JPH11507530A - Cell lines producing analgesic compounds for treating pain - Google Patents

Abstract

(57) Abstract: A latitudinal engineered cell line that produces at least one catecholamine, at least one endorphin, and at least one enkephalin for the treatment of pain. The cells can be provided directly to the patient in need thereof, or can be encapsulated to form a bioprosthesis.

Description

DETAILED DESCRIPTION OF THE INVENTION             Cell lines producing analgesic compounds for treating painField of the invention   The present invention relates to cell lines useful for treating pain. More specifically, the cells of the present invention The strain is a member of the group consisting of endorphin, enkephalin, and catecholamine. Genetically engineered to produce at least one analgesic compound from each I have.Background of the invention   Pain is a common symptom of the disease. Primary afferent fibers convey nociceptive information end The superficial dorsal horn of the connecting spinal cord is composed of enkephalin interneurons and dense Including piate receptor. In addition, the superficial plate of the spinal cord has a dense concentration of noradore. There are narinergic fibers.   Acute pain occurs in response to acute noxious stimuli. Chronic pain is mainly moderate Due to neuropathy of central or peripheral origin. This neuropathic pain is not normal The result of somatosensory processing, which is the increased sensitivity to pain stimuli ( Produces hyperalgesia) and pain (allodynia) usually associated with non-painful stimuli I can do it.   Intradural injection of morphine into the spinal subarachnoid space produces strong analgesia. As well Intradural administration of norepinephrine or noradrenergic agonist Also causes analgesia. For example, Sagen et al.Proc . Natl. Acad. Sci. USA, 83,7522- See page 26 (1986).   Opiates like enkephalin, and catechols like norepinephrine Co-administration of sub-effective doses of olamine may be synergistic to produce analgesia. Ibid. The chromaffin cells of the adrenal medulla are norepinephrine, epinephrine, met- Enkephalin, leu-enkephalin, neuropeptide Y, bathoactive Persistent polypeptide, somatostatin, neurotensin, cholecystocyst Nin, and several neuroactive substances, including calcitonin gene-related peptides Produce and release. For example, Sagen et al.Proc . Natl. Acad. Sci. USA, 83,7 522-26 (1986); Sagen et al.,Jour . Neurochem., 56, pp. 623-27 (1991). When.   Chromaffin cells produce both opioid peptides and catecholamines Therefore, one approach to reducing nociceptive responses or pain sensations is to In an attempt to provide, adrenal medulla tissue as well as isolated adrenal chromaffin We are studying the transplantation of sex cells into the CNS pain control region. For example, Sagen et al.Brain Research 384, 189-94 (1986); Vaguero et al.Neuroreport, 2, pp. 149-51 (1991); Ginzberg and Seltzer,Brain Research, 523 147-50 (1990); Sagen. Et al.,Pain, 42, pp. 69-79 (1990).   Attempts to produce analgesics have included both homologous and xenogeneic chromaffin tissue or Has been done using cell transplants. Allograft tissue is available in limited supply Yes, and not particularly readily available for human pain treatment programs. In addition, allogeneic human tissues carry the risk of pathogenic contaminants. For example, Hama and Sagen ,Brain Research, 651, pp. 183-93 (1994).   Heterologous donors can provide large amounts of material that can be readily obtained. For this reason, Bovine adrenal tissue has been used. For example, Hama and Sagen,Brain Research, 6 51, pages 183-93 (1994).   However, potential serious host consequences, as well as eventual graft rejection, are different species It is an inherent problem in transplanting between. Complete graft of whole or excised tissue Rejection is usually due to the presence of highly antigenic cells in the xenograft, especially endothelial cells. Can even occur in the CNS, which is considered immunologically privileged. further, Donor tissue should be used to avoid introducing viral contaminants or other pathogens into the host. Must be carefully screened. Overcoming graft rejection Typically, immunosuppression using cyclosporin A is required.   Some reduction in pain sensation, especially for the reduction of low intensity chronic pain, It has been reported to arise from planting. In most reports, control and transfer The striking difference between the planted animals is the nicotine for stimulating opioid peptide production. Only shown after tin administration. However, in the rat chronic pain model, How many basal levels of chromaffin tissue allografts have been observed There is a report. For example, Vaquero et al.NeuroReport, 2, pp. 149-51 (1991), and And Hama and Sagen,Brain Research, 651, pp. 183-93 (1994).   Bovine adrenal chromaffin cells can be used in rats for treatment of acute and chronic pain. Encapsulated to form a bioprosthesis ("BAO") for implantation. example Sagen et al.J . Neurosci., 13, 2415-23 (1993), and Hama et al.7th World Congress Pain See Abstruct 982, Paris France (1993). Human subject The first test in was performed using encapsulated bovine chromaffin cells. Was. Aebischer et al.Transplantation, 58, pp. 1275-77 (1994).   Inducing antinociception using other cells, such as AtT-20 cells, can also be used. Attempted. AtT-20 cells were originally derived from a mouse anterior pituitary tumor. this These cells synthesize and secrete β-endorphin. For example, Wu et al.J . Neur al Transpl. & Plasticity , 5, 15-26 (1993). AtT-20 / hENK cells Is an entire human with a 200 base 5 'flanking sequence and a 2.66 kilobase 3' flanking sequence The pro-enkephalin A gene (ie, six met-enkephalin sequences and AtT engineered to have one leu-enkephalin sequence) -20 cells. Wu et al., Supra, Comb et al.EMBO J., 4, 3115-22 (1985). When.   Wu et al.J . Neural Transpl. & Plasticity, 5, 15-26 (1993) describes AtT-20 and Relates to a rat host transplanted with AtT-20 / hENK cells. Unstimulated AtT-20 / hENK cells produce more antinociceptive than is produced by AtT-20 implants (Tail flick test). Conversely, isoproterenol stimulation is associated with AtT-20 / hEN AtT-20 cells produced more antinociceptivity than K cells. Ibid.   In the mouse host, AtT-20 or AtT-20 / hENK implants produce heat nociceptive stimuli It did not affect the basal response to Mice given the AtT-20 transplant had β- This resulted in resistance to endorphins and μ-opioid agonists (DAMGO). Mice given the AtT-20 / hENK implants were given a δ-opioid agonist (DPDPE) Resistance to the disease. For repeated doses of μ opiate agonist, AtT- Mice given the 20 / hENK transplant were mice or mice given AtT-20 cells. Resulted in lower resistance compared to the control.   The antinociceptive effect of isoproterenol treatment was attributable to AtT-20 or AtT-20 / hENK cells. Appeared equally in mice given vesicle implants. Wu et al.J . Neuroscience, 14,480 See pages 6-14 (1994). Wu et al. Transferred enkephalin-producing AtT-20 / hENK cells. One reason for the additional absence of antinociceptive in implanted mice is that It was speculated that this may be due to the lack of sensitivity of the behavioral assay. Other possible reasons Is a known antagonist of met-enkephalin for morphine-induced antinociception. The gonist effect is particularly significant for each leu-enkephalin distribution in pro-enkephalin A. Since there are 6 met-enkephalins per row, a single leu-enkephalin It was to offset possible effects.Summary of the Invention   The present invention is directed to endorphins, enkephalins, and catecholamines. Genetic engineering to produce at least one analgesic compound from each of the groups Provided cell lines. Cell lines can be used for the treatment of pain.   There are advantages to using cell lines over using primary cells. Cell safety Expensive and time-consuming tests to ensure performance and performance standards Must be performed on individual isolates of vesicles. Fewer tests are cell buns Needed about There is no need to isolate primary cells. Run primary cells Desired analgesia as it can depend on the age, gender, health or hormonal status of the animal Drug output may be more stable. To achieve higher yields of the desired product, It is also possible to engineer specific modified peptides into cell lines. This It allows for the delivery of many analgesics at the same time. The expression of one or more analgesics (By using a regulatable promoter to drive the present) obtain. Further, for safety, a "suicide" gene can be integrated into the cell line. Further In addition, for the purpose of encapsulation, cells that are growing are cells that die or die. It has the advantage of splitting to replace dead cells.BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows the vectors pBS-hPOMC-027, pBS-IgSP-hPOMC-028, and pBS-IgSP-hPOM. It is a plasmid map of C-ΔACTH-029.   FIG. 2 shows the vectors pCEP4-hPOMC-030, pCEP4-hPOMC-031, pcDNA3-hPOMC-034, 4 is a plasmid map of pcDNA3-hPOMC-035.   FIG. 3 shows the vectors pCEP4-hPOMC-ΔACTH-032, pCEP4-hPOMC-ΔACTH-033, pcDNA3 FIG. 7 is a plasmid map of -hPOMC-ΔACTH-36 and pcDNA3-hPOMC-ΔACTH-037. .   FIG. 4 shows the vectors pcDNA3-rTH-044, pcDNA3-rTHΔ-045, and pcDNA3-rTHDKS- FIG. 7 is a plasmid map of 075 (also designated as pcDNA3-rTHΔKS-075).   FIG. 5 shows the vectors pcDNA3-rTHΔ-IRES-bDBH-088 and pcDNA3-rTHΔKS-IRES-bD. It is a plasmid map of BH-076.   FIG. 6 is a plasmid map of the vector pZeo-Pcmv-rTHΔKS-IRES-bDBH-088. You.   FIG. 7 is a plasmid map of the vector pBS-Pcmv-rTHΔIRES-bDBH-067.   FIG. 8 shows the plasmid vector pBS-hPOMC-ΔACTH-IRES-rTHΔIRES-bDBH-068. Up.   FIG. 9 shows the plasmid of the vector pcDNA3-hPOMC-ΔACTH-IRES-rTHΔ-IRES-bDBH-069. Domap.   FIG. 10 is a plasmid map of the vector pcDNA3-IRES-Zeocin-072.   FIG. 11 shows the vector pcDNA3-hPOMC-ΔACTH-IRES-rTHΔ-IRES-bDBH-IRES-Zeocin. It is a plasmid map of -073.   FIG. 12 is a plasmid map of the vector pcDNA3-hPROA + KS-091.Detailed description of the invention   In order that the invention may be more fully understood, the following detailed description is set forth.   Any suitable cell can be transformed with a recombinant DNA molecule of the present invention. Intended Some cells are chromaffin cells (e.g., conditioned cells as described in WO 96/02646). Immortalized chromaffin cells, Neuro-2A, PC12, PC12a, SK-N-MC, AtT-20 ), And RIN cells (including RINa and RINb). Preferably, the cells are endogenous Possesses the sex prohormone coonvertase and / or dopa decarboxylase I do.   