI, separa- ted on a 0.8% agarose gel followed by transfer to a nitrocel¬ lulose filter. The RNA was separated on a 1% agarose gel con¬ taining 0.7% formaldehyde and then transferred to a nitrocel¬ lulose filter. Both the DNA and the RNA filters were hybridi¬ zed to the rat NGF 771 bp BstEII/Pstl fragment labelled with 0_-( 32 P)-dCTP by nick-translation, followed by high stringency washing and exposure to X-ray films. EXAMPLE 2 Implantation of NGF transfected cells into the rat brain. Under halothane anesthesia, Sprague-Dawley rats weigh¬ ing 280-350 g received 1 μl ibotenic acid (5μg/μl) into nuc¬ leus basalis magneocellularis using the following stereotaxic coordinates (relative to breg a, in mm): AP 0.8-1.0, L 2.4- -2.6, V 6.4-6.6. Infusions were made over a 2 min interval with an additional 5 min to allow for diffusion prior to withdrawal of the syringe. The 3T3/3E and 3T3 cells were grown in vitro in Dulbecco's modified Eagle's medium contai¬ ning 10% fetal calf serum. For grafting, they were mixed wit a dissociated cell suspension of rat-basal forebrain tissue including nucleus basalis magnocellularis from embryonic day 17 to 18. Three weeks after ibotenic acid lesion, cell mixtu res were implanted at several positions in cholinergically denervated frontal and parietal cortex of immunosuppressed hosts (ciclosporin; lOmg/kg, daily) . Four weeks later, rats were sacrificed and sections prepared for acetylcholine este rase (AChE) histochemiεtry or immunohistochemistry using an- tibodies against AChE. For references to these two technique and how they compare with cholineacetyl transferase in evalu ating cholinergic neurons, see Eriksdotter-Nilsson et al. (Eriksdotter-Nilsson, M. , Shirboll, S., Ebendal, T., Mersch, L., Grassi, J. , Massoulie, J., & Olson, L. (1988) Exp.Brain Res. 74, 89-98) . Other cortically denervated animals receive separate adjacent deposits of fetal cholinergic neurons and 3T3 or 3T3/3E cells, and still others only 3T3 or 3T3/3E cells in the form of cell suspensions or carried in collagen gels. NGF-transfected 3T3/3E cells or parental 3T3 cells were also grown in vitro in a three-dimensional collagen gel. Gel containing 3T3/3E cells was stereotaxically implanted on one side of intact adult striatum, whereas the other side recei¬ ved gel containing parental 3T3 cellε. Additional controls included animals implanted with gel alone. The 3T3/3E cell line has been deposited with the Euro¬ pean Collection of Animal Cell Cultures at the PHLS Centre for Applied Microbiology & Research at Porton Down, Salisbu¬ ry, Wiltshire, England, the accession number for the deposit being 89031401. EXAMPLE 3 Construction and characterization of a stable cell line that produces recombinant rat NGF To enable a study of the effects of NGF on cholinergic neurons in vivo . genetically modified mouse 3T3 fibroblasts producing recombinant NGF were established. For this purpose, a DNA fragment from the 3'-exon of the rat NGF gene encoding a preproNGF protein of 241 amino acids (Whitte ore, S.R., Persson, H., Ebendal, T., Larkfors, L., Larhammar, D. & Eric- sson, A. ( 1987) Nato Advanced Study Institutes Series: Life Sciences (Springer-Verlag, Berlin) 22, 245-256) was inserted into the Pstl site of the mammalian expression vector MRE (4x)-0VEC (Westin, G., Gerster, T., Muller, M.M., Schaffner, G. & Schaffner, W. (1988) Nucl.Acids Res. 15, 6787-6798). In this construct, the rat NGF gene is transcribed from a rabbit β-globin promoter in conjunction with a metal ion-inducible enhancer from the mouse metallothionein-I gene (Fig. la) . This construct was cotransfected with a neo r plasmid into mouse fibroblaεt 3T3 cells. Conditioned medium from cells resistant to the neomycin analogue G-418 was tested for en¬ hanced stimulation of fiber outgrowth from embryonic chicken sympathetic ganglia (Ebendal, T., Olson, L., Seiger, A. & Hedlund, K.O. (1980) Nature 286, 25-28) . Southern blot analy¬ sis of genomic DNA from one transfected cell line (3E) , that produced biologically active NGF, revealed the presence of several hundred copies of the rat NGF gene (Fig. lb) . This cell line also expressed a 2.2kb NGF-hybridizing transcript (Fig. lc) . The size of this