SK167997A3 - Cell line producing analgesic compounds for treating pain - Google Patents

Abstract

A genetically 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 may be provided directly to a patient in need thereof, or encapsulated to form a bioartificial organ.

Description

A cell line producing compounds with analgesic effect for the treatment of pain

Technical field

The present invention relates to a cell line useful for the treatment of pain. More specifically, the cell line of the invention has been engineered by genetic engineering methods to produce at least one analgesic compound of each species from the group of endorphins, enkephalins and catecholamines.

BACKGROUND OF THE INVENTION

Pain is a general symptom of the disease. The posterior spinal cord surface layers, where the primary afferent fibers bearing the nociceptive (ending in painful) information terminate, contain enkephalinergic interneurons and a high opioid receptor density. In addition, there is a high concentration of non-adrenergic fibers in the spinal layers.

Acute pain arises in response to acute harmful stimuli. Chronic pain is mainly caused by neuropathies of central or peripheral origin. This neuropathic pain is the result of an impaired somatosensory process that may result in increased sensitivity to painful stimuli (hyperalgesia) and pain associated with a stimulus that usually does not induce pain (allodinia).

Intrathecal injection of morphine into the subarachnoid spinal cord space leads to strong analgesia (decreased pain sensitivity). Intrathecal administration of norepinephrine or noradrenergic agonists also results in analgesia. See e.g. Sagen et al., Proc. Natl. Acad. Sci. USA 83: 7522-26 (1986).

Co-administration of multiple opioids at doses less than effective, e.g. enkephalins and catecholamines such as norepinephrine can act synergistically to induce analgesia. See above. Chromaffin cells in the adrenal medulla produce and release a variety of neuroactive substances including norepinephrine, epinephrine, met-enkephalin, leu-enkephalin, neuropeptide Y, vasoactive intestinal peptide, somatostatin, neurotensin, cholecystokinin, and calcitonin-related peptide. See Sagen et al., Proc. Natl. Acad. Sci. USA, 83, 7522-26 (1986) and Sagen et al., Jour. Neurochem., 56, 623-27 (1991).

Because chromaffin cells form both opioid peptides and catecholamines, one possible approach to reduce nociceptive response or pain sensitivity has been investigated by transplantation of adrenal tissue or isolated adrenal chromaffin cells directly into the pain modulating CNS region to induce analgesia. See Sagen et al., Brain Research, 384, 189-94 (1986), Vaquero et al., Neuroreport, 2, 149-51 (1991), Ginsberg and Seltzer, Brain Research, 523, 147-50 (1990) Sagen et al., Pain, 42, 69-79 (1990).

Experiments were also performed in which analgesia was to be induced with both allogeneic and xenogeneic grafts of chromaffin tissue or cells. Allograft tissue is only available in limited quantities and not readily accessible, especially for human pain treatment programs. In addition, allogeneic human tissue carries the risk of pathogenic contamination. See Hama and Sagen, Brain Research, 651, 183-93 (1994).

Xenogeneic donors can provide a large amount of material that can be easily obtained. For this reason, bovine adrenal tissue is used. See Hama and Sagen, Brain

Research, 651, 183-93 (1994).

However, in transplants between incompatible biological species, a major problem is the potential serious consequences for the host as well as the ultimate graft rejection. Due to the presence of strongly antigenic cells in the xenograft, especially endothelial cells, complete graft rejection of whole or split tissue may occur even in the CNS, which is considered immunologically privileged. In addition, the donor tissue must be thoroughly examined to prevent the introduction of viral contamination or other pathogens into the host organism. To avoid graft rejection, immunosuppression is required, typically using cyclosporin A.

Using such transplants, according to published results, has shown some reduction in pain sensitivity, especially in low intensity chronic pain. Significant difference between control and transplanted animal was achieved in most published reports only after the addition of nicotine, which stimulated the production of opioid peptides. However, there are several reports that in rat chronic pain model, chromaffin tissue allograft analgesia with a basal level of activity has been achieved. See Vaquero et al., Neuroreport, 2, 149-51 (1991), Hama and Sagen, Brain Research, 651, 183-93 (1994).

Bovine adrenal chromaffin cells have been encapsulated to form a biological artificial organ (BAO) for implantation in rats to treat acute and chronic pain. See e.g. Sagen et al., J. Neurosci., 13, 2415-23 (1993) and Hama et al., 7th World Congress Pain, Abstract 982, Paris, France (1993). The first experiments using encapsulated bovine cells were performed on humans. See Aebischer et al., Transplantation, 58, 1275-77 (1994).

Attempts have also been made to induce a reduced response to painful stimuli using other cells, e.g. AtT-20 cells. AtT-20 cells originally originate from a mouse anterior pituitary tumor. These cells synthesize and secrete β-endorphin. See, e.g., Wu et al., J. Neural. Transplant. & Plasticity, 5, 1526 (1993). AtT-20 / hENK cells are AtT-20 cells that have been engineered by genetic engineering to carry the entire human proenkephalin A gene (ie, contain 6 met-enkephalin sequences and one leu-enkephalin sequence) with 200 bases from the 5'-terminal sequence and 2.66 kilobases from the 3'-ketone sequence. See Wu et al., Supra, Comb et al., EMBO J., 4, 3115-22 (1985).

Wu et al., J. Neural. Transplant. & Plasticity, 5, 15-26 (1993) disclose rat carriers transplanted with AtT-20 or AtT-20 / hENK cells. Unstimulated AtT-20 / hENK cells were more effective at inducing a reduced response to painful stimuli (tail twitch test) than AtT-20 cell implants. In contrast, isoproterenol stimulation was more effective with AtT-20 cells than with AtT-20 / hENK cells above.

In a mouse host, implants of AtT-20 or AtT-20 / hENK cells did not affect the baseline response to thermal nociceptive stimuli. Mice receiving AtT-20 implants developed tolerance to β-endorphin and a µ-opioid agonist (DAMGO). Mice receiving AtT-20 / hENK implants developed δ-opioid agonist (DPDPE) tolerance. In response to repeated doses of µ-opioid agonist, mice receiving AtT-20 / hENK cell implants developed less tolerance compared to mice receiving AtT-20 cells or control animals.

The antinociceptive effect of isoproterenol was the same in both mice receiving ÄtT-20 cell implants and mice with AtT-20 / hENK. See Wu et al., J. Neuroscience, 14, 4806-14, (1994). Wu et al., Believed that one of the causes of the lack of a side-effect in response to pain stimuli in enkephalin-producing AtT-20 / hENK-transplanted mice may be a lack of sensitivity in behavioral tests. Another possible justification was that the known antagonist effect of met-enkephalin on the morphine-induced decreased response to painful stimuli offset the potentiating effect of leu-enkephalin alone, especially since there are 6 met-enkephalin sequences for each leu sequence in pro-enkephalin A. -enkefalínu.

SUMMARY OF THE INVENTION

The present invention provides a cell line that has been engineered by genetic engineering methods to form at least one analgesic compound of each species from the group of endorphins, enkephalins and catecholamines. The cell line can be used to treat pain.

The use of a cell line is preferable to the use of primary cells. To ensure safety and operational criteria for the use of cells, individual primary cell isolates must be costly and time consuming. The cell bank requires less testing. There is no need to isolate primary cells. The production of the desired analgesics may be more stable since the performance of the primary cells may depend on the age, sex, health, or hormone status of the donor animal. It is also possible to achieve higher production of the desired products and also to introduce specifically modified peptides into the cell line. This allows the delivery of several different analgesics simultaneously. The expression of one or more analgesics may be regulated (using a controllable promoter to drive expression). In addition, for safety reasons, a suicide gene may be incorporated into the cell line.

Further, for encapsulation, proliferating cells have the advantage of dividing and thus replacing dying or dead cells.

In order that this invention may be more fully understood, a detailed description follows.

Any suitable cell line can be transformed with the recombinant DNA molecules of the invention. Among the cells in question are chromaffin cells, including conditionally immortal chromaffin cells, such as those described in WO 96/02646, Neuro-2A, PC12, PC12a, SK-N-MC, At-20 and RIN cells including RINa and RINb. Preferably, the cell has endogenous convertorases of prohormone and / or dopa-decarboxylase.

SK-N-MC cells, a neuroepithelioma cell line, co-express several neuropeptides, including enkephalin, cholecystokinin, and gastrin-releasing peptide. See Verbeeck et al., J. Biol. Chem., 265, 18087-190 (1990). The proenkephalin A gene was expressed in SK-N-MC cells. Folkesson et al., Mol. Brain Res., 3, 147-54 (1988). We prefer AtT-20 and RIN cells, most preferably RIN cells.

RIN cells are a pancreatic endocrine cell line derived from a rat. See e.g. Horella et al., J. Physiol., 85, 158-70 (1991). RIN cells are known to produce endogenous GABA and β-endorphin.

Some of the properties of the various cells under consideration are shown in Table 1.

Table 1

Cells analgesic substances Other ingredients chromaffin NE, met-enkephalin TH, DDC, ϋβΗ, PC12, PC12a low NE, met-enkephalin DDC, ϋβΗ, PC AtT-20 β-endorphin DDC, PC RINa β-endorphin, GABA DDC, PC RINb β-endorphin DDC, PC Neuro-2A DDC, ΟβΗ, PC

ΤΗ = tyrosine hydroxylase converts tyrosine-1-dopa

DDC = dopaminedecarboxylase converts 1-dopa-dopamine (DA) ϋβΗ = dopamine p-hydroxylase converts DA - norepinephrine (NE)

PC = prohorraone convertases processing POMC to β-endorphin and Pro-enkephalin A (ProA) to met-enkephalin

AtT-20 = corticotropic mouse pituitary cell line that secretes β-endorphin endogenously via the expression of Proopiomelanocortin (POMC)

RIN = rat insulin

Neuro2A = mouse neuroblastoma

Primary delivery products include at least one endorphine, enkephalin and catecholamine.

Enkephalins and endorphins are endogenous opioid peptides in humans. These opioid peptides comprise about 15 compounds ranging from 5 to 31 amino acids. These compounds bind to and at least partially act through the same opioid receptor µ as morphine, but are not chemically related to morphine. In addition, these compounds stimulate other opioid receptors. Yaksh and Malmberg, Textbook of Pain, 3rd Ed. (Eds. P. Wall and R. Melzack), Central

Pharmacology of Nociceptive Transmission, 165-200, 1994 (New

York).

Opioid peptides share common chemical properties but are synthesized by different pathways.

β-endorphin, the most abundant endorphin, is synthesized as part of a large precursor molecule, proopiomelanocortin (POMC). The POMC molecule contains the entire sequence of adrenocorticotropic hormone (ACTH), α-melanocyte stimulating hormone (α-MSH), β-MSH and β-lipotropin. The POMC precursor molecule also has the potential to produce other endorphins, including α-endorphin and gamma-endorphin. The processing of the POMC precursor is different in different tissues according to the location of the cleavage enzymes, such as prohormone convertases, in these tissues.

In the pituitary gland, POMC is cleaved to form ACTH and β-endorphin, and ACTH is no longer processed. In the hypothalamus, on the other hand, ACTH is converted to β-MSH. While different cell types can synthesize the same primary gene product, the final profile of hormonal secretion varies widely.

The present invention contemplates the use of a DNA sequence that encodes any suitable endorphin with analgesic activity. In addition, analogs or fragments of these endorphins having analgesic activity are contemplated. Thus, the endorphin to be produced by the cells of the invention is characterized by amino acid insertions, deletions, substitutions and modifications at one or more sites of the naturally occurring amino acid sequence of the desired endorphin. We prefer conservative modifications and substitutions (i.e., those that have minimal effect on the secondary or tertiary structure of endorphin and its analgesic properties). Such conservative substitutions include those described

Dayhoff in Atlas of Protein Sequence and Structure, 5, (1987) a

Argos, Embo J., 3, 779-85 (1989).

Techniques for generating such variants of naturally occurring endorphins are well known. For example, codons in the DNA sequence encoding wild-type endorphins may be altered by site specific mutagenesis.

The present invention contemplates a precursor molecule POMC. the host cell enzymes produce a series of endorphins, from analgesic properties.

using an entire DNA coding sequence This embodiment utilizes cleavage (i.e., prohormone convertase 2) on which they have some or all

The present invention also contemplates the use of DNA fragments of the POMC gene that encode a particular endorphin.