SK-N-MC cells are neuroepithelial cell lines, enkephalin, cholecystokinin And several neuropeptides including gastrin-releasing peptide You. For example, Verbeeck et al.J . Biol. Chem., 265, pp. 18087-090 (1990). When. The proenkephalin A gene is expressed in SK-N-MC cells. For example, F olkesson et al.Mol . Brain Res., 3, pp. 147-54 (1988). AtT-20 and RIN cells are preferred, most preferably RIN cells.   RIN cells are a rat-derived pancreatic endocrine cell line. For example, Horellou et al.J . Physiol. , 85, pp. 158-70 (1991). RIN cells contain GABA and β-endo It is known to produce ruffin endogenously.   Some of the characteristics of the various intended cells are shown in Table 1. TH = tyrosine hydroxylase converts tyrosine to l-dopa DDC = dopamine decarboxylase converts l-dopa to dopamine (DA) DβH = dopamine β-hydroxylase converts DA to norepinephrine (NA) PC = prohormone convertase converts POMC to β-endorphin and proenkephalin A             Processing (ProA) to met-enkephalin AtT20 = Endogenous β-endorphin secreted by expression of pro-opiomelanocortin (POMC)             Mouse pituitary corticotropin cell line RIN = Rat insulinoma Neuro 2A = mouse neuroblastoma   Primary delivery products include endorphin, enkephalin, and catecholamine At least one of each.   Enkephalins and endorphins are endogenous opioid pep It is a tide. These opioid peptides have approximately 15 amino acids ranging from 5 to 31 amino acids. Including compounds. These compounds have at least a portion of the same opioid as morphine. Binds and acts through its receptor but is chemically associated with morphine. Do not run. In addition, these compounds stimulate other opiate receptors. Ya ksh and Malmberg,Textbook of Pain, 3rd edition (edited by P. Wall and R. Melzack), " Central Pharmacology of Nociceptive Transmission, pp. 165-200, 1994 (New Yo rk).   Opioid peptides have common chemical properties, but are synthesized by different routes.   Beta-endorphin, the most abundant endorphin, has a larger precursor fraction It is synthesized as part of the pro-opiomelanocortin ("POMC"), a child. POMC molecule Is adrenocorticotropic hormone (ACTH), α-melanocyte stimulating hormone (α-MSH) , Β-MSH, and β-lipotropin. POMC precursor molecules also contain α- Produces other endorphins, including endorphins and gamma-endorphins Has the potential. Processing of the POMC precursor is dependent on prohormone Localization of a cleavage enzyme, such as vertase, occurs differently in various tissues.   In the pituitary, POMC is cleaved to produce ACTH and β-endorphin, and Lever ACTH is not further processed. Conversely, in the hypothalamus, ACTH is β-M Converted to SH. Although different cell types can synthesize the same primary gene product, The final profile of Mon secretion can vary widely.   The present invention relates to a DNA sequence encoding any suitable endorphin having analgesic activity. Intended for the use of columns. In addition, the analysis of these endorphins with analgesic activity Logs or fragments are also contemplated. Therefore, produced by the cells of the invention Is a naturally occurring amino acid sequence of the desired endorphin Characterized by amino acid insertions, deletions, substitutions, and alterations at one or more of the following sites: Can be attached. Conservative modifications and substitutions (i.e., Or minimal effects on tertiary structure and on the analgesic properties of endorphins Are preferred. For such conservative substitutions, see DayhoffAtlas of Protein Se quence and Structure , 5, (1978) and Argos,Embo J., 3, 779-85 (1989) Includes what is described.   Techniques for producing such variants of naturally occurring endorphins are well known. You. For example, codons in the DNA sequence encoding wild-type endorphin are site-specific. It can be altered by mutation.   The present invention contemplates using DNA sequences that encode the entire POMC precursor molecule I do. This embodiment is directed to a host cell cleaving enzyme (i.e., a prohormone convertor). A set of endorphins is produced using the enzyme 2) and some or all of them May have analgesic properties.   The present invention also relates to the DNA fragment of the POMC gene encoding a particular desired endorphin. Intended for use with fragments.   The DNA and amino acid sequences of POMC are well known. Cochet et al.Nature, 297,335-9 Page (1982); Takahashi et al.Nucl . Acids Res., 11, 6847-58 (1983).   DNA sequences encoding POMC in which the ACTH coding region has been deleted are preferred . The preferred endorphin encoded by this construct is β-endorphin It is.   Some enkephalins are large proteins, proenkephalin A Is synthesized in the adrenal gland as part of the Met-enkephalin sequence And one Leu-enkephalin structure. Met-enkephalin and Me t-Enkephalin-Arg-Phe and Met-Enkephalin-Arg-Gly-Leu also have significant anti- Has nociceptive activity. For example, Sagen et al.Brain Res., 502, pp. 1-10 (1989) See also.   Other enkephalins, dynorphin and neoendorphin, From a different molecule, proenkephalin B. Additional "cryptic" Peptides are also encoded within the structure of these precursor proteins, and It can be released by cleavage of the "hormone type". For example, Harrison's "Principi es Of Internal Medicine, 12th edition, pages 1168-69 (1991).   The present invention relates to a DNA sequence encoding any suitable enkephalin having analgesic activity. Intended for the use of columns. Analogs and active fragments with analgesic properties are also Intended. Therefore, such analogs or fragments are naturally occurring. Has an amino acid insertion, deletion, or substitution at one or more sites in the existing amino acid sequence obtain. Such variants can be generated as described above.   The present invention provides for the use of a DNA sequence encoding the desired enkephalin in its "mature" form. Intended for use. Furthermore, the present invention relates to the complete proenkephalin A precursor, or Intended to use DNA sequence encoding the complete proenkephalin B precursor I do. In addition, DNA encoding fusions or fragments of these sequences may be used. It is also intended to provide, when manifested, one or more enkeffes having analgesic properties. To obtain an argin-like molecule.   Preference is given to using a DNA sequence encoding the complete proenkephalin A precursor molecule No. The DNA and amino acid sequences of proenkephalin A are well known.Folkesson, I mentioned earlier. This embodiment comprises a host cell cleavage enzyme such as prohormone convertase. Element is used to produce a set of enkephalins, some or all of which are May have pain characteristics. The preferred enkephalin encoded by this construct is , Met-enkephalin.   Three naturally occurring catechols that function as neurotransmitters in the central nervous system There are amines; norepinephrine ("NE"), epinephrine ("E"), and dopami N. NE is associated with post-sympathetic nerve endings. NE is very close to its release It exerts its effect locally.   Catecholamine is synthesized from the amino acid tyrosine, which is subsequently It is droxylated to form dihydroxyphenylalanine (dopa) and decarboxylated. Oxidized to form dopamine and then by dopamine β-hydroxylase Thus, it is hydroxylated at the β-position of the side chain to form NE. Harrison's, supra, 380. page. NE uses phenylethanolamine-N-methyltransferase ("PNMT") Therefore, it is N-methylated to E.   Hydroxylation of tyrosine by tyrosine hydroxylase ("TH") leads to NE synthesis Is the rate-determining step. Regulation of dopa and NE synthesis in the adrenal medulla It can be accomplished by varying the amount and activity.   In addition, regulation of the synthesis of E from NE is based on phenylethanolamine-N-methyltra It can be caused by changes in the amount and activity of the transferase ("PNMT"). PNMT Inducible by glucocorticoids from the renal cortex. Ibid.   Catecholamines are high in mainly adrenal medullary chromaffin tissues such as E Is maintained in. Opioid peptides are also stored in the adrenal glands.   NE and E are α_TwoHave similar affinities at the receptor, and therefore both May potentially contribute to pain. Bylund,FASEB J., 6, pp. 832-39 (1992). met-enke Enkephalin peptides, mainly containing farin, are delta (δ) opioid receptors. Activate the putter selectively. Reisine and Bell,Trends Neurosci., 16,506 -10 (1993). Α in the spinal cord_TwoAdrenergic agonists and delta opioid receptors Activation, respectively, results in antinociception and potentially synergizes. Yaksh and Malmberg,Progress in Pain Research and Management, Volume 1, F Ed. ields and Lisbeskind, IASP Press, Seattle, pp. 141-71 (1994). For laboratory animals Activation of δ vs. (μ) opioid receptors in rats includes constipation and With few harmful side effects (Lee et al.,J . Pharmacol. Exp. Ther., 267,883-8 7 (1993)). Combined delivery of various opioid and adrenergic agonists Reduces the degree of resistance that occurs to a single drug and leads to sustained pain relief I can do it. Yaksh and Reddy,Anesthesiol., 54, pp. 451-67 (1981).   The present invention relates to catecholamines for obtaining catecholamines having analgesic properties. DNA sequences encoding biosynthetic enzymes or their analogs or fragments Intended for use. Preferred catecholamines in the present invention are NE and E .   In one embodiment, the host cell completes NE or E production, if desired. Transformed with the necessary genes. The selection of the required heterologous gene sequence Catecholamine synthase normally present in the host cell. For example , RIN and AtT-20 cells use tyrosine hydroxylase ("TH") and dopa Lack of min beta hydroxylase ("DBH"). However, RIN and AtT-20 cells Inside Inducible dopa decarboxylase ("DDC"). The desired catecholamine is E In some cases, then, the gene encoding PNMT is also required. Code PNMT Genes to be used are known. Baetge et al.Proc . Natl. Acad. Sci. , 83,5455-58 (1986).   Genes encoding TH are known. For example, U.S. Pat.No. 5,300,436 (reference and And incorporated herein by reference). Modified TH variants are also known. is there. U.S. Patent No. 5,300,436. In addition, a TH short containing the required C-terminal catalytic domain Truncated variants are also known. For example, Daubner et al.Protein Science, 2,145 See pages 2-60 (1993).   AtT-20 cells have been transformed with wild-type TH, as well as various TH variants. An example For example, Wu et al.J . Biol. Chem., 267, pp. 25754-758 (1992).   The sequence of the DBH gene is also well known. For example, Lamoroux et al.EMBO J., 6,3931- See page 37 (1987).   In addition to the preferred DNA sequences described herein, a number encoding a desired analgesic can be used. It is understood that there are many degenerate DNA sequences.   