transcript is consistent with transcription from the upstream TATA box, terminating around the hexanuclθotide sequence (AATAAA) in the rabbit β-globin gene. The level of the 2.2kg transcript was approximately 50 times higher than the level of endogenous NGF mRNA produced in the parental 3T3 cells. Conditioned medium from the 3E cells readily stimulated fiber outgrowth from chicken sympa- thetic ganglia (Fig. Id). A twosite enzyme immunoassay for NGF (Larkfors, L. & Ebendal, T. (1987) J . Immunol .Meth. 97, 41-47) revealed 5 +. lng of NGF protein per ml of medium com¬ pared to 0.2 ± 0.04ng/ml produced by the parental cells. EXAMPLE 4 Implantation of cells producing recombinant NGF into choli- nergicallv denervated cerebral cortex. As a model of the cortical cholinergic deficits of Alzheimer's disease, the frontoparietal cholinergic projec¬ tion from the anterior two-thirds of nucleus basalis to ce- rebral cortex was unilaterally lesioned by stereotaxic injec¬ tion of ibotenic acid. The injection caused a greater than 90% reduction of cholinergic cells in nucleus basalis and a marked unilateral reduction in density of cholinergic fibers in frontal and parietal cortex as revealed by both immunohis- tochemistry using antibodies against acetylcholine esterase (AChE) and AChE histoche istry . 3T3/3E cells, or as control parental 3T3 cells, were mixed with a dissociated cell suspension of fetal basal fore¬ brain tissue including nucleus basalis magnocellularis from the 17th to 18th day of gestation. These mixtures were im¬ planted into denervated cerebral cortices. Counts of choli¬ nergic nerve cell bodies from four weeks after implantation showed that 3T3/3E cells increased survival, as assessed by AChE staining, about 10-fold compared to parental 3T3 cells (Fig. 2a). When cholinergic cells were grafted adjacent to an implant of 3T3/3E cells, survival increased approximately 5-fold, as compared to using parental 3T3 cells. In addition to increased survival, 3T3/3E cells also stimulated fiber outgrowth from grafted cholinergic neurons into host brain parenchyma (Fig. 2a, 3) . The 3T3/3E cells also had a marked stimulatory effect on local intrinsic AChE positive fibers in cerebral cortex. Hence, implants of 3T3/3E cells alone into cholinergically denervated cortex significantly increased density of intrinsic cholinergic innervation in a halo around the implants ( Fig. 3) . No change in nerve density was seen around similar implants of parental 3T3 cells. EXAMPLE 5 NGF responsiveness of adult cholinergic interneurons in stri- a u . To clarify the possible role of NGF in supporting in- trinsic cholinergic neurons in striatum, collagen gels (Eben¬ dal, T., Olson, L., Seiger, A. & Hedlund, K.O. (1980) Nature 286, 25-28) containing 3T3/3E cells were implanted into in¬ tact striatum on one side of the rat brain and similar gels containing the parental cell line on the other side. As in cortical experiments 3T3/3E cells caused a markedly increased density of cholinergic nerve terminals in a zone around im¬ plants, as evaluated by AChE antibody immunohistochemistry (Fig. 2b, c). This was seen in 5 out of 6 cases 3 to 7 weeks after implantation. In addition, AChEpositive nerve fiers often invaded implanted gel containing 3T3/3E cells. Implan¬ tation of a gel containing parental 3T3 cells, or a gel with¬ out any cells, caused no increase in AChE-positive nerve fi¬ bers (Fig. 2d,e). To evaluate viability of grafted 3T3 cells, alternate sections from striatal implants were analyzed using antibodies against fibronectin. Seven weeks after grafting, both parental 3T3 cells and 3T3/3E cells formed dense extra¬ cellular networks of fibronectin-positive fibers. Exocrine sources, in particular the male mouse subman- dibular gland, have for many years been used to obtain puri- fied NGF protein (Levi-Montalcini, R. (1987) Science 237, 1154-1162). In accord with this invention an alternative source for NGF is established and used, namely a genetically modified cell line that produces excess levels of biological ly active recombinant NGF when cultured in vitro and evokes neurotrophic effect in vivo. In the recombinant NGF producin 3T3/3E cell line, the rat NGF-gene is transcribed