POMC amino acid DNA sequences are well known. Cochet et al., Nature, 297, 335-9 (1982); Takahashi et al., Nucl. Acids. Res., 11, 6847-58 (1983).

We prefer a DNA sequence encoding POMC in which the ACTH coding region has been deleted. A preferred endorphin encoded by this construct is β-endorphin.

Some proenkephalins are synthesized in the adrenal glands as part of a large protein, pro-enkephalin A, which contains six repeats of the Met-enkephalin sequence and one Leu-enkephalin structure. Met-enkephalin, as well as Met-enkephalin-Arg-Phe and Met-enkephalin-Arg-Gly-Leu, significantly reduce the response to painful stimuli. See Sagen et al., Brain Res., 502, 1-10 (1989).

Other enkephalins, i. j. dynorphins and neo-endorphins, are derived from a different molecule, proenkephalin B. Other latent peptides are also encoded in the structure of these precursor proteins and can be released by prohormone-type cleavage. See e.g. Harris Principles of Internal Medicine, 12th edition, 1168-69 (1991).

The present invention contemplates the use of a DNA sequence that encodes any suitable enkephalin with analgesic activity. In addition, analogs and active fragments having analgesic properties are also contemplated. Thus, such analogs or fragments have amino acid insertions, deletions, substitutions at one or more sites of the naturally occurring amino acid sequence. Such variants arise as described above.

The present invention contemplates the use of a DNA sequence encoding the desired enkephalin in its mature form. In addition, the present invention contemplates the use of a DNA sequence encoding the entire pro-enkephalin precursor A or the entire pro-enkephalin precursor B. Furthermore, it is contemplated that a DNA encoding a fusion or fragment of these sequences will provide one or more enkephalin-like molecules when expressed. analgesic properties.

We prefer DNA sequences encoding the entire pro-enkephalin A precursor molecule. DNA and pro-enkephalin A amino acid sequences are well known. Folkesson, see above. This embodiment utilizes host cell cleavage enzymes, such as prohormone convertase, to generate a number of enkephalins, some or all of which have analgesic properties. A preferred enkephalin encoded by this construct is Met-enkephalin.

There are three naturally occurring catecholamines that act as neurotransmitters in the central nervous system, norepinephrine (NE), epinephrine (E), and dopamine. NE is associated with postganglionic endings of the sympathetic nerves. NE acts locally in the immediate vicinity of its release.

Catecholamines are synthesized from the amino acid tyrosine, which is progressively hydroxylated to form dihydroxyphenylalanine (dopa), decarboxylated to form dopamine, and then hydroxylated at the beta position of the side chain by dopamine beta hydroxylase to form NE. Harris, supra, p. 380. NE is methylated at the N terminus to epinephrine (E) by phenylethanolamine-N-methyltransferase (PNMT).

Hydroxylation of tyrosine by tyrosine hydroxylase (TH) is a step limiting the rate of NE synthesis. Regulation of dopa and NE synthesis in adrenal pulp can be achieved by varying the amount and activity of TH.

In addition, regulation of E synthesis from NE may occur with changes in the amount and activity of phenylethanolamine-N-methyltransferase (PNMT). PNMT is inducible by glucocorticoids from the adrenal cortex. See above.

Catecholamines are maintained at high concentrations in the adrenal gland marrow tissue, mostly as E. Opioid peptides are also stored in the adrenal gland.

NE and E have similar affinities for O 2 receptors and therefore both contribute potentially to analgesia. Bylund, FEBEB J., 6, 832 39 (1992). Enkephalin peptides, which mainly include met-enkephalin, selectively activate delta (δ) opioid receptors. Reisine and Bell, Trends Neurosci., 16, 506-10 (1993). Activation of both CC2 adrenergic and δ opioid receptors in the spinal cord results in a decreased response to a painful stimulus, and this activation is potentially synergistic. Yaksh and Malmberg, Progress in Pain Research and Management, Volume 1, ed. Fields and Lisbeskind, IASP

Press, Seattle, 141-71 (1994). Activation of δ versus (μ) opioid receptors in experimental animals results in fewer adverse side effects including constipation and the risk of dependence (Lee et al., J. Pharmacol. Exp. Ther., 267, 883-87 (1993)). opioidergic and adrenergic formulations can reduce the extent of tolerance that develops to a single formulation and leads to sustained pain relief Yaksh and Reddy, Anesthesiol., 54, 451-67 (1981).

The present invention contemplates the use of a DNA sequence that encodes catecholamine biosynthetic enzymes, or analogs or fragments thereof, to obtain catecholamines having analgesic properties. Preferred catecholamines of the invention are NE and E.

In one embodiment, the host cell is transformed with genes that are necessary to achieve NE and E production as desired. The choice of heterologous gene sequences of interest depends on the complement of enzymes synthesizing catecholamines that normally occur in the host cell. For example, RIN and AtT-20 cells do not have tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). However, RIN and AtT-20 cells contain endogenous dopecarboxylase (DDC). If catecholamine E is desired, then a gene encoding PNMT is also required. The gene encoding PNMT is known. Baetge et al., Proc. Natl. Acad. Sci., 83, 5455-58 (1986).

The gene encoding TH is known. See e.g. United States Patent 5,300,436, incorporated herein by reference. Modified variants of TH are also known. United States Patent 5,300,436. In addition, shortened versions of TH are also known which contain the necessary C-terminal catalytic domains. See e.g. Daubner et al., Protein Science, 2, 1452-60 (1993).

AtT-20 cells were transformed with wild type TH as well as various TH mutants. See e.g. Wu et al., J. Biol. Chem., 267, 25754-758 (1992).

The sequence of the DBH gene is also well known. See e.g. Lamoroux et al., EMBO J., 6, 3931-37 (1987).

It will be appreciated that in addition to the preferred DNA sequences disclosed herein, there are a number of degenerate DNA sequences that encode the desired analgesics.

Secondary compounds with a potential analgesic effect may also be produced by the cells of the invention. Such compounds include galanin and somatostatin. In addition, neuropeptide Y, neurotensin and cholecystokinin can be produced by the transformed cells of the invention. The cells of the invention may produce some or all of these compounds normally, or may be genetically manipulated by standard techniques to generate them.

Standard methods can be used to obtain or synthesize genes encoding analgesic compounds to be produced by the cells of the invention.

For example, the complete amino acid sequence can be used to construct a gene whose sequence is back-translated from the amino acid sequence. A DNA oligomer containing a nucleotide sequence encoding the desired analgesic compound may be synthesized. For example, several small oligonucleotides encoding portions of each polypeptide of interest can be synthesized and then ligated (linked). For assembly, the individual oligonucleotides typically comprise 5 'or 3' overhangs.

The DNA sequence encoding each analgesic compound of interest may or may not also contain DNA sequences encoding a signal sequence. Such signal sequence, if present, should be recognized by the cell selected for expression of the analgesic compound. It may be prokaryotic, eukaryotic or a combination of both. It may also be a signal sequence of a naturally occurring compound. In general, it is preferred that the signal sequence is encoded and most preferably the use of a signal sequence is naturally occurring.

Once the DNA sequences encoding the desired compound have been assembled, they are inserted into one or more expression vectors and operably linked to appropriate expression control sequences in the desired transformed cell.

Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of the biologically active polypeptide in the transformed cell. As is well known in the art, in order to obtain a high level of expression of a transfected gene in a host, the gene must be operably linked to transcriptional and translational expression control sequences that are functional in the selected expression cell.

The choice of expression control sequence and expression vector depends on the choice of cell. A variety of expression host / vector combinations can be used. Useful expression vectors for eukaryotic hosts, for example, include vectors comprising expression control sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus.

We prefer pcDNA3, pCEP4, pZeoSV (InVitrogen, San Diego) and pNUT.

Any of a variety of expression control sequences can be used in these vectors. Such useful expression control sequences include sequences linked to the structural genes of said expression vectors. Examples of useful sequences controlling the late 3-phosphoglycerate kinase expression promoters include, for example, early and SV40 or adenovirus, promoter or other glycolytic enzymes, acid phosphatase promoters, e.g. Pho5, the promoters of the yeast α-conjugation system and other sequences known to direct the expression of eukaryotic cell genes or their viruses and various combinations thereof.

Of course, it is to be understood that not all vectors and expression control sequences function equally well to express the DNA sequences disclosed herein. Nor is it that all cells function equally well with the same expression effect. However, one of skill in the art can select between these vectors, expression control sequences, and cells without undue experimentation. For example, when selecting a vector, the host cell must be considered because the vector has to reproduce therein. The number of copies of the vector, the ability to control this number, and the expression of any other proteins encoded by the vector, such as antibiotic labels, must also be considered.

A variety of factors must also be considered when selecting the expression control sequence. These include, for example, the relative strength of the sequence, its controllability and compatibility with the actual DNA sequence encoding the desired analgesic compounds, in particular with respect to potential secondary structures. Host cells should be selected for their compatibility with the selected vector, the toxicity of the product encoded by the DNA sequences, their secretory properties, their ability to fold the polypeptides correctly, and their culture requirements. If the host cell is to be encapsulated, the viability of the cell in encapsulation and implantation into the recipient should also be considered.

Within these parameters, one of ordinary skill in the art can select different vector / expression control / host combinations that will express the desired DNA sequences in tissue culture.

In one embodiment, cells (e.g., RIN cells) are sequentially transformed with 4 separate expression vectors containing the POMC gene, the pro-enkephalin A gene, the TH gene, and the DBH gene. In such a transformed host cell, it is more difficult to achieve amplification of the copy number of heterologous genes. Thus, it is preferred to use less expression vectors. Most preferred is the use of a single expression vector containing all 4 heterologous genes.

In a more specific embodiment, RIN cells are sequentially transformed with 3 expression vectors. The first vector contains the POMC gene operably linked to the CMV promoter. Preferably, a truncated version of the POMC gene is used that has the coding region for ACTH removed. The second vector contains the pro-enkephalin A gene operably linked to the CMV promoter. The proA construct preferably comprises a consensus Cossack sequence located immediately upstream of the start (initiation) codon. The third vector contains both the TH genes (preferably truncated and having a Kozak consensus sequence upstream of the start codon) and DBH. In this embodiment, the TH gene is operably linked to the CMV promoter. The DBH gene is operably linked to the promoter sequence of the internal ribosome entry site. RIN cells are then sequentially transformed with each expression vector according to known protocols.

In another embodiment, one expression vector is constructed comprising a pro-enkephalin A gene, a POMC gene, a TH gene, and a DBH gene. Preferably, the ACTH region of the POMC gene is deleted. Preferably, the truncated gene is TH.

Multiple gene expression from a single transcript is preferred as compared to expression from multiple transcriptional units. One approach to achieving expression of many genes from a single eukaryotic transcript utilizes picorna virus mRNA sequences known as internal ribosome entry sites (IRES). These sites function to facilitate translation of the protein from sequences located downstream of the first AUG to mRNA.

Macejak and Sarnow report that the mRNA for the 5 'untranslated immunoglobulin heavy chain binding protein sequence (BiP, also known as CRP 78, a glucose-driven protein of 78,000 molecular weight) can directly cause internal binding of the ribosome to mRNA in mammalian cells in a cap-independent manner at the 5'-end. This suggests that this cellular mRNA utilizes an internal ribosome entry site binding mechanism to initiate translation. Nature 353, 90-94 (1991).

WO 94/24870 discloses the use of more than two IRESs to initiate translation from a single transcript, as well as the use of multiple copies of the same IRES in a single construct.

The invention also contemplates the use of suicide genes in transformed cells. Most preferably, the cells carry the TK (thymidine kinase) gene as a precautionary measure, allowing the host cell to be killed in vivo by treatment with ganciclovir.

The use of a suicide gene is known in the art. See e.g. Anderson, published PCT application WO 93/10218, Hamre, published PCT application WO 93/02556. The first level of protection against adverse reactions to implanted cells provides the recipient's own immune system. In encapsulation, the self-contained polymeric sleeve may be immunologically insulating. The presence of the TK gene (or other suicide gene) in the expression construct adds an additional degree of safety to the recipient of the implanted cells.