Secondary compounds with potential analgesic action are also produced by the cells of the invention. obtain. Such compounds include galanin and somatostatin It is. Additionally, neuropeptide Y, neurotensin, and cholecystokini Can be produced by the transformed cells of the present invention. The cells of the present invention Some or all of these compounds can usually be produced or Can be genetically engineered to use   Obtaining a gene encoding an analgesic compound produced by the cells of the invention; or For synthesis, standard methods can be used.   For example, the complete amino acid sequence of the desired compound constitutes a back-translated gene. Can be used to Contains the nucleotide sequence encoding the desired analgesic compound DNA oligomers can be synthesized. For example, encoding a portion of each desired polypeptide Some small oligonucleotides can be synthesized and then ligated. Teens Representatively, individual oligonucleotides have 5 'or 3' overhangs for assembly. including.   The DNA sequence encoding each desired analgesic compound is a DNA sequence encoding a signal sequence. Columns may or may not be included. Such a signal sequence must not exist. Is recognized by cells selected for expression of the analgesic compound Should. It can be prokaryotic, eukaryotic, or a combination of the two possible. It can also be the signal sequence of the original compound. Generally, Signa Preferably, the original signal sequence is used. Is most preferred.   Once assembled, the DNA sequence encoding the desired compound may contain one or more DNA sequences. Inserted into an expression vector and suitable for expression in the desired transformed cells. Operably linked to an expression control sequence.   Proper assembly involves nucleotide sequencing, restriction mapping, and transformation. Can be confirmed by expression of a biologically active polypeptide in the transformed cells . As is well known in the art, high expression of transfected genes in a host To obtain levels, the gene must be transcribed and functional in the selected expression cell. And operably linked to a translation expression control sequence.   The choice of expression control sequences and expression vectors depends on the choice of cells. Broad species Various expression host / vector combinations can be used. Useful sources for eukaryotic hosts Current vectors include, for example, SV40, bovine papillomavirus, adenovirus, And vectors containing expression control sequences from cytomegalovirus.   pcDNA3, pCEP4, pZeoSV (InVitorgen, San Diego), and pNUT are preferred.   Any of a wide variety of expression control sequences can be used in these vectors. Such useful expression control sequences include those associated with the structural genes of the aforementioned expression vectors. Expression control sequences. Examples of useful expression control sequences include, for example, SV40 and Or adenovirus early and late promoters, 3-phosphoglycerate quina Promoter for lyase or other glycolytic enzymes, promoter for acid phosphatase (E.g., Pho5), yeast alpha mating promoter, and eukaryotic cells or Other sequences known to control the expression of the genes of these viruses, as well as their species Various combinations are mentioned.   All vectors and expression control sequences are compatible with the DNA sequences described herein. It is, of course, understood that they do not function equally well to express Should be. Also, not all cells function equally well with the same expression system. is not. However, one of ordinary skill in the art can, without undue experimentation, use these vectors, Expression control sequences and selection among cells can be made. For example, in selecting a vector The host cell, since the vector must be replicated in the cell. Must be done. The number of copies of the vector, the ability to control that copy number, and And any other proteins encoded by the vector, such as antibiotic markers Quality expression should also be considered.   Various factors should also be considered in selecting an expression control sequence. this These include, for example, the relative strength of the sequence, its ability to control, and the desired analgesic compound. Compatibility with the actual DNA sequence to be loaded, especially with regard to potential secondary structure Can be Host cells are subject to their compatibility with the chosen vector, DNA sequence. Thus, the toxicity of the encoded products, their secretory characteristics, and the exact folding of the polypeptide The choice should be made in consideration of the ability to fold and their culture requirements. Inn If the primary cell is to be encapsulated, it is encapsulated and the recipient Cell viability when transplanted into a cell should also be considered.   Within these parameters, one skilled in the art will recognize species that express the desired DNA sequence in culture. Different vector / expression control sequence / host combinations may be selected.   In one embodiment, the cell (eg, a RIN cell) contains the POMC gene, proenkeffe Four separate expression vectors containing the argin A gene, the TH gene, and the DBH gene And is continuously transformed. In the host cell thus transformed, Amplification of the copy number of the seed gene is more difficult to achieve. Therefore The use of less expression vectors is preferred. Most preferably, all four heterogeneous A single expression vector containing the gene is used.   In certain embodiments, RIN cells are sequentially transformed with the three expression vectors. It is. The first vector is a POMC gene operably linked to a CMV promoter. including. Preferably, a shortened version of the POMC gene is used, which is Having a storage region. The second vector was operably linked to the CMV promoter Contains the proenkephalin A gene. Preferably, the proA construct has an initiation codon. Contains the Kozak sequence immediately upstream. The third vector is the TH gene (preferably abbreviated And has a Kozak consensus sequence immediately upstream of the start codon) and the DBH gene Including both children. In this embodiment, the TH gene is operable on the CMV promoter. Be linked. DBH gene can operate on internal ribosome entry site promoter sequence Linked to Next, the RIN cells were transformed with each expression vector according to a known protocol. And transformed continuously.   In other embodiments, the proenkephalin A gene, POMC gene, TH gene, A single expression vector is constructed containing the and DBH genes. Preferably, POMC genetics The child's ACTH region is deleted. Preferably, the TH gene is truncated.   Multiple gene expression from a single transcript is better than expression from multiple transcription units. Good. One to achieve expression of multiple genes from a single eukaryotic transcript Approach uses a picornavirus known as an internal ribosome entry site ("IRES"). Utilizes the sequence of mRNA. These sites are located downstream of the first AUG of the mRNA It functions to facilitate protein translation from the sequence.   Macejak and Sarnow discuss immunoglobulin heavy chain binding proteins (BiP, CRP 78 and Also known, a glucose-regulated protein with a molecular weight of 78,000) 5 'translation of mRNA The untranslated sequence is expressed in mammalian cells in a 5'-cap independent fashion. Report that it can directly provide internal ribosome binding to RNA, That translation initiation by a cellular binding mechanism is used by this cellular mRNA. Show.Nature353, 90-94 (1991).   Wo 94/24870 describes the use of more than two IRESs for initiating translation from a single transcript, As well as the use of multiple copies of the same IRES in a single construct.   The present invention also contemplates the use of "suicide" genes in transformed cells. Most preferably, the cells have the TK (thymidine kinase) gene as a measure of safety Kills host cells in vivo by treatment with ganciclovir.   The use of "suicide" genes is known in the art. For example, Anderson, published See PCT application WO 93/10218; Hamre, published PCT application WO 93/02556. Les Scipient's own immune system is the first level from adverse reactions to the transplanted cells. Provide protection. When encapsulated, polymer capsules are themselves immune Can be isolation. The presence of the TK gene (or other suicide gene) in the expression construct is Add an additional level of safety to the recipient of the implanted cells.   Preferred vectors for use in the present invention include DNA encoding an analgesic compound. Includes vectors that are amplified by copy number. Such an amplifiable vector is It is well known in the art. These include, for example, vectors that can be amplified by DHFR amplification. (E.g., Kaufman, U.S. Pat.No. 4,470,461, Kaufman and Sharp, "Cons. truction Of A Modular Dihydrafolate Reductase cDNA Gene: Analysis Of Sig nals Utilized For Efficient Expression ",Mol . Cell. Biol., 2, 1304-19 ( 1982)), or glutamine synthetase (`` GS '') amplification (e.g., US (See Patent 5,122,464 and European Published Application 338,841). Can be Such amplification is used to increase the production of the desired analgesic compound. obtain.   Other techniques for increasing the output of the desired analgesic compound are contemplated. For example, It is contemplated that existing polyclonal cell lines will be subcloned. cell Is cloned by limiting dilution to a single cell in each well. Cell clones are cultures and clones are cloned with the highest production of analgesics. Tested to select an option.   Other techniques for increasing the production of desired analgesic compounds are higher than wild-type forms. Cloning of a modified form of a biosynthetic enzyme having high activity (Ie, shortened TH 1-155). Some truncated forms of TH are better than wild-type forms of TH. It has a 4-6 fold increased activity. For example, Daubner et al., "Expression and cha racterization of catalytic and regulatory domains of rat tyrosine hydrox ylase ",Protein Science, 2, 1452-60 (1993).   In addition, select to increase tetrahydrobiopterin cofactor levels The use of tyrosine-free media for potential tyrosine hydroxylase activity Can be increased. For example, Horellou et al., "Retroviral transfer of a human ty rosine hydroxylase cDNA in various cell lines; regulated release of dopa mine in mouse anterior pituitary AtT-20 cells ",Proc . Natl. Acad. Sci. U SA 86, 7233-37 (1989).   Preferably, the production of β-endorphin is between 1 and 10,000 pg / 10<sup>6</sup>Between cells / hour Range. Preferably, the production of met-enkephalin is between 1 and 10,000 pg / 10<sup>6</sup>Fine Range between cells / hour. Preferably, the production of catecholamines is between 1 and 1,0 00 pmol/10<sup>6</sup>Range between cells / hour.   