from a rab bit β-globin promoter which has been modified by insertion o a heavy metal inducible enhancer motif (MRE) . However, when cultured in vitro the 3T3/3E cells express NGF RNA and pro- tein apparently independent of the concentrations of heavy metals, suggesting that the MRE motif has no effect on NGF expression in 3T3/3E cells. However, we have isolated OVEC- -NGF cell lines in which NGF mRNA and protein expression can be induced several-fold by adding ZnSU4 to the cell culture medium. When implanted into intact rat brain, either as a cell suspension or embedded in a collagen gel, the 3T3/3E cells markedly stimulated central cholinergic neurons. An apparent¬ ly similar enhancement of nerve fiber formation was seen when AChE positive neurons from three different regions of rat brain were analyzed: basal forebrain (in the form of fetal grafts), intrinsic neurons in cerebral cortex, and instrinsic neurons in striatum. Thus, it has been shown that NGF func¬ tions in vivo as a neurotrophic factor for several polula- tions of central cholinergic neurons, including immature fe¬ tal cells. The effect on the intrinsic cholinergic neurons both in the cerebral cortex and striatum suggests the presen¬ ce of functional NGF receptors on these cells, at least after perturbation caused by implantation. The fact that newly formed cholinergic nerve fibers produced a gradient of increasing density towards 3T3/3E cells, and that such fibers even invaded the collagen gel containing 3T3/3E cells, suggests that NGF in brain can also function as a neurotropic factor for central cholinergic neurons. Hence, locally high concentrations of extracellular NGF protein will not only enhance survival of cholinergic neurons but also direct their growing fibers and maintain the adult pattern of cholinergic terminals. The marked neurotrophic effects demonstrated by im¬ plantation of 3T3/3E cells into the rat brain indicates that implantation of genetically modified cell lines that produce functional recombinant gene products (transgeneε) into the brain can be used as a tool to unravel new functional aspects of the product encoded by the transgene . In this disclosure, the neurotrophic effects of NGF in vivo on instrinsic choli¬ nergic neurons in cerebral cortex and striatum provide an example of this . The results disclosed herein also indicate that recom¬ binant NGF, administered continuously into the brain by the use of genetically modified cell lines will prevent accelera¬ ted death and/or atrophy of basal forebrain cholinergic neu¬ rons, such as in Alzheimer-type dementia.

Claims: CLAIMS 1. A plasmid DNA construct comprising essentially DNA sequences endogenous to mammalian cells excluding DNA sequences of viral origin including the gene encoding a mammalian β-nerve growth factor (β-NGF) or an active polypep¬ tide fragment thereof, and DNA sequences necessary for repli¬ cating the plasmid in a bacterial cell. 2. A plasmid DNA construct according to claim 1, con¬ taining a DNA sequence encoding the gene for a human β-nerve growth factor (β-NGF). 3. A transfected mammalian host cell, selected by re¬ sistance to an antibiotic marker and therefore a stable cell line harbouring a recombinant mammalian expression plasmid vector based on the DNA construct of claim 1 or 2. 4. A transfected mammalian host cell according to claim 3, which is a human cell. 5. A method for the treatment of neurodegenerative diseases in mammals including man, based on implantation into the brain of a mammal including man of an effective amount of the transfected and selected stable mammalian cell defined in claim 3 or 4. 6. A method according to claim 5, wherein said amount of biologically active recombinant β-NGF is effective to sti¬ mulate central cholinergic neurons in said mammal. 7. A method according to claim 5 or 6, wherein said cells are implanted into the mammalian brain including man either as a cell suspension or embedded in a collagen gel.

Inventors: EBENDAL TED, OLSON LARS, PERSSON HAAKAN, EBENDAL, TED, OLSON, LARS, PERSSON, HAAKAN
Applicants: LOPE MEDICINE AB
Application year: 1990
Main classification: C12N15
IPC codes: A61K35, A61K38, A61K45, A61P25, A61P43, C07K14, C12N5, C12N15