Vectors preferred for use in the present invention include those that allow DNA encoding analgesic compounds to be amplified in multiple copies. Vectors capable of such multiplication are well known in the art. They include, for example, vectors that are capable of multiplying by the amplification of DHFR (see, e.g., Kaufman, U.S. Patent 4,470,461, Kaufman and Sharp,

Construction Of A Modular Dihydrafolate Retuctase eDNA Gene: Analysis Of Signals Utilized For Efficient Expression, Mol. Cell. Biol., 2, 1304-19 (1982)) or by amplification of glutamine synthetase (GS) (see, e.g., U.S. Patent 5,122,464 and European Published Application 336,841). Such amplification can be used to increase production of the desired analgesic compounds.

Other methods of increasing the production of the desired analgesic compounds are contemplated. For example, subcloning of existing polyclonal cell lines is contemplated. Cells are cloned by final dilution to one cell in each well. Cell clones are tissue cultures, and clones are tested to select the clone with the greatest production of analgesic compounds.

Another method of increasing the production of the desired analgesic compounds relates to the cloning of altered forms of biosynthetic enzymes with higher activity than the wild type (i.e., truncated TH 1-155). Some truncated forms of TH have 4 to 6 times higher activity than wild type TH. See e.g. Daubner et al., Expression and Characterization of Catalytic and Regulatory Domains of Rat Tyrosine Hydroxylase, Protein Science, 2, 1452 60 (1993).

In addition, the use of tyrosine-free culture media to selectively increase the level of tetrahydrobiopterin cofactor can potentially increase tyrosine hydroxylase activity. See e.g. Horellou et al., Retroviral transfer of human tyrosine xydroxylase cDNA in various cell lines, regulated release of dopamine in mouse anterior pituitary AtT-20 cells, Proc. Natl. Acad. Sci. USA, 86, 7233-37 (1989).

Preferably, the β-endorphin production ranges between 1 and 10,000 pg / 10 ⁶ cells / h. Preferably, the formation of met-enkephalin ranges between 1 and 10,000 pg / 10 ⁶ cells / h. Preferably, catecholamine formation is in the range of between 1 and 10,000 pg / 10 ⁶ cells / h.

The cells of the invention may be implanted in a mammal, including a human, to treat pain. If implanted unencapsulated, any suitable implantation protocol may be used, including those suggested by Sagen et al., U.S. Patent 4,753,635, incorporated herein by reference.

It may be desirable to encapsulate the genetically modified cells of the invention prior to implantation. Such a biological artificial organ (BAO)

The BAO encapsulated cells formed may be designed for implantation into a recipient or may be designed to function extracorporally. typically has at least one

A BAO useful in the present invention is a semipermeable outer surface membrane or sheath surrounding a core containing cells. The sheath allows diffusion of nutrients, biologically active molecules and other selected products through the BAO. BAO is biocompatible.

In some cases, the membrane can also serve to immunologically isolate cells by blocking the cellular and molecular effectors of immunological rejection. The use of immunologically insulating membranes allows implantation of allogeneic and xenogeneic cells by individuals without the use of immunosuppression.

A variety of different types of membranes have been used in the BAO design. Generally, the membranes used in the BAO are either microporous or ultrafiltration. Various types of membrane materials have been proposed for use in BAOs, including PAN / PVC, polyurethanes, polysulfones, polyvinylidenes and polystyrenes. A typical membrane assembly comprises flat sheets from which sandwich-type structures may be assembled having a living cell layer positioned between two planar parallel membranes with joints formed around the periphery of the device. Alternatively, a hollow fiber device may be used where living cells are located within the tubular membrane. Hollow fiber BAOs may be formed sequentially by inserting living cells into the lumina of the hollow fiber and sealing the ends of the fiber. Hollow fiber BAOs can also be produced by a co-extrusion process where living cells are extruded together with a polymer solution that forms a membrane around the cells.

BAOs have been described, for example, in U.S. Pat. 4,892,538, 5,106,627, 5,156,844, 5,158,881 and 5,182,111 and PCT applications no. PCT / US / 94/07015, WO 92/19195, WO 93/03901 and WO 91/00119, all of which are incorporated by reference.

BAOs may contain other ingredients that promote the long-term survival of the encapsulated cells. For example, WO 92/19195 relates to implantable immunologically isolating biocompatible carriers having a hydrogel culture medium to enhance cell viability.

The encapsulating BAO membrane may be made of a material that is the same as the core material, or may be of any other material. In either case, a boundary or peripheral membrane region is formed that is selectively permeable and biocompatible. If desired, the membrane can also be designed to be immunologically insulating. The nucleus contains isolated cells, either suspended in a liquid medium or immobilized on a hydrogel broth.

The choice of materials used to construct the BAO is determined by a number of factors and is described in detail in Dionne WO 92/19195. Briefly, various polymers and blends of polymers can be used to produce the sheath. The BAO-forming polymer membranes and internal growth areas may include polyacrylates (including acrylic copolymers), polyvinylidenes, polyvinyl chloride copolymers, polyurethanes, polystyrenes, polyamides, acetate celluloses, nitrate celluloses, polysulfones, polyphosphazenes, polyacrylonitrile, polyacrylonitrile, polyacrylonitrile, polyacrylonitrile, derivatives, copolymers and mixtures thereof.

BAOs can be generated by any suitable method known in the art. One such method relates to co-extrusion of a polymeric flow solution and a precipitant, which may include fragments of biological tissue, organelle or cell suspension, and / or other therapeutic agents as described in Dionne, WO 92/19195 and US Patents 5,158,881, 5 283 187 and 5 284 761, incorporated herein by reference.

The sheath may be single or bilayer. The monolayer hollow fiber may be formed by rapidly cooling only one of the surfaces of the polymer solution when extruded. The bilayer hollow fiber may be formed by rapidly cooling both surfaces of the polymer solution.

Different outer housing shapes are possible, such as cylindrical shape, disc shape or spherical.

The BAO sheath has pores of a size that ensures that the selective permeable membrane behaves as a molecular sieve with a nominal molecular weight limit (nMWCO). Molecules larger than nMWCO are physically prevented from passing through the membrane. A molecular sieve with a nominal molecular weight limit is defined by 90% rejection under convection conditions. In situations where it is desirable for BAO to be immunologically isolating, the pore size of the membrane is selected to allow specific factors produced by the cells to diffuse out of the carrier, but does not allow the entry of host immune response factors into the BAO. Typically, the nMWCO is in the range between 50 and 200 kD, preferably between 90 and 150 kD. The most suitable membrane composition also minimizes reactivity between host immune effector molecules known to be present at the selected implantation site and the outer membrane components of the BAO.

The BAO core is designed to provide a suitable local environment for the particular cells isolated therein. The core may comprise a liquid medium sufficient to maintain cell growth. The liquid core is particularly suitable for maintaining transformed cell lines such as PC12 cells. Alternatively, the core may comprise a gel broth. The gel broth may consist of a hydrogel (alginate, Vitrogen ™, etc.) or extracellular constituents of the broth. See e.g. Dionne WO 92/19195.

The hydrogel-forming compositions fall into three general classes. First class hydrogels carry a net negative charge (eg alginate). In the second class there are hydrogels carrying a net positive charge (e.g. collagen and laminin). Examples of commercially available extracellular broth components are Matrigel ™ and Vitrogen. The third class is charge neutral (e.g., highly crosslinked polyethylene oxide or polyvinyl alcohol).

Any suitable method may be used to seal the BAO, including the use of polymer adhesives and / or flanging, kinking and welding. Such sealing techniques are known in the art. In addition, any suitable dry sealing method may be used. Such methods use a substantially non-porous fitting through which a cell-containing solution is fed. After filling, the BAO is sealed. Such a method is described in the pending U.S. application Ser. 08/082 407, incorporated herein by reference.

Prior to in vivo implantation, it is preferred to use one or more in vitro assays to determine BAO functionality. Assays or diagnostic tests well known in the art may be used for this purpose. See e.g. Methods In Enzymology, Abelson (Ed.), Academic Press, 1993. For example, an enzyme-linked immunosorbent assay (ELISA), a chromatographic or enzymatic assay, or a product-specific bioassay may be used. If desired, implant secretory function can be monitored over time by collecting appropriate samples (e.g., serum) from the recipient and testing them. If the recipient is a primate, microdialysis may be used.

The number of BAOs and their size sufficient to provide therapeutic effect upon implantation is determined by the amount of biological activity required for a particular application. For drug-releasing secretory cells, standard dosing considerations and criteria known in the art are used to determine the amount of secretory required. Factors to be considered are discussed in Dionne, WO 92/19195.

BAO implantation is performed under sterile conditions. Generally, the BAO is implanted at a site in the host organism that allows adequate delivery of the secreted product or function to the host and the delivery of nutrients to encapsulated cells or tissue, and also allows access to the BAO for removal and / or replacement. The preferred host is a primate, most preferably a human.

Several different implant sites are envisaged. These implant sites include the central nervous system, including nucieus basalis cerebrospinal fluid, brain, spinal cord and aqueous humor, and vitreous humor of the eye. Preferred sites in the brain include striatum, cerebral cortex, subtalamic nuclei, and Meynerti. Other preferred sites are most preferably subarachnoid space and lateral ventricles. The present invention also contemplates implantation into other sites, such as under the renal capsule, intraperitoneally, subcutaneously, or other medically beneficial site.

The following examples are given in order to better understand the invention. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention in any way.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a plasmid map of the vector pBS-hPOMC-027, pBS-IgSP-hPOMC-028, and pBS-18SP-hPOMC-AACTH-029.

Fig. 2 is a plasmid map of vectors pCEP4-hPOMC-030, pCEP4-hPOMC-031, pcDNA3-hPOMC-034, and pcDNA3-hPOMC-035.

Fig. 3 is a plasmid map of vectors, pCEP4-hPOMC-AACTH-032, pCEP4-hPOMC-AACTH-033, pcDNA3-hPOMC-AACTH-036, and pcDNA3-hP0MC-AACTH-037.

Fig. 4 is a plasmid map of the vectors pcDNA3-rTH-044, pcDNA3-rTHA-045, and pcDNA3-rTHDKS-075 (also referred to as pcDNA3-rTHAKS-075).

Fig. 5 is a plasmid map of pcDNA3-rTHA-IRES-bDBH-088 and pcDNA3-rTHAKS-IRES-bDBH-076 vectors.

Fig. 6 -bDBH-088th is a plasmid map vector pZeo-pCMV-rTHAKS-IRES- Fig. 7 -067. is a plasmid map vector pBS-Pcmv-rTHÁIRES-bDBH- Fig. 8 is -rTHAlRES-bDBH- plasmid 068th map vector pBS-hPOMC-AACTH-IRES- Fig. 9 is a plasmid map vector pcDNA3-hPOMC-AACTH-IRES-

-rTHA-IRES-bDBH-069th

Fig. 10 is a plasmid map of pcDNA3-IRES-Zeocin-072 vector.

Fig. 11 is a plasmid map of pcDNA3-hPOMC-ACACTH-IRES-rTHA-IRES-bDBH-IRES-Zeocin-073 vector.

Fig. 12 is a plasmid map of pcDNA3-hPROA + KS-91 vector.

DETAILED DESCRIPTION OF THE INVENTION

Construction of a polycistronic expression vector

Construction of IgSP-POMC fusion

The DNA fragment between the SmaI and SalI restriction sites (hereinafter the SmaI-SalI fragment) containing the 3rd exon from the human POMC gene was sub-cioned into the pBS vector (Stratagene). See Takahashi, supra, Cochet, supra. The resulting plasmid was named pBS-hPOMC-027 (Fig. 1).

The PCR fragment (DNA fragment generated in the polymerase chain reaction -PCR) was synthesized using two oligo26 nucleotide primers oCNTF-003 (SEQ ID NO: 1) and oIgSP-018 (SEQ ID NO: 2) from a plasmid pNUT containing the human CNTF gene. See Baetge et al., Proc. Natl. Acad. Sci. USA, 83, 5454-58 (1986). Both primers oCNTF-003 and oIgSP-018 contained synthetic BamHI and SmaI restriction sites at their 5 'ends.

The 196 base pair (bp) PCR fragment was digested with BamHI and XmaI (which is the SmaI isoschoisomer) and then electrophoretically separated on a 1% SeaPlaque agarose gel. The HindIII / Xmal DNA fragment was excised from the gel and purified using a SpinBind purification kit (FMC BioProducts, Rockland, ME).