The cells of the invention can be transplanted into mammals, including humans, for the treatment of pain. If an unencapsulated one is to be transplanted, any suitable transplant protocol may be used. Sagen et al., U.S. Pat.No. 4,753,635, which is incorporated herein by reference. ).   It is desirable to encapsulate the genetically modified cells of the present invention prior to transplantation. It can be. Such encapsulated cells are used as bioprosthetic devices ("BAO"). To form BAO can be designed for transplantation in recipients, or Can be created to function extracorporeally. BAOs useful in the present invention are typically A jacket surrounding at least one semipermeable outer surface membrane or cell-containing core I do. This jacket is designed for nutrition, biologically active molecules, and other selected products. Enables the diffusion of objects through BAO. BAO is biocompatible.   In some cases, the membrane is a cellular and molecular effector of immunological rejection. Blocking the vector may also help to immunoisolate cells. The use of immunoisolating membranes allows the transfer of allogeneic and xenogeneic cells to an individual without the use of immunosuppression. Enable transplantation. When biologically active molecules are released from the isolated cells, They pass through the surrounding semipermeable membrane and into the recipient's body. Metabolic function isolated When provided by a dead cell, the substance to be metabolized It enters the BAO from the recipient's body through the membrane to be removed.   Various types of membranes have been used in the construction of BAO. Generally used for BAO The membrane is either a microporous membrane or an ultrafiltration grade membrane. Various membrane materials Has been suggested for use in BAO, including PAN / PVC, polyurethane, Lishon (polysufone), polyvinylidiene (polyvinylidiene), and polystyrene Included. Representative membrane geometries include planar sheets, which are described as " It can be manufactured into a "sandwich" type construction, which is shaped around the perimeter of the device. A layer of living cells disposed between two essentially planar membranes with a seal formed Having. Alternatively, a hollow fiber device may be used, where the live cells are a tubular membrane Located inside. Hollow fiber BAO fills the living cells into the hollow fiber lumen , And by providing a seal at the end of the fiber. Hollow fiber BAO can also be formed by a co-extrusion process, where live cells are It is co-extruded with a polymer solution that forms a membrane around the vesicle.   BAO is disclosed, for example, in U.S. Pat.Nos. 4,892,538, 5,106,627, 5,156,844, 5,158,881, and 5,182,111, and PCT Application No.PCT / US / 94/07015, WO 92/19195, WO 93/03901, and WO 91/00119, all of which are Incorporated herein by reference.   BAO may include other components that promote long-term survival of the encapsulated cells. For example, WO 92/19195 discloses a hydrogel matrix for enhancing cell viability. And an implantable immunoisolating biocompatible vehicle having:   The BAO encapsulation membrane can be manufactured from the same material as the core, or It can be manufactured from raw materials. In each case, BAs that are permselective and biocompatible A film area around or around O is formed. The membrane can also be immunoisolated, if desired. Can be constructed to be sexual. The core is suspended in liquid medium or Contains isolated cells, either immobilized within a gel matrix.   The choice of materials used to construct a BAO is determined by many factors. And in Dionne WO 92/19195. Briefly, various polymers And polymer blends can be used to make capsule jackets. You. The polymer film that forms the BAO and growth surface therein includes polyacryl (Including acrylic acid copolymer), polyvinylidene, polyvinyl chloride copolymer ー, polyurethane, polystyrene, polyamide, cellulose acetate, cellulose nitrate Polysulfone, polyphosphazene, polyacrylonitrile, poly (acryloyl Nitrile / vinyl chloride) and its derivatives, copolymers, and mixtures. Can be done.   BAO can be formed by any suitable method known in the art. like this One method involves co-extrusion of a polymer casting solution and a coagulant. This is described in Dionne, WO 92/19195 and U.S. Pat.Nos. 5,158,881, 5,283,187. , And 5,284,761 (hereby incorporated by reference). Tissue fragments, organelles, or cells and / or other therapeutic agents It may include a suspension.   Jackets can be single or double skin n). Single-skin hollow fibers are polymer-extruded when co-extruded. It can be produced by quenching only one of the surfaces of the liquid. Double ski When the hollow fibers of the polymer are co-extruded, they quench both surfaces of the polymer solution. Can be generated by   Many capsule shapes are possible, such as cylindrical, disc-shaped, or spherical .   BAO jacket determines nominal molecular weight cut-off (nMWCO) of permselective membrane Have a pore size of Molecules larger than nMWCO physically cross the membrane Be disturbed. Nominal molecular weight cutoff defined as 90% rejection under convective conditions Is done. In situations where it is desired that BAO be immunoisolated, the pore size of the membrane will Certain factors produced by the cells diffuse out of the vehicle, but host immunity into BAO It is chosen so as to allow the ingress of the response factor to be ruled out. Typically, nM The WCO ranges between 50 and 200 kD, preferably between 90 and 150 kD. Most appropriate A novel membrane composition is also known to be a host immune effect known to be present at a selected implantation site. Minimize the reactivity between the receptor molecule and the outer membrane components of BAO.   The BAO core provides an appropriate local environment for the particular cells isolated within it. Is built. The core may contain enough liquid medium to maintain cell growth . Liquid cores are particularly suitable for maintaining transformed cell lines such as PC12 cells. It is off. Alternatively, the core may include a gel matrix. The gel matrix is Drogel (alginate, `` Vitorogen^TM) Or from extracellular matrix components Can be configured. See, for example, Dionne WO 92/19195.   Compositions that form hydrogels fall into three general classes. The first class Have a net negative charge (eg, alginate). The second class is net It has a positive charge (eg, collagen and laminin). Commercial extracellular matrix Examples of ingredients include Matrigel^TMAnd Vitrogen^TMIs included. The third class is charge Is net neutral (e.g., highly crosslinked polyethylene oxide, or Vinyl alcohol).   Any suitable method of sealing the BAO can be used, including polymer bonding and And / or the use of crimping, knotting, and heat sealing. this These sealing techniques are well known in the art. In addition, any suitable "dry" seal The method can also be used. In such a method, a cell-containing solution is passed through A substantially non-porous fitting into which is introduced is provided. After filling, the BAO Is controlled. Such a method is described in co-pending US patent application Ser. No. 08 / 082,407. Which is incorporated herein by reference.   One or more in vitro assays establish BAO functionality before transplantation in vivo It is preferably used for Assays or diagnostic tests well known in the art Can be used for these purposes. For example,Methods In Enzymology, Edited by Abelson, See Academic Press, 1993. For example, an ELISA specific for the secreted product ( Enzyme-linked immunosorbent assay), chromatographic assay Or an enzymatic assay, or a bioassay may be used. If desired The secretory function of the implant is to collect the appropriate sample (e.g., serum) from the recipient, And by assaying them, they can be monitored over time. recipe If the ent is a primate, microdialysis can be used.   The number and size of BAOs are sufficient to produce a therapeutic effect during transplantation. Should be determined by the amount of biological activity required for a particular application . For secretory cells releasing therapeutic substances, consider standard dosages known in the art. And criteria are used to determine the amount of secreted substance required. Take into account Factors of power are discussed in Dionne, WO 92/19195.   BAO implantation is performed under sterile conditions. In general, BAO is a secreted product or Properly delivers function to the host and nutrients to the encapsulated cells or tissues Access to BAO for repair and / or replacement To be implanted at a site in the host. Preferred hosts are primates, most Is also preferably human.   Many different implantation sites are contemplated. These transplant sites include the central nervous system This includes the brain, spinal cord, and aqueous and vitreous humor of the eye. Preference in the brain New areas include the striatum, cerebral cortex, hypothalamus, and Meynert basal ganglia (nucleus Ba salis of Meynert). Other preferred sites are cerebrospinal fluid, most preferred Is the subarachnoid space and the lateral ventricle. The invention also relates to the subcapsular site of the kidney, as well as the abdomen. Intended for endoluminal and subcutaneous sites, or any other therapeutically beneficial site .   In order that the present invention may be better understood, the following examples are set forth. these The examples are for illustrative purposes only and in any way limit the scope of the invention Should not be construed asExample Construction of polycistronic expression vector   Construction of IgSP-POMC fusion   The SmaI-SalI fragment containing human POMC exon 3 was transferred to a pBS cloning vector. (Stratagene).Takahashi, Supra;CochetThe above See also. The resulting plasmid was named pBS-hPOMC-027. See FIG. thing.   The PCR fragment was combined with oCNTF-003 (SEQ ID NO: 1) and oIgSP-018 (SEQ ID NO: 2). PNUT containing two oligonucleotide primers and human CNTF gene Produced using a plasmid. Baetge et al.Proc . Natl. Acad. Sci. USA, 83, See pages 5454-58 (1986). Both oCNTF-003 and oIgSP-018 primers Contains synthetic BamHI and SmaI restriction sites at the 5 'end, respectively.   The 196 base pair (bp) PCR fragment was digested with the restriction endonucleases BamHI and SmaI- Digested with the isoschizomer XmaI and electrophoresed on 1% SeaPlaque agarose . A 193 bp HindIII / XmaI DNA fragment was excised and FMC SPinBind DNA Purification was performed using a purification kit (FMC BioProducts, Rockland, ME).   pBS-hPOMc-027 is also digested with BamHI and XmaI and FMC SPinBind DNA purification kit. 1% SeaPlaque agarose using FMC BioProducts, Rockland, ME And purified. The ligation mixture was added to E. coli. coli DH5α (Gibco BRL, Gaithersbur g, MD).   Positive subclone is subjected to cracking gel procedure (Promega Protocols and Applications Guide, 1991). Then, the minilysate DNA was purified using the FMC SpinBind DNA Purification Kit (FMC BioProducts , Rockland, ME) and subjected to BamHI and SmaI restriction digests. The positive subclone was named pBS-IgSP-hPOMC-028. See FIG. . The nucleotide sequence of the fusion junction in pBS-IgSP-hPOMC-028 was Kit (USBC, Cleveland) using dideoxynucleotide sequencing. Decided. The sequence of the IgSP-hPOMC fusion is shown in SEQ ID NO: 3.   Construction of IgSP-POMC expression vector   The IgSP-hPOMC DNA fragment in pBS-IgSP-hPOMC-028 was converted to pcDNA3 (Invitrogen Corp., San Diego, CA) and pCEP4 (Invitrogen Corp., San Diego, CA). Subcloned in sense and antisense orientation.   NotI-SalI IgSP-hPOMC fragment from pBS-IgSP-hPOMC-028 was converted to NotI-XhoI Ligation with the digested pCEP4 yielded a sense-oriented clone named pCEP4-hPOMC-030 . FIG. The BamHI-SalI IgSP-hPOMC fragment from pBS-IgSP-hPOMC-028 was Antisense orientation ligated with HI-XhoI digested pCEP4 and named pCEP4-hPOMC-031 A clone was obtained. FIG. The orientation of the insert in pCEP4-hPOMC-030 and-031 was amHI, NotI, SalI, and NotI / SalI restriction digestion, and the Sequenase kit. (USBC, Cleveland) using dideoxynucleotide sequencing confirmed.   The BamHI-SalI IgSP-hPOMC fragment from pBS-IgSP-hPOMC-028 was After ligation with pcDNA3 digested with I, a sense-oriented clone named pcDNA3-hPOMC-034 was Obtained. FIG. NotI-HindIII IgSP-hPOMC fragment derived from pBS-IgSP-hPOMC-028 Was ligated with NotI-HindIII-digested pcDNA3 and named antisense pcDNA3-hPOMC-035. A sense orientation clone was obtained. FIG. SmaI, BamHI, EcoRI, and BamHI / EcoRI Confirm the orientation of the insert in pcDNA3-hPOMC-034 using the restriction digest used, On the other hand, HindIII, NotI and SalI were used for pcDNA3-hPOMC-035.   Construction of ACTH deleted IgSP-POMC   The ACTH coding region in the POMC gene in pBS-IgSP-hPOMC-028 was deleted. pB S-IgSP-hPOMC-028 was first digested with Xmal restriction enzyme, and pfu DNA polymerase ( Promega, Madison, WI). Then, it was treated with XmaI-pfu DNA polymerase. PBS-IgSP-hPOMC-028 was digested with StuI restriction enzyme and FMC SpinBind DNA purified 1% SeaPlaque agarose using the kit (FMC BioProducts, Rockland, ME) And purified. The self-ligation mixture was added to E. coli. coli DH5α (Gibco BRL, Gaith ersburg, MD). BamHI / HindIII restriction of positive subclones Identified by digestion and named pBS-IgSP-hPOMCΔACTH-029. See FIG. That. Nucleotide sequence of ACTH deletion region in pBS-IgSP-hPOMC-ΔACTH-029 Was confirmed by dideoxynucleotide sequencing. IgSP-hPOMC-ΔACTH The sequence of the fusion is shown in SEQ ID NO: 4.   Construction of ACTH-deleted IgSP-POMC expression vector   The IgSP-hPOMC-ΔACTH DNA fragment in pBS-IgSP-hPOMC-ΔACTH-029 was A3 (Invitrogen Corp., San Diego, CA) and pCEP4 (Invitrogen Corp., San Die go, CA) in the sense and antisense orientation. pBS-IgS NotI-SalI IgSP-hPOMC-ΔACTH fragment derived from P-hPOMC-ΔACTH-029 was Ligated to XhoI-digested pCEP4 and labeled with pCEP4-hPOMC-ΔACTH-032 I got a loan (Figure 3). BamHI-SalI IgSP-hPOMC from pBS-IgSP-hPOMC-ΔACTH-029 The -ΔACTH fragment was ligated into BamHI-XhoI digested pCEP4, pCEP4-hPOMC-ΔAC An antisense orientation clone named TH-033 was obtained (FIG. 3). pCEP4-hPOMC-ΔACTH The orientation of the inserts in -032 and -033 was determined by BamHI and EcoRI restriction digestion. And dideoxynucleotides using the Sequenase kit (USBC, Cleveland). Confirmed by DNA sequencing.   BamHI-SalI IgSP-hPOMC-ΔACTH fragment derived from pBS-IgSP-hPOMC-ΔACTH-029 Was ligated to BamHI-XhoI digested pcDNA3 and named pcDNA3-hPOMΔACTH-036 A sense orientation clone was obtained (FIG. 3). NotI-Hind derived from pBS-IgSP-hPOMC-ΔACTH-029 The III IgSP-hPOMC-ΔACTH fragment was ligated to NotI-HindIII digested pcDNA3. , An antisense-oriented clone named pcDNA3-hPOMC-ΔACTH-037 was obtained (FIG. 3).   Using restriction digestion with PvuII and EcoRI, in pcDNA3-hPOMC-ΔACTH-036 The orientation of the insert was confirmed, whereas for pcDNA3-hPOMC-ΔACTH-037, SalI and And EcoRI were used.   Cloning of full-length and truncated TH cDNA   Total RNA from PC12 cells was converted to guanidium thiocyanate-based TRI reagent (Molec ular Research Center, Inc., Cincinnati, OH). 500ng PC 12 Total RNA, 10 mM Tris.HCl (pH 8.3), 50 mM KCl, 4 mM each dNTP, 5 mM MgCl_Two, 1.25 μM oligo (dT) 15mer, 1.25μM random hexamer, 31 units RNase Guar d RNase Inhibitor (Pharmacia, Sweden) and 200 units of SuperScript II In a reaction volume of 20 μl containing reverse transcriptase (Gibco BRL, Gaithersburg, MD) at 42 ° C For 30 minutes. 2 μl of the above reverse transcribed cDNA was added to 10 mM Tris.HCl (pH 8.3). ), 50 mM KCl, 800 nM each dNTP, 2 mM MgCl_Two, 400nM primers # 1 and # 2, if 2.5 unitsThermus aquaticus(Taq) DNA polymerase (Boehringer Mann heim, Germany) was added to 25 μl of the PCR reaction mixture.   Oligonucleotide primers orTH-052 (sequence No. 5) and orTH-053 (SEQ ID NO: 6) were used. For shortened TH, The markers orTH-054 (SEQ ID NO: 7) and orTH-053 (SEQ ID NO: 6) were used instead. This These oligonucleotides, Grima et al.,Nature, 326, pp. 707-11 (1987); No. 5,300,436, andDaubnerBased on published TH sequence information Built.   The primers orTH-052 (SEQ ID NO: 5) and orTH-054 (SEQ ID NO: 7) It has a mature HindIII restriction site, while orTH-053 has a BamHI at the 5 'end. PCR reaction mix The compound was subjected to 30 amplification cycles consisting of: denaturation, 30 seconds at 94 ° C (first sample). Annealing, 50 ° C for 1 minute; and elongation, 72 ° C for 3.5 minutes (final sample). Icle is 5 minutes). 1537 bp full length and 1087 bp truncated rat TH PCR fragment Is digested with the restriction endonucleases BamHI and HindIII and 1% SeaPla Separated by que agarose gel. 1531-bp and 1081-bp HindIII / BamHI DNA Fragments, and use the FMC SPinBind DNA purification kit (FMC BioProducts, Ro (ckland, ME).   The pcDNA3 expression vector was also digested with BamHI and HindIII and FMC SPinBi nd DNA purification kit (FMC BioProducts, Rockland, ME) with 1% SeaPlaque Purified from galose. The ligation mixture was added to E. coli. coli DH5α (Gibco BRL, G aithersburg, MD).   Cracking gel procedure (Promega Protocols and Applications Guide, 1991) Used to screen positive subclones. The same clone Assay was further confirmed by BamHI / HindIII double digestion.   positive subclone for full-length and truncated rat TH in pcDNA3 Were designated as pcDNA3-rTH-044 (FIG. 4) and pcDNA3-rTHΔ-045 (FIG. 4), respectively. . Nucleotide sequences of both full-length and truncated rat TH PCR clones were Using dideoxynucleotide sequencing with an ase kit (USBC, Cleveland) Was decided. The sequence of the rTHΔ construct is shown in SEQ ID NO: 16.   Oligonucleotide primers to optimize translation efficiency of truncated rat TH -OrTH-078 (SEQ ID NO: 8) with consensus Kozak sequence just above the start codon ATG Designed to be in the flow. pcDNA3-rTHΔ-45, oligonucleotide primer Is replaced by orTH-078 (SEQ ID NO: 8) and orTH-053 (SEQ ID NO: 6) In a 50 μl PCR reaction mixture having the same reagent composition as described above, Used. The 1097 bp PCR product was cloned into pcDNA3 in the same manner as described above. Was. The obtained subclone was named poDNA3-rTHΔKS-75 (FIG. 4). rTHΔKS construction The sequence of the product is shown in SEQ ID NO: 17.   Construction of rTH-IRES-bDBH fusion gene   The rTH-IRES-bDBH fusion gene was generated using recombinant PCR methodology. Oligonuk Leotide oIRES-057 (SEQ ID NO: 9) and obDBH-065 (SEQ ID NO: 10) And bDBH gene sequences, and synthetic BamHI and And NotI restriction sites. Oligonucleotides oIRES-bDBH-064 (SEQ ID NO: 11) and oIRES-bDBH-066 (SEQ ID NO: 12) is complementary to each other. In addition, oligonucleoti Primer IIRES-bDBH-064 (SEQ ID NO: 11) has its 5 '16 nucleotides identical to the IRES sequence. Has the same 3 ′ 18 nucleotides as the leotide and bDBH sequences; and oIRES- The opposite is true for bDBH-066 (SEQ ID NO: 12).   The two first PCR reactions were each performed using the template pCTI-001 (shown in SEQ ID NO: 30). PBS-bDBH-006 (with insert containing IRES sequence) and bovine adrenal chromaffin Lamoroux et al., Including the bovine DBH gene cloned from cells,EMBO J., 6,393 Oligonucleotide pair oIRES-057 / oIRES-bDB Performed using H-066 and oIRES-bDBH-064 / obDBH-065. 100ng template The DNA was treated with 10 mM Tris.HCl (pH 8.3), 50 mM KCl, 800 nM of each dNTP, 2 mM MgCl_Two, 400nM Primer # 1 and # 2 and 2.5 unitsThermus aquaticus(Taq) DNA port Add 50 μl of the PCR reaction mixture containing remelase (Boehringer Mannheim, Germany) Added.   The PCR reaction mixture was subjected to 30 amplification cycles consisting of: denaturation, 94 ° C. 30 Seconds (first cycle 2 minutes); annealing, 50 ° C. for 1 minute; and extension, 72 ° C. 30 Seconds (last cycle is 5 minutes). Separate the PCR product with 1% TrivieGel 500 (TrivieGen) Was. Two agarose plugs containing each of the first PCR products were 50 μl of the same PCR reaction as above except that oIRES-057 and obDBH-065 were used. The mixture was transferred to a tube containing the reaction mixture.   The second PCR reaction was subjected to 30 amplification cycles consisting of: denaturation, 94 ° C. 30 Seconds (first cycle is 2 minutes); annealing, 60 ° C. for 30 seconds (second to fourth cycles 37 ° C for 2 minutes); and extension, 72 ° C for 30 seconds (last cycle is 2 minutes). 2407bp IRES-bDBH The fusion PCR product and the cloning vector pcDNA3-rTHΔ-45 were converted to BamHI and NotI Digestion with restriction enzymes, followed by FMC SpinBind DNA Purification Kit (FMC BioProducts, Rock land, ME) using a 1% SeaPlaque agarose gel.   Ligation of IRES-bDBH / BamHI / NotI and pcDNA3-rTHΔ-045 / BamHI / NotI Is an rTHΔ-IRES-bDBH expression vector named pcDNA3-rTHΔ-IRES-bDBH-066 (Fig. 5), whereas IRES-bDBH / BamHI / NotI and pcDNA3-rTHΔKS-075 / BamHI / NotI The ligation of rTHΔKS-IRES named pcDNA3-rTHΔKS-IRES-bDBH-076 -bDBH expression vector (FIG. 5), where the start codon ATG in rTHΔ There is a consensus Kozak sequence. The sequence of the rTHΔ-IRES-bDBH construct is shown in SEQ ID NO: 18 You. The sequence of the rTHΔKS-IRES-bDBH construct is shown in SEQ ID NO: 19. Ligation mix The product was transformed into DH5α (Gibco BRL, Gaithersburg. MD). Positive claw The cracking gel procedure (Promega, Madison, WI) as well as HindIII, BamHI , HindIII / BamHI, SmaI, and NotI.   4114 bp NruI-XhoI fragment containing CMV promoter-rTHΔKS-IRES-bDBH Excised from pcDNA3-rTHΔKS-IRES-bDBH-076 and multiple cloning sites PZeoSV cloning vector digested with ScaI and XhoI in Invitrogen Corp. , San Diego, CA). The obtained expression vector was ligated with pZeo-P It was named cmv-rTHΔKS-IRES-bDBH-088 (FIG. 6).   