Plasmid pBS-hPOMC-027 was also digested with BamHI and Xmal, isolated and purified from 1% agarose using a SpinBind purification kit (FMC BioProducts, Rockland, ME). E. coli DH5α cells (Gibco BRL, Gaitherburg, MD) were then transformed with the ligation mixture.

Positive subclones were first identified by the cracking gel procedure (Promega Protocols and Application Guide, 1991). Minilysates were then prepared using a SpinBind purification kit (FMC BioProducts, Rockland, ME) and digested with BamHI and SmaI. The positive subclone was named pBS-IgSP-hPOMC-028 (Fig. 1). The nucleotide sequence of the fusion gene junction site in plasmids pBS-IgSP-hPOMC-028 was determined by sequencing by the dideoxynucleotide method using the Sequenase kit (USBC, Cleveland). The sequence is shown in FIG. 3 as the sequence of identification number 3.

Construction of IgSP-POMC expression vectors

The IgSP-hPOMC DNA fragment in plasmid pBS-IgSP-hPOMC-028 was subcloned into plasmid pcDNA3 (Invitrogen Corp., San Diego, CA) and pCEP4 (Invitrogen Corp., San Diego, CA) in a sense of sense. and in an orientation opposite to the reading direction (anti-sense).

The IgSP-hPOMC fragment delimited by the NotI-SalI sites of pBS-IgSP-hPOMC-028 was ligated (linked) to the NotI-XhoI fragment from the appropriately digested pCEP4, resulting in a clone with a sense orientation of the cloned gene termed pCEP4-hPOMC-030 ( Fig. 2). The BamHI-SalI fragment of IgSP-hPOMC from plasmid pBS-IgSP-hPOMC-028 was ligated with the plasmid pCEP4 digested with BamHI and XhoI to form a clone with the anti-sense orientation of the cloned gene called pCEP4-hPOMC-031 (Fig. 2). The orientation of the insert (inserted fragment) in the pCEP4-hPOMC-030 and pCEP4-hPOMC-031 vectors was verified by digestion with BamHI, Note, SalI and NotI / SalI as well as dideoxynucleotide sequencing using the Sequenase kit (USBC, Cleveland).

The BamHI-SalI fragment of IgSP-hPOMC from plasmid pBS-IgSP-hPOMC-028 was ligated with BamHI-XhoI digested pcDNA3, resulting in a clone with a sense orientation of the cloned gene called pcDNA3-hPOMC-034 (Fig. 2). The NotI-HindIII fragment of IgSP-hPOMC from pBS-IgSP-hPOMC-028 was ligated with NotI-HindIII digested pcDNA3 to form a clone with the anti-sense orientation of the cloned gene called pcDNA3-hPOMC-035 (Fig. 2). Restriction with SmaI, BamHI, EcoRI and BamHI / HindIII was used to confirm the orientation of the insert in pcDNA3-hPOMC-034, while HindIII, Niti and SalI were used to confirm the orientation of the pcDNA3-hPOMC-035 vector.

Construction of the IgSP-POMC fusion gene with the ACTH gene region removed

The ACTH coding region was deleted (deleted) in plasmid pBS-IgSP-hPOMC-028. Plasmid pBS-IgSP-hPOMC-028 was first digested with XmaI and then treated with Pfu DNA polymerase (Promega, Madison, WI). The treated pBS-IgSP-hPOMC-028 was digested with StuI, then isolated from 1% SeaPlaque agarose and purified using the FMC SpinBind purification kit (FMC BioProducts, Rockland, ME). After self-ligation (joining the free ends of the linear plasmid to each other to form a circular molecule again), E. coli DH5α bacteria (Gibco BRL, Gaithersburg, MD) were transformed with the ligation mixture. Positive subclones were identified by BamHI / HindIII digestion and then a clone named pBS-IgSP-hPOMCACACTH-029 was selected (Fig. 1). The nucleotide sequence of the deleted (deleted) ACTH region in plasmid pBS-IgSP-hPOMCÁACTH-029 was an oxynucleotide method. IgSP-hPOMC-AACTH is number 4.

confirmed by sequencing the fusion gene dide sequence as sequence with identification

Construction of IgSP-hPOMC gene expression vectors without ACTH coding region

The DNA segment containing IgSP-hPOMC-AACTH from plasmid pBS-IgSP-hPOMCAACTH-029 was subcloned into plasmids pcDNA3 (Invitrogen Corp., San Diego, CA) and pCEP4 (Invitrogen Corp., San Diego, CA) both in sense and anti. -sense orientation. The NotI-SalI fragment of IgSP-hPOMC-AACTH from plasmid pBS-IgSP-hPOMCAACTH-029 was ligated with plasmid pCEP4 digested with NotI-XhoI, resulting in a sense clone called pCEP4-hPOMC-AACTH-032 (Fig. 3). The BamHI-SalI fragment of IgSP-hPOMC-AACTH from plasmid pBS-IgSP-hPOMCAACTH-029 was ligated with BamHI-XhoI digested pCEP4, resulting in an anti-sense clone called pCEP4-hPOMC-AACTH-033 (Fig. 3). The orientation of the inserts (inserted fragments) in plasmids pCEP4-hPOMC-AACTH-032 and pCEP4-hPOMC-AACTH-033 was confirmed by both BamHI and EcoRI digestion and sequencing using the Sequenase kit (USBC, Cleveland).

The BamHI-SalI fragment of IgSP-hPOMC-AACTH from plasmid pBS-IgSP-hPOMCAACTH-029 was ligated with pcDNA3 digested with BamHI-XhoI to form a sense clone called pcDNA3-hPOMCACATH-036 (Fig. 3). The NotI-HindIII fragment of IgSP-hPOMC-AACTH with pBS-IgSP-hPOMCAACTH-029 was ligated with pcDNA3 digested with NotI-HindIII and an anti-sense clone called pcDNA3-hPOMC-AACTH-037 was generated (Fig. 3).

The insert orientation of plasmid pcDNA3-hPOMC-AACTH-036 was verified using PvuII and EcoRI, while plasmid pcDNA3-hPOMC-AACTH-037 was restricted with SalI and EcoRI.

Full and truncated TH gene cDNA cloning

Total RNA was prepared from PC12 cells by a guanidium thiocyanate-based procedure with the TRI Chemical Kit (Molecular Research Center, Inc., Cincinnati, OH). Five hundred ng of total RNA from PC12 cells was subjected to reverse transcriptase transcription at 42 ° C for 30 minutes in a 20 μΐ reaction mixture containing 10 mM Tris.HCl (pH 8.3), 50 mM KCl, 4 mM each of dNTPs, 5 mM MgCl ₂ , 1.25 μΜ oligo (dT) 15-mer, 1.25 μΜ random hexamers, 31 units of Rnase Guard Rase inhibitor (Pharmacia, Sweden) and 200 units of SuperScript II reverse transcriptase (Gibco BRL, Gaithersburg, MD). Two microliters of the above cDNA were added to a 25 μΐ PCR reaction mixture containing 10 mM Tris.HCl (pH 8.3), 50 mM KOI, 800 nM each of dNTPs, 2 mM MgCl ₂ , 400 nM each of primers # 1. 1 and no. 2 and 2.5 units of Taq (Thermus aquaticus) DNA polymerase (Boehringer Manheim, Germany).

Oligonucleotide printers orTH-052 (SEQ ID NO: 5) and orTH-053 (SEQ ID NO: 6) were used to synthesize the full-length TH cDNA gene. The primers orTH-054 (SEQ ID NO: 7) and orTH-053 (SEQ ID NO: 7) were used to synthesize the truncated TH gene.

6). Primer sequences were taken from the TH gene sequence published by Grima et al., Nature, 326, 707-11 (1987), and U.S. Pat. 5,300,436, and also Daubner, supra.

Primers orTH-052 (SEQ ID NO: 5) and orTH-054 (SEQ ID NO: 7) contain a synthetic HindIII site at their 5 'end, while orTH-053 has a BamHI site at the 5' end.

The PCR reaction mixture was subjected to 30 amplification cycles consisting of a 30 second denaturation at 94 ° C (2 minutes in the first cycle), primer hybridization at 50 ° C for 1 minute and synthesis (chain extension) at 72 ° C for 3.5 minutes (last 5 minutes in the last cycle). The PCR fragment (a fragment synthesized in PCR) representing a 1537 bp full length TH gene and a 1087 bp truncated fragment was digested with BamHI and HindIII, and then resolved on a 1% SeaPlaque agarose gel. HindIII / BamHI fragments of 1531 bp and 1081 bp in length were excised and purified using the FMC SpinBind purification kit (FMC BioProducts, Rockland, ME).

PcDNA3 expression vector was also digested with BamHI and HindIII and isolated and purified from 1% SeaPlaque agarose using FMC SpinBind Purification Kit (FMC BioProducts, Rockland, ME). Ligation mixtures were then transformed with E. coli DH5α (Gibco BRL, Gaithersburg, MD).

A cracking gel procedure was used to search for positive subclones (Promega Protocols and Application Guide, 1991). The identity and correctness of the selected clones was verified by BamHI / HindIII digestion.

Positive subclones containing the full length or truncated rat TH gene in pcDNA3 were named pcDNA3-rTH-044 (Figure 4) and pcDNA3-rTHÄ-045 (Figure 4). The sequence of both full-length and truncated PCR-derived rat TH gene in the above clones was verified by sequencing with the Sequenase kit (USBC, Cleveland). The sequence of the rTHA construct is shown herein as SEQ ID NO: 16.

In order to optimize translation efficiency (translation of DNA sequence into protein) in the truncated TH gene, the oligonucleotide primer orTH-078 (SEQ ID NO: 8) was designed such that the consensus Kozak sequence is immediately upstream (ie upstream) ATG codon. Plasmid pcDNA3-rTHA-045 was used as a template in a 50 μ PCR PCR reaction with the composition identical to the PCR reaction mixture above, except that the oligonucleotide primers were replaced with primers orTH-078 (SEQ ID NO: 8) and orTH-053 (SEQ ID NO: 6). The resulting PCR reaction product of 1097 bp in length was cloned into the pcDNA3 vector in the same manner as described above. The resulting subclone was called pcDNA3-rTHACKS-75 (Fig. 4). The sequence of the pcDNA3-rTHACKS-75 construct is designated as SEQ ID NO: 2. 17).

Construction of the rTH-IRES-bDBH fusion gene

The recombinant DNA method using PCR was used to generate the fused rTH-IRES-bDBH gene.

Oligonucleotides oIRES-057 (SEQ ID NO: 9) and obDBH-065 (SEQ ID NO: 10) are specific for the IRES and bDBH gene sequences, wherein oIRES-057 contains a synthetic BamHI restriction site at its 5 'end and obDBH-065 contains Noci site (sequence with identification (sequence with

Oligonucleotides oIRES-bDBH-064 # 11 and oIRES-bDBH-066 # 12) are complementary to each other. In addition, the oligonucleotide primer oIRES-bDBH-064 (SEQ ID NO: 11) has a 16-nucleotide region at its 5 'end identical to the IRES sequence and an 18-nucleotide region at its 3' end identical to the bDBH sequence. The same, however, applies in reverse order to the oligonucleotide oIRES-bDBH-066 (SEQ ID NO: 12).

The first two PCR reactions were performed with the pairs of the oligonucleotides oIRES-057 / oIRES-bDBH-066 and oIRES-bDBH-064 / obDBH-065 and the plasmids pCTI-001 (insert containing the IRES sequence given herein under identification number 30) and pBS-bDBH- 006 (contains the bovine DBH gene cloned from the bovine adrenal chromaffin cells of Lamoruox et al., EMBO J., 6, 3931-37 (1987)) as templates. One hundred ng of template DNA was added to a 50 μΐ PCR reaction mixture containing 10 mM TRIS.HCl (pH 8.3), 50 mM KCl, 800 nM each of dNTPs, 2 mM MgCl 2, 400 nM each of primers # 1. Lac. 2 and 2.5 units of Taq (Thermus aquaticus) DNA polymerase (Boehringer Manheim, Germany).