Construction of IgSP-hPOMC ACTH-rTHD-IRES-bDBH fusion gene   CMV promoter, rTHD-IRES-bDBH fusion gene, and BGH polyadenylation sequence Was cut from pcDNA3-rTHΔ-IRES-bDBH-066. And subcloned into pBS (Stratagene, La Jolla, CA).   The resulting plasmid pBS-Pcmv-rTHΔ-IRES-bDBH-067 (FIG. 7) The R IgSP-hPOMCDACTH-IRES fragment was used as an intermediate construct to be inserted.   Oligonucleotide oIgSP-068 (SEQ ID NO: 13) containing a synthetic EcoRV restriction site is an IgSP Specific to the sequence.   Oligonucleotide primer orTHΔ-073 (SEQ ID NO: 14) is specific for rTHΔ And contains an endogenous SmaI restriction site.   Oligonucleotide primers ohPOMC-IRES-069 (SEQ ID NO: 15) and ohPOMC-IR ES-070 (SEQ ID NO: 20) is complementary to each other. In addition, oligonucleotide ply The mer ohPOMC-IRES-069 has its 5 ′ 18 nucleotides identical to the hPOMC sequence, and IR Has its 3 ′ 12 nucleotides identical to the ES sequence; and for ohPOMC-IRES-070 Is the opposite.   Oligonucleotide primers oIRES-rTHΔ-071 (SEQ ID NO: 21) and oRIRES-rT HΔ-072 (SEQ ID NO: 22) is complementary to each other. In addition, oligonucleotide The immersion oIRES-rTHΔ-071 has its 5 ′ 15 nucleotides identical to the rTHΔ sequence, and Has its 3 ′ 18 nucleotide sequence identical to the IRES sequence; and oRIRES-rTHΔ-072 The opposite is true.   Three sets of first PCR reactions were performed.   PCR reaction A: template pBS-IgSP-hPOMCDACTH-029, oligonucleotide oTgS P-068 / ohPOMC-IRES-069;   PCR reaction B: template pCTI-001, oligonucleotide ohPOMC-IRES-070 / oIR ES-rTHΔ-071; and   PCR reaction C: template pcDNA3-rTHΔ-045, oligonucleotide orIRES-rTH Δ-072 / or THΔ-073.   Three primary PCR reactions were performed with 100 ng of template DNA, 10 mM Tris.HCl (pH 8.3), 5 0 mM KCl, 800 nM each dNTP, 2 mM MgCl_Two, 400 nM of primers # 1 and # 2, and 2.5 unitsThermus aquaticus(Taq) DNA polymerase (Boehringer Mannheim , Germany) in a 50 μl PCR reaction mixture.   The PCR reaction mixture was subjected to 30 amplification cycles consisting of: denaturation, 94 ° C. 30 Seconds (first cycle 2 minutes); annealing, 50 ° C. for 1 minute; and extension, 72 ° C. 30 Seconds (last cycle is 5 minutes).   PCR products were separated on a 1% TrivieGel 500 (TrivieGen). From PCR reactions B and C Two agarose plugs containing each of the PCR products of the oligonucleotides ohPOMC- The same 50 μl PCR reaction as above except using IRES-070 and orTHΔ-073 Transferred to tube containing mixture.   The second PCR reaction was subjected to 30 amplification cycles consisting of: denaturation, 94 ° C. 30 Seconds (first cycle: 2 minutes); annealing, 60 ° C. for 30 seconds (37 cycles for second to fourth cycles) And extension, 72 ° C. for 30 seconds (last cycle is 2 minutes).   The PCR product was processed as described above. Second PCR reaction and PCR product from PCR reaction A Align the agarose plug with the stuff and use oIgSP-068 / rTHΔ-073 3 was subjected to PCR amplification. The 1203 bp IgSP-hPOMC-IRES-rTHΔ fusion PCR product and Vector pBS-Pcmv-rTHΔ-IRES-bDBH-067 with EcoRV and XmaI restriction enzymes With FMC SpinBind DNA Purification Kit (FMC BioProducts, Rockla (nd, ME) using a 1% SeaPlaque agarose gel. Ligation The mixture was transformed into DH5α (Gibco BRL, Gaithersburg, MD).   Positive clones, if cracking gel procedure (Promega, Madison, WI) And by restriction digestion with EcoRI, KpnI, and NotI. Got The clone was named pBS-IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES-bDBH-068. FIG. . The sequence of this construct is shown in SEQ ID NO: 23.   Construction of IgSP-hPOMCACTH-IRES-rTHΔ-IRES-bDBH expression vector   4449 bp NotI fragment containing IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES-bDBH gene From pBS-IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES-bDBH-068, and At the NotI site in the multiple cloning site, pcDNA3 (Invitrogen Corp., San Diego, (CA). Restriction digestion using NotI and SmaI was performed using IgSP-hP OMCΔACTH-IRES-rTHΔ-IRES-bDBH gene is inserted in the sense orientation, pcDNA3-IgSP- It was confirmed that hPOMCΔACTH-IRES-rTHΔ-IRES-bDBH-069 was obtained. See FIG. 9 That.   Construction of IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES-bDBH-IRES-Zeocine expression vector   An IRES-Zeocine fusion gene was generated using recombinant PCR methodology. Oligonu The nucleotides oIRES-074 (SEQ ID NO: 24) and oZeocin-077 (SEQ ID NO: 25) Specific to the IRES and Zeocin gene sequences, and at each 5 'end a synthetic NotI And a XhoI restriction site. Oligonucleotide oIRES-Zeocin-075 (SEQ ID NO: 26) And oIRES-Zeocin-076 (SEQ ID NO: 27) are complementary to each other. In addition, Oligonu The nucleotide oIRES-Zeocin-075 is its 5 '15 nucleotides identical to the Zeocin sequence, And its 3 '18 nucleotides identical to the IRES sequence; and oIRES-Zeocin-0 The opposite is true for 76.   The two first PCR reactions were performed with the templates pCTI-001 and pZeoSV (Invi trogen Corp., San Diego, Calif.) Performed using S-074 / oIRES-Zeocin-075 and oIRES-Zeocin-076 / oZeocin-075 .   100 ng of template DNA was added to 10 mM Tris.HCl (pH 8.3), 50 mM KCl, 800 nM of each dNT. P, 2mM MgCl_Two, 400 nM of primers # 1 and # 2, and 2.5 unitsThermus aquaticus (Taq) 50 μl containing DNA polymerase (Boehringer Mannheim, Germany) Was added to the PCR reaction mixture.   The PCR reaction mixture was subjected to 30 amplification cycles consisting of: denaturation, 94 ° C. 30 Seconds (first cycle 2 minutes); annealing, 50 ° C. for 1 minute; and extension, 72 ° C. 30 Seconds (last cycle is 5 minutes).   PCR products were separated on a 1% TrivieGel 500 (TrivieGen). Each of the first PCR products Containing two agarose plugs, oligonucleotides oIRES-074 and oZeocin-0 In the tube containing the same 50 μl PCR reaction mixture as described above except that 77 was used. Moved.   The second PCR reaction was subjected to 30 amplification cycles consisting of: denaturation, 94 ° C. 30 Second (2 minutes for the first cycle); annealing, 50 ° C. for 30 seconds (37 cycles for the second to fourth cycles) And extension, 72 ° C. for 30 seconds (last cycle is 2 minutes).   The 974 bp IRES-Zeocin fusion PCR product and the cloning vector pcDNA3 were And XhoI restriction enzyme, followed by FMC SpinBind DNA purification kit (FMC Bio Products, Rockland, ME) using a 1% SeaPlaque agarose gel. .   The ligation of IRES-Zeocin / NotI / XhoI and pcDNA3 / NotI / XhoI Generate an intermediate cloning vector named IRES-Zeocin-072. FIG.   Positive clones, if cracking gel procedure (Promega, Madison, WI) By restriction digestion with HindIII, SmaI, XhoI, NotI, and NotI / XhoI. Identified.   Final IgSP-hPOMCDACTH-IRES-rTHD-IRES-bDBH-IRES-Zeocine expression vector 4951 bp containing the IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES-bDBH gene to generate The NotI fragment was ligated with pBS-IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES-bDBH-068 (FIG. 8; SEQ ID NO: 23) and pcDNA at the NotI site in the multiple cloning site It was subcloned into 3-IRES-Zeocin-072 (FIG. 10).   Restriction digestion using NotI and SmaI was performed using IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES-b The DBH gene was inserted in the sense orientation, pcDNA3-IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES It was confirmed that -bDBH-IRES-Zeocin-073 was obtained. Sequence number of this construct No. 28. FIG.   Construction of ProA + KS fusion   A human proenke with a consensus Kozak sequence immediately upstream of the start codon ATG A construct comprising the coding region of the Farin A gene. The sequence of this construct is set forth in SEQ ID NO: 29. Show.   Construction of hProA + KS expression vector   The HindIII / BamHI fragment containing the hProA + KS fusion was substantially as described above. , BamHI and HindIII digested into pcDNA3 expression vector. as mentioned above After screening, the positive subclone was named pcDNA3-hProA + KS-091 did. FIG. Construction of the pBS-CMV Pro A vector is described in Mothis, J. and Lindberg, I. .,Endocrinology, 131, pp. 2287-96 (1992).   Cell transformation   RIN and AtT-20 cells were transformed as follows.   A cell line based on RINa and AtT-20 was prepared using 10% fetal bovine serum and pen-strep-fung. Grow in DMEM (Gibco) with izone (Gibco) basal medium. Transfer cells to 10 ml The seeds were seeded underground in P100 Petri dishes (750,000 cells / dish). 18-24 hours The cells were then harvested using the calcium phosphate method with a kit from Stratagene (San Diego, CA). And transfected. Remove 10 μg of plasmid vector DNA into 450 μl Diluted with sterile ionized water. Then, 50 μl of 10 × buffer (solution # 1) was Added to sumid DNA. Immediately add 500 μl of solution # 2 to the DNA-containing solution and add And mixed gently. This is incubated for 20 minutes at room temperature, then 1.0 ml The solution was added to the cells in the Petri dish. Incubate the cells overnight and allow After 18-24 hours, the cells are washed with calcium and magnesium free Hanks plates. Washed twice with balanced salt solution. The cells were then cultured in basal medium + selective drug . Cells were harvested with 600 μg / ml geneticin (Gibco) or 400 μg / ml hygromycin (Boeh ri nger Mannheim) or 500 μg / ml Zeocin (In Vitrogen, San Diego, CA) I chose it. Then transfect the cells and get the final cell line Selected.   RINa cells were transfected with plasmid pCEP4-hPOMC-030 containing the POMC gene. I did it. This is a hygromycin resistance vector. Cells can also be It was transformed with pcDNA3-hProA + KS-091. This is a geneticin resistance vector. Finally, cells were transformed with plasmid pZeo-PCMV-rTHΔKS-IRES-bDBH-, which confers Zeocin resistance. Transfected at 088.   AtT-20 cells were harvested from plasmids conferring geneticin and hygromycin resistance, respectively. Transfected with the pmids pBS-CMV-ProA and pCEP4-POMC-ΔACTH-32. last Next, cells are transfected with the plasmid pZeo-Pcmv-rTHΔKS-IRES-bDBH-088 did.   We have tested a number of media for cell growth. Surprisingly, The authors reported that in certain serum-free media, the cell line It has been found that it has enhanced the production of transtransmitters. CH for AtT-20 cell proliferation O-Ultra (Biowhitaker) and Ultra-Culture (Biow hitaker) is preferred.   Production of various analgesics from one transformed RINa cell (RINa / ProA / P030 / P088) The results are shown in Table 2. All values indicate unstimulated cells. β-endorphin And the production of met-enkephalin is pg / 10⁶Cells / hour. β-endorphy And met-enkephalin using Incstar kit (Stillwater, Minnesota) It was determined by radioimmunoassay. Catecholamine production is pmol /Ten⁶Cells / hour. Numbers in parentheses are 100 μM tetrahydrobiopterin Values from cells previously incubated for 18 hours are shown. Catecholamine, Lavoie et al., `` Two PC12 pheochromocytoma lines sealed in hollow fiber-based capsules tonically release l-dopa in vitro ",Cell transplantation, 2,16 It was measured by high performance liquid chromatography as described on page 3-73 (1993). This The production of GABA from these RINa cells is 28 ng / 10⁶Cells / hour. Encrypted, poorly processed from proenkephalin precursor molecule Enkephalin fragment. These encrypted enkephalins Has opioid receptor binding activity. To measure opioid activity Cleaved these encrypted enkephalins. Trypsin cleavage protocol Is as follows. Add 2 μg / ml trypsin (Worthington # 34E470) solution to ice Add to the above medium sample. Vortex the sample and then in a 37 ° C water bath for 20 minutes Incubate. After a 20 minute cut, return the sample to ice and remove 100 ng / ml Boxypeptidase B (Sigma # C-7011) is added. Vortex the sample And return to a 37 ° C. water bath for 15 minutes. Place the sample on ice again And 10 ug / ml trypsin inhibitor is added. At this stage, the sample is met- Extract for enkephalin or freeze immediately for future extraction Do either one. This means that all longer encrypted fragments This results in complete enzymatic cleavage to release met-enkephalin. Cut sample The meta-enkephalin radioimmunoassay is used to measure total met-enkephalin from the supernatant. give. Transformed RINa cells are fully processed met-enkefu Seems to have more than 5 times more encrypted enkephalin compared to is there.Fiber capsule formation and characterization   The hollow fiber is made from 12.5-13.5% poly (acrylonitrile vinyl) by wet spinning technique. (Chloride) spin from solution. Cabasso,Hollow Fiber Membranes, Volume 12,Kirk-O thmer Encyclopedia of Chemical Technology , Wiley, New York, 3rd edition, 492- 517 (1980), U.S. Patent No. 5,158,881, which is incorporated herein by reference.   The resulting membrane fibers can be either double or single skin PAN / PVC fibers . To produce an implantable capsule, the length of the fiber must first be And the distal end of each segment is sealed with acrylic glue. Cap The cellized hub assembly can be provided with a membrane of the above length and LCM 24 (light curable acryl Seal one end of the fiber with a drop of (available from ICI) and cure the glue with blue light. And by repeating this process with a second drop. Opposite The end is pre-attached to the fragile neck hub assembly, which allows the cell solution to It has a silicone septum that can be introduced through it. LCM 22 outside of hub assembly By applying to the diameter, pulling the fiber over it, and curing with blue light. To bond the fibers to the hub assembly. Hub / fiber assembly in sterile bag And sterilize with ETO.   After sterilization with ethylene oxide and evacuation, first 70% EtOH, then H Ultrafiltration of the EPES buffered saline solution through the fiber wall under vacuum To deglycerine the fibers.Preparation and encapsulation of transformed cells   Transformed cells are prepared and encapsulated as follows:   Mix the matrix solution with commercially available alginate, collagen, or other suitable matrices. Prepared using Rix material. Add 2 parts (matrix solution) to 1 part cell solution (Diluted at a ratio of cell solution 9 containing the transformed cells as described above. As the Trix, Vitrogen (Celtix, Santa Clara) is preferred.   Organogen (Organogenesis, Canton, MA) is a good matrix for RINa cells. Good. RINa-based cells are prepared for encapsulation by the following method. The cells are grown in a basal medium of DMEM + 10% fetal calf serum during the growth phase. these Cells in Hanks balanced salt solution without calcium and magnesium With two washes, it can be removed from the tissue culture flask. The cells are then Incubate for 1 minute in 0.25% trypsin + EDTA. Take this out, The cells using a Hanks balanced salt solution without calcium and magnesium. And rinse from flask. Place cells in 10 ml of basal medium and add 10 Centrifuge at 0 × g for 2 minutes. Cells are grown in 10 ml of a preferred serum-free medium (Ultra culture , Biowhitaker, Walkersville, MD). Surprisingly, RINa cells When cultured in this serum-free medium, more culture medium is Secretes earth materials.   Before concentrating the cells 1: 1 with the preferred Organogen matrix, the cells are Centrifuge twice at 100 g in serum medium. Organogen is obtained as a sterile solution 1% bovine tendon collagen. Eight parts of this solution are mixed with one part of 10 × DPBS. 0.5 N sodium hydroxide is added until physiological pH is reached (about 250 μl).   The final concentration of cells + matrix solution used for encapsulation is 20,000-50,000 It can be in the range of cells / μl. Cells are counted in a hemacytometer by standard methods.   Place the cell / matrix suspension in a 1 ml syringe. Hamilton 1800 Series 50 Set the μl syringe in a 15 μl bubble and insert into a 1 ml syringe containing the cell solution And raise 30 μl. Transfer the cell solution to the hub / fiber assembly silicone Through the seal, a model with a molecular weight cutoff of about 50,000 to 100,000 daltons. Inject into the lumen of the acrylic acrylic fiber membrane. Ultrafiltration along the entire length of the fiber Should be observed. After one minute, break the hub off the sub-hub and remove it from the cell solution. Expose fresh, unsurfaced surfaces. Apply one drop of LCM 24 and remove the sticky with blue light To cure. The device was first placed in a HEPES buffered NaCl solution, then CaCl 2_TwoIn solution To crosslink the alginate. Each implant is approximately 5 cm long and 1 mm in diameter. And contains about 2,500,000 cells.   After filling and sealing the device, the silicone tether (Speciality Sil icone Fabrication, Paso Robles, CA) (ID: 0.69, OD: 1.25), followed by fiber Place on the proximal end of the Radiopaque titanium plug with radiographic marker Insert into the lumen of the silicone tether to act as Then, the device Place the chairs in 1.5 ml PC-1 medium in a 100 mm tissue culture dish and incubate 5% CO at 37 ° C. for toro analysis and storage until transplantation_TwoIncube Store in a mortar.   The encapsulated cells are then implanted into the human subarachnoid space as follows:Surgical procedure   Establish IV access and prophylactically treat antibiotics (cefazolin sodium, 1 g After administration of Lamb IV), the patient is bent forward with the lumbar spine approximately Place it on the operating table. Surgical area is aseptically prepared and covered, midline Exposed the dorsal lumbar region from S-1 level to L-1 and use C-arm fluoroscopy And perform lumbar spine surgery. Skin using local infiltration with 1.0% lidocaine And anesthesia to the periosteum and other deep connective tissue structures, including the yellow ligament Stand up.   A 3-5 cm skin incision is made using an electric scalpel to stop bleeding, one to the right or left A 矢 2 cm lateral sagittal plane was created and continued to the thoracolumbar fascia. Iliac crest and lumbar spine Using traditional bone landmarks, including projections, and fluoroscopy indices, 18 gauge A Touhy needle was placed into the subarachnoid space between L-3 and L-4 via an oblique median approach Introduce. The injection needle should be oriented so that it enters the subarachnoid space shallowly, preferably in a sagittal plane or Is at an angle not exceeding 30-35 ° with respect to the spinal cord in any of the cross sections. Injection needle tip Proper position of catecholamine, enkephalin, glucose, human CNTF and a few ml of cerebrospinal fluid (CSF) for protein level and cell number Confirm by punching.   Re-examine the Touhy needle hub and make sure the opening is in the desired direction at the tip. (The orientation of the opening is indicated by the indicating notch for the obturator on the needle hub. Until the guidewire extends 4-5 cm into the subarachnoid space (measure in advance). Determined) to pass along the lumen of the injection needle. During the passage of the wire, the injection No resistance to needle-to-wire advancement, and patient does not complain of significant neurological symptoms Note that Neither of these observations can indicate a guidewire misoperation. And may indicate an imminent possibility of nerve root or spinal cord injury.   After the guidewire appears to be properly positioned in the subarachnoid space, the Touhy injection needle Is pulled separately and removed from the wire. Then the wire in the midline of the spinal canal Position (forward of the expected position of the cauda equina) and no torsion or unexplained flexion This is confirmed by fluoroscopy. After removing the Touhy needle, guidewire Very little resistance as the wire surface travels through the dense, fibrous yellow ligament It should be able to move freely into and out of the subarachnoid space.   The 7 French dilator is then placed on a guide wire and the dilator is Following the wire trajectory, towards the subarachnoid space, fascia, paraspinal muscle, and yellow Gently but firmly pushed through the ligament. 7 French Extender Stops progressing and detects that resistance is gone after passing through the yellow ligament Remove the dilator from the wire immediately. This is because of the relatively rigid nature of the Move the dilator to any significant degree to avoid advancing and manipulating it. Will be   After the wire trajectory is "overextended" by the 7 French extender, the 6 French extender and The cannula sheath was assembled and placed over the guidewire. 6 French extension The cannula with the open tip of the cannula at 7 cm in the subarachnoid space. Proceed carefully until deployed. Assembling as with the 7 French extender 6French dilator and cannula with wire inside dilator lumen . The position in the subarachnoid space was determined by pre-measuring the device, and the total Physically, it is confirmed by X-ray fluoroscopy. Intrathecal dilator and cannula Great care should be taken in the operation to avoid misdirection and possible neurological damage.   