The PCR reaction mixture was subjected to 30 amplification cycles consisting of a 30 minute denaturation at 94 ° C (2 minutes in the first cycle), primer hybridization at 50 ° C for 1 minute and synthesis (chain extension) at 72 ° C for 3.5 minutes (last 5 minutes in the last cycle). PCR products were resolved in a 1% agarose gel from TrivieGel 500 (TrivieGen). Two gel blocks containing PCR products were used in the following PCR reaction in which the reaction mixture was identical to the above except that the oligonucleotides oIRES-057 and obDBH-065 were used. The second PCR reaction was performed in 30 amplification cycles consisting of a 30 second denaturation at 94 ° C (in the first cycle of 2 minutes), hybridization at primers at 60 ° C for 30 seconds (in the second to fourth cycle of 37 ° C for 2 minutes ) and synthesis (chain extension) at 72 ° C for 30 seconds (last 2 minutes in the last cycle). The 2407 bp PCR product representing the IRES-bDBH fusion gene and the pcDNA3-rTHA-45 cloning vector were digested with BamHI and Noti and then purified after separation in 1% SeaPlaque agarose using the FMC SpinBind Purification Kit (FMC BioProducts, Rockland ME, ME).

Ligation of IRES-bDBH / BamHI / NotI and pcDNA3-rTHA-45 / BamHI / NotI fragments gave rise to an expression vector called pcDNA3-rTHA-IRES-bDBH-066 (Fig. 6), while ligation of IRES-bDBH / BamHI / NotI and pcDNA3-rTHAKS-075 / BamHI / NotI resulted in an expression vector called pcDNA3-rTHAKS-IRES-bDBH-076 (Fig. 5), with a Kozak sequence in the rTHA region prior to the ATG start codon. The sequence of the rTHAKS-IRES-bDBH construct is shown herein under ID No. 18. The ligation mixture was then transformed with E. coli DH5α (Gibco BRL, Gaithersburg, MD). Positive clones were screened by the cracking gel procedure (Promega Protocols and Application Guide, 1991) and verified by digestion with HindIII, BamHI, BamHI / HindIII, SmaI and Note.

The 4114 bp Nrul-XhoI fragment containing the CMV-rTHAKS-IRES-bDBH promoter was selected from the pcDNA3-rTHAKS-IRES-bDBH-076 plasmid and was then subcloned into the pZeoSV cloning vector (Invitrogen Corp., San Diego, CA). digested with Seal and XhoI at multiple cloning sites. The resulting vector was named pZeo-Pcmv-rTHAKS-IRES-bDBH088 (Fig. 6).

Construction of IgSP-hPOMCACTH-rTHD-IRES-bDBH fusion gene

The 4100 bp NruI-NotI fragment containing the CMV promoter, the rTHD-IRES-bDBH fusion gene and the BGH polyadenylation sequence was selected from plasmid pcDNA3-rTHA-IRES-bDBH-066 and subcloned into the pBS cloning vector (Stratagene, La Jolla) CA).

The resulting plasmid pBS-Pcmv-rTHA-IRES-bDBH-067 (Fig. 7) was used as an intermediate construct into which a recombinant PCR fragment containing IgSP-hPOMCACTH-IRES was inserted.

The oligonucleotide oIgSP-068 (SEQ ID NO: 13) containing a synthetic EcoRV restriction site is specific for the IgSP sequence.

The oligonucleotide primer orTHA-073 (SEQ ID NO: 14) is specific for the rTHA sequence and contains an internal SmaI restriction site.

The oligonucleotide primers ohPOMC-IRES-069 (SEQ ID NO: 15) and ohPOMC-IRES-070 (SEQ ID NO: 20) are complementary to each other. In addition, the oligonucleotide primer ohPOMC-IRES-069 has an 18-nucleotide region at its 5 'end identical to the hPOMC sequence and a 12-nucleotide region at its 3' end identical to the IRES sequence. However, the same is true for the ohPOMC-IRES-070 oligonucleotide.

The oligonucleotide primers oIRES-rTHA-071 (SEQ ID NO: 21) and oRIRES-rTHA-072 (SEQ ID NO: 22) are complementary to each other. In addition, the oligonucleotide primer oIRES-rTHA-071 has a 15-nucleotide region at its 5 'end identical to the rTHA sequence and an 18-nucleotide region at its 3' end identical to the IRES sequence. However, the same is true for the oRIRES-rTHA-072 oligonucleotide.

Three different primary PCR reactions were performed.

PCR Reaction A: The template was pBS-IgSP-hPOMCDACTH-029, primers were oligonucleotides oTgSP-068 / OhPOMC-IRES-069.

PCR Reaction B: The template was pCTI-001, primers were oligonucleotides ohPOMC-IRES-070 / oIRES-rTHA-071.

PCR reaction C: The template was pcDNA3-rTHA-045 and the primers were oligonucleotides orIRES-rTHA-072 / orTHA-073.

The three 50 µΐ primary PCR reaction mixtures contained 100 ng of template DNA, 10 mM TRIS.HCl (pH 8.3), 50 mM KCl, 800 nM each of dNTPs, 2 mM MgCl 2, 400 nM of each of primers # 1. 1 and no. 2 and 2.5 units of Taq (Thermus aquaticus) DNA polymerase (Boehringer Manheim, Germany).

The PCR reaction mixture was subjected to 30 amplification cycles consisting of a 30 second denaturation at 94 ° C (2 minutes in the first cycle), primer hybridization at 50 ° C for 1 minute and synthesis (chain extension) at 72 ° C for 3 seconds ( last 5 minutes).

The PCR products were resolved in a 1% TrivieGel 500 agarose gel (TrivieGen). Two gel blocks containing the PCR products of reactions B and C were used in the following PCR reaction with the same reaction mixture as above except that the oligonucleotides ohPOMC-IRES-070 and orTHA-073 were used.

The second PCR reaction mixture was subjected to 30 amplification cycles consisting of a 30 second denaturation at 94 ° C (2 minutes in the first cycle), primer hybridization at 60 ° C for 30 seconds and synthesis (chain extension) at 72 ° C for 30 seconds (2 minutes last cycle).

PCR products were isolated as described above. Gel blocks containing the PCR products from the second reaction and from reaction A were combined in the following third PCR amplification reaction with the oligonucleotides oIgSP-068 / rTHA-073. The PCR fragment containing the IgSP-hPOMC-IRES-rTHA fusion gene and the pBS-Pcmv-rTHA-IRES-bDBH-067 cloning vector were digested with EcoRV and Xmal and then purified after digestion in 1% SeaPlaque agarose using the EMC SpinBind EMC Purification Kit , Rockland, ME). The ligation mixture was then transformed with E. coli DH5ct (Gibco BRL, Gaithersburg, MD).

Positive clones were screened by a cracking gel procedure (Promega, Madison, WI) and verified by digestion with EcoRI, KpnI and Noti. The resulting clone was called pBS-IgSP-hPOMCACAC-IRES-rTH-IRES-bDBH-068 (Fig. 8). The sequence of this construct is shown herein as SEQ ID NO: 23.

Construction of IgSP-hPOMCAACTH-IRES-rTHA-IRES-bDBH Expression Vector

The 4444 bp fragment containing the IgSP-hPOMCAACTH-IRES-rTHA-IRES-bDBH gene was selected from plasmid pBS-IgSP-hPOMCAACTH-IRES-rTHA-IRES-bDBH-068 and subcloned into plasmid pcDNA3 (Invitrogen Corp., San Diego). , CA) at the N-position of the multiple cloning site. Restriction digestion Note and SmaI confirmed that the IgSP-hPOMCACAC-IRES-rTHA-IRES-bDBH gene was inserted in sense orientation and the resulting plasmid was named pcDNA3-IgSP-hPOMCAACTH-IRES-rTHA-IRES-bDBH-069 (Fig. 9) ).

Construction of IgSP-hPOMCÁACTH-IRES-rTHA-IRES-bDBH-Zeocin Expression Vector

The IRES-Zeocin fusion gene was generated by recombinant PCR. Oligonucleotide oIRES-074 (SEQ ID NO: 24) is specific for the IRES gene sequence and contains a synthetic Nose restriction site at its 5 'end, oligonucleotide oZeocin-077 (SEQ ID NO: 25) is specific for the Zeocin gene sequence, containing the restriction place Xhol. Oligonucleotides oIRES-Zeocin-075 (SEQ ID NO: 26) and oIRES-Zeocin-076 are complementary to each other. In addition, the IRES-rTHA-071 oligonucleotide has a 15-nucleotide region at its 5 'end identical to the Zeocin gene sequence and an 18-nucleotide region at its 3' end identical to the IRES sequence. Similarly, but in reverse order, this is also the case with the oligonucleotide oIRES-Zeocin-076.

The first two PCR reactions were performed with pairs of oIRES-074 / oIRES-Zeocin-075 and oIRES-Zeocin-076 / oZeocin-075 oligonucleotides and plasmids pCTI-001 and pZeoSV (Invitrogen Corp., San Diego, CA) as templates.

One hundred ng of template DNA was added to a 50 μΐ PCR reaction mixture containing 10 mM TRIS.HCl (pH 8.3), 50 mM KCl, 800 nM each of dNTPs, 2 mM MgCl ₂ , 400 nM each of primers # 1. 1 and no. 2 and 2.5 units of Taq (Thermus aquaticus) DNA polymerase (Boehringer Manheim, Germany).

The PCR reaction mixture was subjected to 30 amplification cycles consisting of a 30 second denaturation at 94 ° C (2 minutes in the first cycle), primer hybridization at 50 ° C for 1 minute and synthesis (chain extension) at 72 ° C for 3.5 minutes (last 5 minutes in the last cycle).

PCR products were resolved in a 1% agarose gel from TrivieGel 500 (TrivieGen). Two gel blocks containing the first PCR products were transferred to a tube containing a reaction mixture identical to the above mixture except that oligonucleotides oIRES-074 and oZeocin-077 were used.

The second PCR reaction was performed in 30 amplification cycles consisting of a 30 second denaturation at 94 ° C (first cycle 2 minutes), primer hybridization at 50 ° C for 30 seconds (second to fourth cycle 37 ° C for 2 minutes) and synthesis at 72 ° C for 30 seconds (last 2 minutes).

The 974 bp PCR product representing the IRES-Zeocin fusion gene and the pcDNA3 cloning vector were digested with Nôti and XhoI, and then purified after separation in 1% SeaPlaque agarose using the FMC SpinBind Purification Kit (FMC BioProducts, Rockland, ME).

Ligation of IRES-Zeocin / NotI / XhoI and pcDNA3 / NotI / XhoI fragments resulted in a transient cloning vector called pc.DNA3-IRES-Zeocin-072. Fig. 10th

Positive clones were screened by cracking gel (Promega, Madison, WI) and restriction digestion with HindIII, SmaI, XhoI, Noti and NotI / XhoI.

To generate the final expression vector IgSP-hPOMCACTH-IRES-rTHD-IRES-bDBH-IRES-Zeocin, it was from plasmid pBS-IgSP-hPOMCACAC-IRES-rTHA-IRES-bDBH-068 (Fig. 8, SEQ ID NO: 23) selected N91 fragment of 4491 bp in length containing the gene

IgSP-hPOMCAACTH-IRES-rTHA-IRES-bDBH and was subcloned into pcDNA3-IRES-Zeocin-072 (Fig. 10) at the Nine multiple cloning site.

Restriction digestion Note and SmaI confirmed that the IgSP-hPOMCACACTH-IRES-rTHA-IRES-bDBH gene was inserted in sense orientation and the resulting plasmid was named pcDNA3-IgSP-hPOMCAACTH-IRES-rTHA-IRES-bDBH-IRES-Zeocin-3 . The sequence of this construct is shown in SEQ ID NO: 28. FIG. 11th

ProA + KS fusion design

A construct comprising the coding region of human proenkephalin A with a consensus Kozak sequence immediately upstream of the ATG initiation codon. The sequence of this construct is set forth in SEQ ID NO: 29.

Construction of hProA + KS expression vector

The HindIII / BamHI fragment containing the hProA + KS fusion was inserted into the expression vector pcDNA3 digested with BamHI and HindIII in a manner essentially as described above. Following selection as above, the positive subclone was called pcDNA3-hProA + KS-091. Fig. 12. Construction of the vector pBS-CMV ProA is detailed in Mothis, J. A. Lindberg, I., Endocrinology, 131, 2287-96 (1992).

Cell transformation

RIN and AtT-20 cells were transformed as described below.