When it is confirmed that the cannula has been properly positioned, the guidewire and 6 Slowly remove the French dilator sequentially from the cannula lumen. On the operating table Depending on the position of the patient, the CSF flow through the cannula at this point should be perceived And can be very active and cannulated with a cannula to avoid excessive CSF loss. Very quick placement of the capsule or implant is required.   The encapsulated (transformed cells) are bathed in a transport medium, and Fully assembled, sterile, double jacket container with tubular silicone tether Can be provided throughout the country. Through the cannula and prior to implantation into the subarachnoid space, The capsule can be transferred to an insertion kit tray where the capsule is damaged or damaged. Placed in a location maintained in the transport medium during a comprehensive inspection for major defects But the silicone tether adjusts the length of the pusher and blocks the outer ends. Hemaclip^TMIs arranged to remove   The tether of the capsule is attached to the membrane of the device by the small diameter The ear part is inserted on the stainless steel extruder by inserting Carefully introduced into the cannula. The capsule, the tip of the membrane, Proceed until reaching a point 2-10 mm within the cranial tip of the cannula in the subarachnoid space You. This arrangement allows for a cannula and capsule-tether-extruder assembly. Measurement, which is accomplished by measuring the position of the membrane portion of the capsule. Ensure that it is protected by the cannula for the entire time that it is advanced to.   After the capsule has been placed in the cannula, the cannula is While completely withdrawn from the tool, the capsule is moved to a position in the subarachnoid space (Without or without being pulled out). Then Slide the extruder out of the silicone ties and the wire portion of the extruder. Remove from capsule by letting. Using this method, capsules can be Membrane completely lies on the ventral side of the cauda equina in the subarachnoid space containing CSF. To the final position. It has silicone tethers from the dura and yellow ligament Previously, the tether was passed through the subarachnoid space and was approximately 1-2 cm long. And is moored at its caudal end. From this level, the connection Available to attach to device, continuing through and out of thoracolumbar fascia Leave a good 10-12cm free splicing material. Inject into the runway and use a purse string suture to tightly close the superficial fascia opening of the runway. To minimize it. The free end of the tether is then engaged using a non-absorbable suture. Retain and completely cover with a two-layer closure of skin and subcutaneous tissue.   The patient is then transferred to a neurosurgical recovery area and placed in bed for 24 hours after surgery. Lying on top, keep strictly at rest. Antibiotic prophylaxis is also a transplant procedure Continue for 24 hours.Array   The following is a summary of the sequences listed in the sequence listing: SEQ ID NO: 1--DNA sequence of oligo oCNTF-003 SEQ ID NO: 2-DNA sequence of oligo oIgSP-018 SEQ ID NO: 3-DNA sequence of IgSP-hPOMC fusion SEQ ID NO: 4-DNA sequence of IgSP-hPOMC-ΔACTH fusion SEQ ID NO: 5--DNA sequence of oligo orTH-052 SEQ ID NO: 6--DNA sequence of oligo or TH-053 SEQ ID NO: 7--DNA sequence of oligo or TH-054 SEQ ID NO: 8--DNA sequence of oligo or TH-078 SEQ ID NO: 9--DNA sequence of oligo oIRES-057 SEQ ID NO: 10--DNA sequence of oligo obDBH-065 SEQ ID NO: 11--DNA sequence of oligo oIRES-bDBH-064 SEQ ID NO: 12--DNA sequence of oligo oIRES-bDBH-066 SEQ ID NO: 13--DNA sequence of oligo oIRE-068 SEQ ID NO: 14--DNA sequence of oligo or THΔ-073 SEQ ID NO: 15--DNA sequence of oligo ohPOMC-IRES-069 SEQ ID NO: 16--DNA sequence of rTHΔ1-155 SEQ ID NO: 17--DNA sequence of rTHΔ + KS SEQ ID NO: 18--DNA sequence of rTHΔ-IRES-bDBH SEQ ID NO: 19--DNA sequence of rTHΔKS-IRES-bDBH SEQ ID NO: 20--DNA sequence of oligo ohPOMC-IRES-070 SEQ ID NO: 21--DNA sequence of oligo oIRES-rTHΔ-071 SEQ ID NO: 22--DNA sequence of oligo orIRES-rTHΔ-072 SEQ ID NO: 23-DNA sequence of IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES-bDBH-068 fusion SEQ ID NO: 24-DNA sequence of oIRES-074 SEQ ID NO: 25--DNA sequence of oligo oZeocin-077 SEQ ID NO: 26--DNA sequence of oligo oIRES-Zeocin-075 SEQ ID NO: 27--DNA sequence of oligo oIRES-Zeocin-076 SEQ ID NO: 28--of IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES-bDBH-IRES-Zeocin-073               DNA sequence SEQ ID NO: 29--DNA sequence of proA + KS SEQ ID NO: 30--DNA sequence of IRES fragmentDeposit   RINa / ProA / POMC / TH-IRES-DBH cells were cultured as described above in the Examples (and Table 2). Traits to produce techolamine, enkephalin, and endorphin Converted and named RINa / ProA / P030 / P088, which has been deposited. Bye Commissioning is performed in accordance with the Budapest Treaty, and the American Type Culture Collection Was deposited with Rockville, Maryland, U.S.A. on June 7, 1995. Deposit Received accession number CRL 11921.   The above description has been made for the purposes of illustration and description only. This description is an example It is not intended to limit the invention to the precise form in which it is performed. The scope of the present invention is as follows: It is intended to be defined by the appended claims.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12N 5/10 A61K 37/24 (81) Designated country EP (AT, BE, CH, DE, DK, ES, FI, FR, GB , GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, SZ, UG), UA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BB, BG , BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IL, IS, JP, KE, KG, KP, KR, KZ, LK LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR , TT, UA, UG, US, UZ, VN (72) Inventor Wong, Shaw United States Rhode Island 02903, Providence, Park Row West 50, Number 209, Center Place

Claims (1)

[Claims]   1. Of the group consisting of endorphins, enkephalins and catecholamines Stably transformed to produce at least one analgesic compound from each Cells.   2. The cell according to claim 1, wherein the endorphin is β-endorphin. .   3. 2. The cell of claim 1, wherein said enkephalin is met-enkephalin. .   4. Wherein the catecholamine is norepinephrine or epinephrine. The cell according to claim 1.   5. The cell according to any one of claims 1 to 4, wherein the cell is a RIN cell.   6. The cell according to any one of claims 1 to 4, wherein the cell is an AtT-20 cell.   7. The cells may be composed of galanin, somatostatin, neuropeptide Y, neuron, Further produce a compound selected from the group consisting of tensin or cholecystokinin The cell according to any one of claims 1 to 6, which grows.   8. DNA encoding POMC, DNA encoding TH, DNA encoding DBH, and A cell transformed with DNA encoding ProA, wherein each DNA molecule has an expression control sequence. A cell operably linked to a cell.   9. The cells are pCEP4-POMC-030, pcDNA3-hproA + KS-091, and pZeo-pCMV-r 9. The cell according to claim 8, which has been transformed with THΔKS-IRES-bDBH-088.   10. The cells are pCEP4-hPOMC-ΔACTH-032, pBS-CMV-proA, and pZeo-pCM The cell according to claim 8, which has been transformed with V-rTHΔKS-IRES-bDBH-088.   11. The cells, pcDNA3-hPOMCDACTH-IRES-rTHD-IRES-bDBH-IRES-Zeocin-07 The cell according to claim 8, which has been transformed with 3 and pcDNA3-proA + KS-091.   12. At least one enkephalin, one endorphin, and one A transformed cell producing two catecholamines, wherein the cell comprises:   First vector containing DNA encoding POMC operably linked to an expression control sequence Tar,   DNA encoding proenkephalin A operably linked to an expression control sequence A second vector, comprising:   DNA encoding TH operably linked to expression control sequences and expression control sequences Contains DNA encoding dopamine β-hydroxylase operably linked to The third vector, The cells, which are transformed with   13. 13. A therapeutically effective number of any of claims 1 to 12 at the patient's implantation site. A method for treating pain, comprising transplanting cells.   14． Wherein the cells are encapsulated in a semi-permeable membrane to form a bioprosthesis. 14. The method according to claim 13.   15. 15. The method of claim 14, wherein the bioprosthesis is immunoisolated.   16. The method according to any one of claims 13 to 15, wherein the implantation site is the CNS. .   17． 16. The transplantation site of any one of claims 13 to 15, wherein the implantation site is a subarachnoid space. the method of.   18. At least one enkephalin, one endorphin, and one A method for producing cells secreting two catecholamines, comprising: DNA encoding POMC operably linked to a sequence, operable to a second expression control sequence DNA encoding proenkephalin A operably linked, and a third expression DNA encoding TH operably linked to a control sequence and a fourth expression control sequence DNA encoding dopamine β-hydroxylase operably linked to A method comprising the step of transforming a cell.   19. The first, second, third, and fourth expression control sequences are identical. Item 19. The method according to Item 18.   20. 13. A method according to any of claims 1 to 12, for the manufacture of a medicament for the treatment of pain. Use of the cell according to the above.   21. 21. The cell of claim 20, wherein said cell is transplanted.   22. Wherein the cells are encapsulated in a semi-permeable membrane to form a bioprosthesis. 23. The cell according to any one of claims 21 to 22.   23. 23. The cell of claim 22, wherein said bioprosthesis is immunoisolated.   24. The cell according to any one of claims 21 to 23, wherein the transplantation site is the CNS. .   25. 24. The method according to claim 21, wherein the implantation site is a subarachnoid space. Cells.   26. (a) a biocompatible permeable jacket surrounding the core; and   (b) the group consisting of endorphin, enkephalin and catecholamine Transformed to produce at least one analgesic compound from each of the The core comprising at least one living cell And a biological prosthesis.   27. 27. The living human of claim 26, for use in treating pain. Engineering organ.   28. Group consisting of endorphin, enkephalin, and catecholamine Transformed to produce at least one analgesic compound from each of the The core containing at least one living cell is encapsulated with a biocompatible permeable jacket. A method for producing a biological prosthesis, comprising the step of   29. 13. The method according to claim 1, wherein the medicament is used for the treatment of pain. Use of bioprostheses containing vesicles.