Cell lines based on RIN and AtT-20 were grown in DMEM medium (Gibco) with 10% fetal bovine serum and a pen-strep-fungisone mixture medium (Gibco). Cells were plated on Petri dishes P100 (750,000 cells / dish) in 10 ml of basic medium. 18-24 hours later, cells were transfected with a Stratagene kit (San Diego, CA) based on the calcium phosphate method. 10 μg of plasmid vector DNA was diluted with 450 μΐ of sterile deionized water. Then, 50 μΐ10x buffer (Solution 1) was added to the plasmid DNA. 500 μΐ of solution no. 2a gently mixed. The mixture was incubated at room temperature for 20 minutes and then 1.0 ml of the solution was added to the cells on Petri dishes. Cells were incubated overnight and 18-24 hours later, cells were washed twice with Hanks Balanced Saline without calcium and magnesium. Thereafter, the cells were grown in basic nutrient medium + selective drugs. Cells were selected in either 600 µg / ml geneticin (Gibco) or 400 µg / ml hygromycin (Boehringer Mannheim) or 500 µg / ml Zeocin (InVitrogen, San Diego, CA). Cells were sequentially transfected and selected to obtain a final cell line.

RIN cells were transfected with plasmid pCEP4-hPOMC-030 containing the POMC gene. This is a hygromycin resistant vector. Cells were also transformed with plasmid pcDNA3-hProA + KS-091. This is a geneticin resistant vector. Finally, cells were transfected with plasmid pZeo-PMCV-rTHAKS-IRES-bDBH-088 with demonstrable Zeocin resistance.

AtT-20 cells were transfected with plasmids pBS-CMV-ProA and pCEP4-POMC-AACTH-32, which showed resistance to geneticin and hygromycin, respectively. Finally, the cells were transfected with plasmid pZeo-Pcmv-rTHAKS-IRES-bDBH-088.

We tested media volumes for cell growth. We have surprisingly found that in certain serum-free nutrient media, the above cell lines had a higher neurotransmitter formation compared to serum-containing nutrient media. We prefer CHO-Ultra (Biowhitaker) to grow AtT-20 cells and Ultra-Culture to grow RIN cells.

The generation of various analgesics by a single transformed RINα cell line (RINα / ProA / PO30 / PO88) is presented in Table 2. All values represent unstimulated cells. The production of β-endorphin and met-enkephalin is in pg / 10 ⁶ cells / h. β-endorphine and met-enkephalin were measured by radioimmunoassay using Incstar kits (Stillwater, Minnesota). Catecholamine production is in pmol / 10 ⁶ cells / h. The numbers in parentheses represent values from cells that were preincubated for 18 hours with 100 μΜ tetrahydrobiopterin. Catecholamines were measured by HPLC as reported by Lavoie et al., Two PC12 pheochromocytoma lines sealed in hollow fiber-based capsules tonically release 1-dopa in

in vitro, Cell transplantation, 2, 163-173 (1993). GABA production these cells The RIN was 28 ng / 10 ⁶ cells / h. Table 2 Cell line endogenous β-endorphin Met-enk WILL GIVE E RINa / Proa / analgesic substances β-endorphin 22 17 3 0 POMC / GABA (6) (2)

TH-IRES-ϋβΗ

There are latent fragments of enkephalin that do not result from complete processing of the pro-enkephalin precursor molecule. These latent enkephalins have binding activity for opioid receptors. We cleaved these latent enkephalins to alleviate their opioid activity. The trypsin cleavage protocol is described below. 2 μς / ιηΐ of trypsin (Worthington # 34E470) solution is added to the nutrient medium samples on ice. The samples were shaken and then incubated for 20 minutes in a water bath at 37 ° C. After a cleavage time of 20 minutes, the samples are returned to ice and 100 ng / ml carboxypeptidase B (Sigma C-7011) is added. The samples were shaken and returned to a 37 ° C water bath for 15 minutes. The samples are then placed on ice once more and 10 μg / ml trypsin inhibitor is added. At this stage, either met-enkephalin is extracted from the samples or immediately frozen for future extraction. This results in complete enzymatic cleavage of all free met-enkephalins from long latent fragments. The radioimmunoassay for the determination of met-enkephalin in the digested sample gives the total met-enkephalin from the supernatant. Transformed RINα cells appear to have more than 5x more latent enkephalins than fully processed met-enkephalins.

Origin and properties of fibrous casing

The hollow fibers are made of 12.5 (acrylonitrile vinyl chloride) wet Cabasso, Hollow Fiber Membranes, up to 13.5% solution by the poly-fiber method, vol. 12, Kirk-Othmer

Encyclopedia of Chemical Technology, Wiley, New York, 3rd Ed., 492-517, (1980), U.S. Patent 5,158,881, incorporated herein by reference.

The resulting membrane fibers may be either bilayer or monolayer PAN / PVC fibers. To form implantable sheaths, the long fiber is first cut into segments 5 cm long and one end of each segment is sealed with acrylic adhesive. Filling sleeve with filler neck is formed by one end of the fiber being sealed with one drop of LCM 24 (a light-curable acrylic adhesive available from ICI), then the adhesive is cured with blue light and the step repeated with the other drop. The opposite end of the fiber is pre-attached to a brittle filling neck with a silicone baffle through which a cell solution can be introduced. The fiber is adhered to the neck by applying LCM 22 to the outer diameter of the neck and passing the fiber over it, then the adhesive is cured by blue light. The fibers connected to the filler neck are placed in sterilization bags and sterilized by ETO.

After sterilization with ethylene dioxide and degassing, the fibers are deglycerinized by ultrafiltration first with 70% EtOH and then with HEPES buffered saline through the fiber walls in vacuo.

Preparation and encapsulation of transformed cells

Transformed cells are prepared and encapsulated as described below:

A broth solution is prepared using commercially available alginate, collagen or other suitable broth material. The cell solution is diluted in a ratio of two parts of the broth solution to one part of the cell solution containing the transformed cells described above. We prefer Vitrogen

(Celtix, Santa Čiara) than nutrient soil for AtT-20 cells Than nutrient soil for cells RIN we give priority Organogen (Organogenesis, Canton, MA). Cells RINa are on encapsulation

prepared by the following method. Cells are grown in DMEM base medium with 10% fetal bovine serum during the proliferation phase. These cells are removed from tissue culture vials by double washing in Hanks Balanced Salt Solution without calcium and magnesium. The cells are then incubated for 1 minute in 0.25% trypsin + EDTA. The solution is discarded and the cells are rinsed off the vial using Hanks Balanced Salt Solution without calcium and magnesium. Cells are placed in 10 ml of basic nutrient medium and centrifuged at 100 x g for minutes. The cells are then resuspended in 10 ml of the preferred serum-free nutrient medium (Ultra culture, Biowhitaker, Walkersville, MD). Surprisingly, RIN cells secrete more analgesic substances when grown in serum-free nutrient media than in serum-containing basal media.

The cells are centrifuged twice at 100 g in the preferred serum free medium before the cells are concentrated 1: 1 with the preferred Organogen broth. Organogen is 1% bovine tendon collagen accessible as a sterile solution. 8 parts of this solution are mixed with 1 part 10x DPBS. 0.5 N sodium hydroxide is added until physiological pH is reached (approximately 250 μos).

The final concentration of cell solution + culture medium used for encapsulation may vary from 20,000 to 50,000 cells / μΙ. Cells are counted in a standard manner on a hemocytometer.

The cell suspension / culture broth is placed in a 1 ml syringe. The Hamilton 1800 Series 50 microliter syringe is set to 15 microliter air bubbles, is placed in a 1 ml syringe containing the cell solution and 30 microliters are withdrawn. The cell solution is injected through a silicone baffle in the filler neck into the lumina of an acrylic membrane hollow fiber, which acts as a molecular sieve with a limit of approximately 50,000 to 100,000 daltons. Ultrafiltration can be observed along the full length of the fiber. After one minute, the throat breaks, revealing a clean surface not wetted by the cell solution. One drop of LCM 24 is applied and the adhesive is cured with blue light. The filled fiber is then placed first in a HEPES buffered NaCl solution and then in a CaCl2 solution for 5 minutes to cross-link the alginate. Each implant is 5 cm long, 1 mm in diameter, and contains approximately 2.5 million cells.

After the hollow fibers are filled and sealed, a silicone tie cable (Special Silicone Fabrication, Paso Robles, CA) (ID: 0.69, OD: 1.25) is placed at the proximal end of the thus formed sleeve. In the lumina of the silicone tie cable is inserted a radio-contrast titanium plug, which acts as a radiograph. The filled and treated sheaths are then placed on 100 mm diameter tissue culture dishes in 1.5 ml of PC-1 nutrient medium and stored at 37 ° C in a 5% CO 2 incubator for in vitro analysis and storage until implant.

The encapsulated cells are then implanted into the human subarachnoid space as described in the following section.

Surgical procedure

After securing i. in. access and filed. For prophylactic antibiotics (cefazoline sodium, 1 gram i. v.), the patient is placed on an operating table, generally either in the lateral position in the orthosis or in the gene-pectoral position, with the lumbar spine bent forward. The surgical field is sterile prepared and purged to expose the midline dorsal lumbar region from S-1 to L-1, and intraoperative imaging of the lumbar spine is possible by X-ray x-ray. Local infiltration of 1.0% lidocaine is used to ensure anesthesia of the skin, periosteum and other deep connective tissue structures up to and including flavum ligaments.

In the parasagital plane, a 3 to 5 cm long skin incision is made 1 to 2 cm to the right or left of the midline that continues down to the lumbodorous fascia using electrocautery to stop bleeding. Using traditional bone landmarks, including the loin crest and lumbar spine, as well as fluoroscopic guidance, it is introduced into the subarachnoid space between

L-3 and L-4 of Caliber 18 Touhy's Needle via the Oblique Paramedic Approach. The needle is directed so that it enters the space in a shallow upwardly directed angle of not more than 30 to 35 ° with respect to the spinal cord in each of the sagittal and transverse planes. The appropriate needle tip position is confirmed by withdrawing a few ml of CSF to determine catecholamine, enkephalin, glucose, protein and cell count levels prior to implantation.

The Needle Throat is re-examined to confirm that the tip opening is oriented upwards (the direction of the opening is marked with a groove on the needle throat) and the lead wire is «pushed down the needle lumen until it reaches 4 to 5 cm into the subarachnoid space. (determined by preliminary measurement). Care should be taken during the passage of the wire that there is no resistance to the progress of the needle wire and that the patient does not complain of significant neurogenic symptoms, each of these observations could indicate a bad lead wire direction and possible threat to the nerve root or spinal cord injury.

After the lead wire is placed in the subarachnoid space, the Touhy needle is withdrawn separately and removed from the wire. Position of the wire in the midline of the spinal canal, before the expected location of cauda equina and

Even without loops or unexplained bends, it is then confirmed by fluoroscopy. After removal of the Needle Needle, the guide wire should be capable of moving freely into and out of the space with only very weak resistance caused by the rough surface of the wire going through the rigid and fibrous ligamentum flavum.

A French dilator no. 7 and the wire is used to guide a dilator that is gently but firmly pushed through the fascias, paraspinal muscles and ligamentum flavum, following the wire path toward the subarachnoid space. As soon as the loss of resistance after the transition /

ligamentum flavum, is the procedure of French dilator no. 7 and the dilator is removed from the wire. This is done to prevent the progress and manipulation of this relatively rigid dilator in the subarachnoid space to any significant degree.

After the wire path is expanded to a greater extent using the French dilator no. 7, the French dilator no. 6 and the cannula housing and are located on the inserter wire. French dilator no. 6 and the cannula are carefully advanced forward into the subarachnoid space until the cannula tip opening is located 7 cm in the space. As with French »dilator no. 7 is the French dilator no. 6 connected to a cannula directed by a wire into the dilator of the dilator. The location in the subarachnoid space is determined by previous measurement of the device and is approximately confirmed by fluoroscopy. Manipulators of the dilators and cannula in the subarachnoid space are given great care to avoid the wrong direction and possible neurological injury.

When the appropriate placement of the cannula is confirmed, the guide wire and the French dilator no. 6 gradually removed gently from the lumina cannula. Depending on the patient's position on the operating table, there may be significant to very significant flow rates at this point

Even cerebrospinal fluid via cannula, which requires plugging of the cannula or very rapid placement of the implanted capsule to prevent excessive loss of cerebrospinal fluid.

The encapsulation (transformed cells) is provided in a sterile double-walled container, immersed in a transport medium and fully folded including a tubular silicone tie-wire. Prior to cannula implantation into the subarachnoid space, the sleeve is transferred to an advertising kit tray where it is positioned to allow the sleeve to be held in the transport medium while being roughly examined for damage or major defects, while the silicone tie cable and its length are shortened. adapted for the displacement device and removing the Hemaclip ™ clamp that closes the outer end of the housing.

When the membrane portion of the device is carefully inserted into the cannula, the sheath portion of the housing is placed on the stainless steel shunting device by inserting a small diameter wire forming part of the shunting device. The capsule moves forward until it reaches the membrane tip at a point that is 2-10 mm at the cranial tip of the cannula in the subarachnoid space. This placement is achieved by measuring the cannula and the connection of the sleeve-tie cable-shifting device, and ensures that the diaphragm portion of the sleeve is protected by the cannula all the time as it is moved into place.

After the housing is placed in the cannula, the displacement device is used to hold the housing in place (without moving forward or backward) in the subarachnoid space while the cannula is completely withdrawn from the housing and the displacement device. The shifting device is then removed from the housing such that its wire portion slips off the silicone tie-wire. Using this method, the final placement of the capsule is such that the 5 cm long membrane portion of the capsule lies entirely within the subarachnoid space containing the cerebrospinal fluid ventral to cauda equina. At its caudal end, an approximately 1 to 2 cm silicone tie rope is anchored, which passes through the subarachnoid ´ space before the rope leaves the space through the dura and ligamentum flavum. The silicone lashing rope extends from this level externally through the paraspinal muscles and emerges from the lumbar dorsal fascia leaving generally 10 to 12 cm of loose material which is accessible to secure the sheath.

Cerebrospinal fluid leakage is minimized by injecting fibrin glue (Tissel®) into the pathway that the tether cable occupies in the paraspinal muscle and by firmly closing the pathway opening in the superfacial fascia with a tobacco stitch. The loose end of the lanyard is then anchored by a non-absorbable suture and completely covered by a 2-layer closure of the skin and subcutaneous tissue.

The patient is then transported to the recovery area after neurosurgery and kept in bed under strict rest on the bed for 24 hours after surgery. Antibiotic prophylaxis is also continued for 24 hours after implantation.

sequences

The following is a summary of the sequences listed in the Sequence Listing:

Sequence s identification number: 1 sequence DNA oligonucleotide oCNTF-003 Sequence s identification number: 2 sequence DNA oligonucleotide oIgSP-018 Sequence with identification number: - DNA sequence mergers IgSP-hPOMC Sequence with identification number: - DNA sequence mergers

IgSP-hPOMC-AACTH

Sequence s identification number: 5 sequence DNA oligonucleotide Ortho-052 Sequence s identification number: 6 - sequence DNA oligonucleotide Ortho-053 Sequence s identification number: 7 sequence. DNA oligonucleotide Ortho-054 Sequence s identification number: 8 sequence DNA oligonucleotide Ortho-078 Sequence s identification number: 9 sequence DNA oligonucleotide oIRES-057 Sequence s identification number: 10 - sequence DNA oligonucleotide obDBH-065

Sequence with identification number: oligonucleotide oIRES-bDBH-064 11 sequence DNA Sequence with identification number: oligonucleotide oIRES-bDBH-066 12 sequence DNA Sequence with identification number: oligonucleotide oIRE-068 13 sequence DNA Sequence with identification number: orTHA-073 oligonucleotide 14 sequence DNA Sequence with identification number: ohPOMC-IRES-069 oligonucleotide 15 sequence DNA Identification number: 16 - sequence RTHA1 DNA -155 Identification number: 17 - sequence RTHA + KS DNA Sequence with identification number: RTHA-IRES-bDBH 18 - - sequence DNA Sequence with identification number: rTHAKS-IRES-bDBH 19 - - · sequence DNA Sequence with identification number: of the oligonucleotide ohPOMC-IRES-070 20 - ~ sequence DNA Sequence with identification number: oligonucleotide oIRES-rTHA-071 21 - - sequence DNA Sequence with identification number: of the oligonucleotide orIRES-rTHA-072 22 ”“ · sequence DNA Sequence identification number: IgSP-hPOMCAACTH-IRES-RTHA-IRES-bDBH-068 - ~ DNA sequence mergers Sequence with identification number: oIRES-074 24 - sequence DNA Sequence with identification number: oligonucleotide oZeocin-077 25 - sequence DNA Sequence with identification number: oligonucleotide oIRES-Zeocin-075 26 - - sequence DNA Sequence with identification number: oligonucleotide oIRES-Zeocin-076 27 sequence DNA Sequence with identification number: 28 - sequence DNA

IgSP-hPOMCAACTH-IRES-RTHA-IRES-bDBH-IRES-Zeocin-073

SEQ ID NO: 29 - proA + KS DNA sequence SEQ ID NO: 30 - IRES DNA fragment sequence

deposits

RINα / ProA / POMC / TH-IRES-DBH cells were transformed to produce catecholamine, enkephalin and endorphin as described above in RINα / ProA / PO030 / PO88.

in the case (and in Table 2), called Deposit was made in accordance with the Budapest Agreement and was deposited with the American Type Culture Collection, Rockville, Maryland, U.S.A., June 7, 1995. The deposit received accession number CRL 11921.

The foregoing description has been given for purposes of illustration and description only. The present invention is not intended to be limited to the exact form exemplified. It is to be understood that the scope of the invention is defined by the claims which follow.

Industrial usability

The present invention provides a cell line that has been engineered by genetic engineering methods to produce analgesic compounds. These compounds belong to the groups of endorphins, enkephalins and catecholamines. The cell line can be used to treat pain, long-term when implanted into the host's body as a biological artificial organ.

52, 53

LIST OF SEQUENCES (2) INFORMATION ON SEQUENCE WITH IDENTIFICATION NUMBER 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULA TYPE: cDNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no ( (vii) DIRECT RESOURCES:

(B) CLONE: oCNTF-003 (xi) SEQUENCE DESCRIPTION: IDENTIFICATION NO. SEQUENCES: 1

CCCGGATCCG CGTCACCCCT AGAGTCGAGC TGT (2) INFORMATION ON SEQUENCE IDENTIFICATION NUMBER 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULA TYPE: cDNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vii) DIRECT SOURCE:

(B) CLONE: oIgSP-018 (xi) SEQUENCE DESCRIPTION: IDENTIFICATION NO. SEQUENCES:

TTTCCCGGGA AAGCCGAATT CAC (2) SEQUENCE INFORMATION WITH IDENTIFICATION NUMBER (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 849 base pairs (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (genomic) (iii) HYPOTHETICAL: no (iv) ANTI-SENSE : not

(Vii) DIRECT SOURCE: (B) CLONE: IgSP-hPOMC (Ix) SIGN: (A) NAME / BRAND: 5'UTR (B) LOCATION: 1..43 (Ix) SIGN: (A) NAME / BRAND: exon (B) LOCATION: 44..89 (Ix) SIGN: (A) NAME / BRAND: intron (B) LOCATION: 90..168 (Ix) SIGN: (A) NAME / BRAND: 3'UTR (B) LOCATION: 807..849 (Ix) SIGN: (A) NAME / BRAND: different characters (B) POSITION: 43 ... 186 (D) ADDITIONAL INFORMATION: product (Ix) SIGN: (A) NAME / BRAND: different characters (B) LOCATION: 187..806 (D) ADDITIONAL INFORMATION: product.

IgSp region hPOMC region (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION NO. SEQ ID NO: 3: C334GG3333IT 363AIC3AAT G3-G3IG33T 60 irtlUIliil. 3333 ^^ 03 T3A.aaá-'L d-AjxU_i-r. 33C331rGj3 —0

TUJAffinPCT C333j £ ¾3O3 G t. ~ AllAd 'lllaJJi'Hul 33313CG33 0313333333 1SC

ΤΟΠΤΟ33333 Μ £ 'Π · ίΐΤ? Ο GrijJrtjJJL. 333O313SA C33333jä3 ´LUCU.-J.'jj 240 iLLAt. ' * S r S S S f j 14 j / n n-i. ti-i ··!., (aJACii-i-G) GG3G3333 AG33O33333 300

CTi-In-fG3A t-i-t-yCl1L-1. I-G GIA ^ GrĽlU Lí.í.t. il.UUHj Li — i.C? iaaJJ 360 x-t. ί x i-ijAJJJ 0 '' s4 Α; ~ τ ί — T G3333333G3 0333303333 03G33333G 0333031303 420

ClAIGjAíA 3333333333 G333O3333 33333PffA G3333333ľA. G3333G3333 480 • 3C33I3AC33 G333? G3FC Ώ33333333 3G333T3333 C333SG33C A3C3G333G3 540

IGA-llaJ-LA G333O33333 G-liASIG G3333SO33 G3333333T (? O33333A3 600

G333333O33 0303333 = 0 C = £ 3O33333 3333333333 G3®A3A3 GG3G3333 660

031303330 G3G3CI33 O33333333A O33333333A G3CÄ3333 Ι3Ο33333ΓΓ 720

TCAiLrtJJIC 03 = 0 = 33 = 03 CTgQjJJj- 33333 = 0333 GIlUAàAfiC (3333JLAILA 780

ASňjjJLiA G3SSG333 3Gleri-i · ---.- 3Ώ3333333 023033330 031033330 840

SG3IG3G ', 849 (2) INFORMATION ON SEQUENCE WITH IDENTIFICATION NUMBER 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 525 base pairs (B) TYPE: nucleic acid (C) FIBER TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (genomic) (iii) HYPOTHETICAL: no (iv) ANTI-SENSE : no (vii) DIRECT SOURCE:

(B) CLONE: IgSP-hPOMCDACTH '(ix)

(A) NAME / BRAND: 5'UTR (B) POSITION: 1..43 (ix) BRAND:

(A) NAME / BRAND: exon (ix) (ix) (ix) (ix) (ix) (B) POSITION: 44..89 SIGN:

(A) NAME / BRAND: intron (B) POSITION: 90..168 BRAND:

(A) NAME / BRAND: exon (B) LOCATION: 169..482 BRAND:

(A) NAME / BRAND: 3'UTR (B) POSITION: 483..525 BRAND:

(A) NAME / BRAND: various characters (B) LOCATION: 44..188 (D) ADDITIONAL INFORMATION: the product is an IgSp region BRAND:

(A) NAME / BRAND: various characters (B) LOCATION: 189..482 (D) ADDITIONAL INFORMATION: product is hPOMC area (xi)

DESCRIPTION OF THE SEQUENCE: IDENTIFICATION NO. SEQUENCE: 4:

GSTZILĽi CSTCTS. GľCSAmGľ GGSZUľr JOàOCFàAT GTGľlGCCľ 60

CIGKIGZAS 'IjjTPgg ggΑΑΞΞΞΞΣΟ GUAGICnZA ΑΑΖΠΞΏΞΞ 120

TCEKISSSCr CIGIGCGľ GZTAICkľ ΤΊΚΓΓΠΟΓ l CCUOGSS GKSKTIGjj 180 crrimis: cmín ggtictkä gsghsc tghzähä ciu ^ & gs 240 ggmss-ĽL QPi'm'¾.? (znsiGns gctgtit: czszrcm: cigszka 300

GXIGCIGST Gznznsc ίΓΟΚΞΏΞ Α33ΖΧΠΣΆ CAľľlUľGľľ 360 360ττιτ Αΐ P / ¹ , ti nttftntť Jňj.X3-irC3 GOSSTUdľ ΛΑΞτΞΖΣλ? ·. 420

11-i3: i3l3G3ZIGHC iSSPAjj-CA T333SrA G3UZECASG AA.i.in. .j-iSI 480

SSSZKTG ίΠΐΐΠΤΤ, GZ2ÄHX CEXSGjGj ΊΠ? £

525 (2) INFORMATION ON SEQUENCE WITH IDENTIFICATION NUMBER 5:

(I) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (Ii) MOLECULA TYPE: cDNA (Iii) HYPOTHETICAL: no (Iv) ANTI-SENSE: no (Vii) DIRECT SOURCE: (B) CLONE: orTH-052 (Xi) ť SEQUENCE DESCRIPTION: IDENTIFICATION NO. SEQUENCES: CCCAAGCTTG CACTATGCCC ACCCCCAGCG

(2) INFORMATION ON SEQUENCE WITH IDENTIFICATION NUMBER 6:

(I) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (Ii) MOLECULA TYPE: cDNA (Iii) HYPOTHETICAL: no (Iv) ANTI-SENSE: no (Vii) DIRECT SOURCE: (B) CLONE: orTH-053 (Xi) SEQUENCE DESCRIPTION: IDENTIFICATION NO. SEQUENCES: CCCGGATCCT ATGCATTTAG CTAATGGCAC

(2) INFORMATION ON SEQUENCE WITH IDENTIFICATION NUMBER 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULA TYPE: eDNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vii) DIRECT SOURCE:

(B) CLONE: orTH-054 (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION NO. SEQUENCES: 7:

CCCAAGCTTA TGGTCCCCTG GTTCCCAAGA (2) INFORMATION ON SEQUENCE IDENTIFICATION NUMBER 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULA TYPE: eDNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vii) DIRECT SOURCE:

(B) CLONE: orTH-078 (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION NO. SEQUENCE: 8:

CCCAAGCTTC GCCACCATGG TCCCCTGGTT CCC

NUMBER 9:

(2) SEQUENCE IDENTIFICATION (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULA TYPE: cDNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vii) DIRECT SOURCE:

(B) CLONE: oIRES-057 (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION NO.

AAAGGATCCG CCCCTCTCCC TCCCCCCCCC

SEQUENCE: 9:

(2) SEQUENCE IDENTIFICATION (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULA TYPE: cDNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vii) DIRECT SOURCE:

(B) CLONE: obDBH-065

NUMBER 10:

(xi) SEQUENCE DESCRIPTION: IDENTIFICATION NO. SEQUENCE: 10:

AAAGCGGCCG CCCACGTTCA GCCTTTGCCC (2) SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULA TYPE: cDNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vii) DIRECT SOURCE:

(B) CLONE: oIRES-bDBH-064 (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION NO. SEQUENCE: 11:

CTTGCCACAA CCATGTACGG CACCGCGGTG (2) INFORMATION ON SEQUENCE WITH IDENTIFICATION NUMBER 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULA TYPE: cDNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vii) DIRECT SOURCE:

(B) CLONE: oIRES-bDBH-066 (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION NO. SEQUENCE: 12:

CGCGGTGCCG TACATGGTTG TGGCAAGCTT (2) INFORMATION ON SEQUENCE IDENTIFICATION NUMBER 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULA TYPE: cDNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vii) DIRECT SOURCE:

(B) CLONE: oIgSP-068 (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION NO. SEQUENCE: 13:

AAAGATATCG CGGCCGCGTC ACCCCTAGAG (2) INFORMATION ON SEQUENCE WITH IDENTIFICATION NUMBER 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULA TYPE: cDNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vii) DIRECT SOURCE:

(B) CLONE: orTHD-073 (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION NO. SEQUENCE: 14:

ATACACCTGG TCAGAGAAGC CCGGG (2) INFORMATION ON SEQUENCE WITH IDENTIFICATION NUMBER 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULA TYPE: cDNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vii) DIRECT SOURCE:

(B) CLONE: ohPOMC-IRES-069 (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION NO. SEQUENCE:

GGGGGGAGGG AGAGGGGCCC GCTGTGCCCT (2) INFORMATION ON SEQUENCE IDENTIFICATION NUMBER 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1030 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULA TYPE: DNA (genomic) (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vii) DIRECT SOURCE :

(B) CLONE: rTHD (ix)

(A) NAME / BRAND: 5'UTR (B) POSITION: 1..6 (ix) BRAND:

(A) NAME / BRAND: exon (B) LOCATION: 7..1017 (ix) BRAND:

(A) NAME / BRAND: 3'UTR (B) POSITION: 1018..1030 (xi) SEQUENCE DESCRIPTION: IDENTIFICATION NO. SEQUENCE: 16:

AňCZľKIGC It :; : íl íl <1 IG IG IG IG IG IG «« «« «

GT3CZ? FGr 'lil-ll-ciIj · llCJriJJlG GC ^ CCSjS GOlTCIUIGA G3GCIG3Z 120 aznmzc tztsgzt σζππτ £ τ ataczaocs igaztätt iso crrrAjcľGG ípsccaz: G5? αςαοοίαία larTccľľ 240 wmsľľicr AIGZTCZA TĽĽOláJJL »GtGTCZGj Α322ΓΓΧΣΑ GľľCIG ^ Ä 300

GSdTdGIG CLrOGLAI Í A3 A3ľ? GCľdC GZľxľľ-dai 360

AAaAjJJjA t-‘n-in .i lil and GZIGC5C33 GIQjOOjjIC UCIGIOCCľ CľCIG-Jm 420

CIG333GIC TGZCTTG UCiCľľUA IG3 £ HACT ΑΓΚΚΗΠΑ TCCľICľľA 480

CTTroSKIT GGľľGGľľ GTOCľlGľ CÄIGmGr IGZ? CAIGľ ÄHKGľl 540

CTGUIZA σΠΤΖΙΣΑ GTICIOX = G C CSTIG? C TIGľAICľ GSiá.i: .lL-. 600 © K? Ft3 «A CIO7C2ZG TTCICClCA ClCPZPAl CCSl1AJUl 660

TAPCŤffàĹG GGTGľHTA aSraSTOZ ΤΌΧΠΟΣΑ QCJffGZC 720

CK33Ľ1UX IGIOffGA GĽCLSLC'IL CI ?iáJLTTIC ÄZLPG = C? C 3333GZIGIG 780

CA23CTC: MKKTČrC CTOTCDľ GOCZAZTIG IGIQSffG CTIUAIG £ 840

O33A33CAÄZICASA CIÄIGOľCC ΟΖΚΚΠΏΖ GľľľICICľC TSIGAZľT 900

GdEUCA ΟΟΕΖΣλΓ ΊσΠόΠΟΞ ggíLUľTC X3CDUUCA QľCľICCľlG 960

GgnSHCĽ SSGTCZ · GZPCCCľGG G3ZPCZ7C Τ3 £ ΐαΧΑΓ TSCCTňAIG 1020 cmcsocc 1030 (2) INFORMATION ON SEQUENCE IDENTIFICATION NUMBER 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1037 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULA TYPE: DNA (genomic) (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vii) DIRECT SOURCE :

(B) CLONE: rTHDKS (ix)

(A) NAME / BRAND: 5'UTR (B) POSITION: 1..13 (ix) BRAND:

(A) NAME / BRAND: exon (B) LOCATION: 14..1024 (ix) BRAND:

(A) NAME / BRAND: 3'UTR (B) POSITION: 1025..1037 (xi) SEQUENCE DESCRIPTION: IDENTIFICATION NO. SEQUENCE: 17:

mznnzz axaigzcs (πεζίοχ ίπξ ^ αγτζ; ääggr 6o

O3LĽ1LĽ1U ALAASArnC AH'IffilCT CTGIlGSC CAtiliLC-T ICICTTCaA 120 (jJIG'lÄilLC CAXGIOSGk ÄZIGZIGZ JOSITOX TIC3GIXÄ AZTLUZd - 180

AZPÄTICX CSGIG3ÄT JOCGZS. ASGmGľľ ΚΠΒ5ΚΕ ΑΞΠΚλΠΖΓ 240

ΖΙΘΚ3ΖΙΘΚ3 G3XKZKIG CDUIMG: CIGIHSG CCCIG ^ GS GnnTGOr 300

TOGSMX ΈΟΖΕΖΓ ÄI5SG3Í CHSOaXA. GSG3IG3GG AJCTZOLX 360 cuctkjk; ggzztog cznraGzr gzsczsig ctszck: τξππζπχ 420

IGETTICIG GXAZZES UJiHĽSXT GTľlCrŕTCZ ÄZX7GIÄDÍ ICJCJAIGľ 480

COOOTCCľ A323TK3C 03GOSSA CIG3G03T 30007322 3ι333ΖηΟ 540

33233220 330303 0227320 003330 £ 323332 0300222 600

G23Z33I3I 37i377LIL 7777723232 OCG2323C 32200C32 UjúpäTIuís 660

G233237A OO732222 ΑΞ273Α22: T3322IG3. G222I332ľ 03223022 720

A3G232232 O27C2222T 3333322 I3iiil Litri. L-L111 / 1H2C2 03012 = 32,780

A3232333G 022022772 ÄIOKC2IA 032223333 Ί ~ ΠΤ3333Γ C033 <22¾¾ 840

OTlďí í i .1 ,. 77330732 liALž-Ti-'AA l.LÍLiLti 7Iit7tLí3-L L.-i.-LLLiLi 900

3Ä3ITB3C 023300C 32227333. 0370230 ALW. 0330702 960

C322OG3G G222322723 TltTmlLi-A 302232222 3C227C33. LiLU —LL-O 1020

03773230 702 = 30 1037 (2) INFORMATION 0 SEQUENCE WITH IDENTIFICATION NUMBER 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3245 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULA TYPE: DNA (genomic) (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vii) DIRECT SOURCE :

(B) CLONE: rTH-IRES-bDBH (ix)

(A) NAME / BRAND: 5'UTR (B) POSITION: 1..6 (ix) BRAND:

(ix) (ix) (ix) (ix) (Xi) (A) NAME / BRAND: exon (B) LOCATION: 7..1017 BRAND:

(A) NAME / BRAND: intron (B) LOCATION: 1018..1617 BRAND:

(A) NAME / BRAND: exon (B) LOCATION: 1618..3411 BRAND:

(A) NAME / BRAND: 3'UTR (B) POSITION: 3412..3425 BRAND:

(A) NAME / BRAND: various characters (B) LOCATION: 1025..1617 (D) ADDITIONAL INFORMATION: product is IRES area SEQUENCE DESCRIPTION: IDENTIFICATION NO. SEQUENCE: 18:

The imxTGZT gzpaswa gtgigsät icsczaxiG 60 is a 1CG3ClIG GluZCICI®. GZAjjIGKľ 120

OZUGOGIC GSČGZGS TZXSffiST UXTUlAGr JGAZTCGS Ί ^ ΐΣΡΑΓΓ 180 rrrrftlGltxI MK3CGZ GSVSSTľ uJAJCÍía TCTľAľGCIG 240

ÄIGZEGZA IGXľGZGS Α233ΓΠΟΞΑ GľľllIĽďA. 300 (jjjTfXliijili G-iXXrr CXTGlTjS C3a C3IGIC QajJľľCľlb 360 ctztdza iscwsm: Gľsznzzc Eozmz: ccsrsTľr 420

CI03D3GIC IGSjHIGZj GjIGTnCAA ILLA-LC-GT .-. ‘LAlt: 1 A IGZCIOdCA 480 ζΠΑΤΧΚΓΓ OCnSGX GSJU_TtĽ C??? GľIGľ TGSOuGT ÄDZXIGnG 540

S37CH ?. CTTIGZZA. GľlCICZTG G £ XľIG? C TIĽĽAILTCľ ΠΤ ^ ΏΊΓΆ 600

O ^ CPrGa TIGPSPrCr CICDCjSIG TJCUjuTKa CIGIG ^ HT OSSZKIG 1 660

APPÍTGÄIG GSJGZIOA (XJJlÄIGSr SJň'ii.'ÍU. IGICnCZZA CCA? GľIC 720

CinTCIOZ TGICffiGS. GTTGGľC CEúxiTiu ÄXDGOC ASľAHTOIG 780 cwxrax ŕarospc οκώζςγ gigcotíg ightosg ctzsapsc 840 (ΞΙΤΑΞΞΏ. ÄS2CS5A CZSTOZZCľ GGIrCOľASľ CIZKľICľC GľGVľ

Cjíj. i, ijatCä CY'-i · .-! Γ * ίΤ iGrCĽôTCIG ffuAVi.í. ILLCLLruliA 2223332732

Sti-i · ΐ3.ι1k, JA -i -A'i 1 -4 <, í ,. j ¹ G3CC3ZZG xunJÍtaXl-j¾ Ζ? .233? Αη22 omcsicz gxzzcc: cmnm: Gľzvcsm οξξπξαξ cezthsa

ΈΑΙ-ί.ι iinx t.n-t i-ίί! 'Ii.? L CíäíAíGí'ía Ti'ľlixAlA 1 ?¾IG333ľC TZTIGj ^ ftT

GISGZm: G? ØGZG3 CTTIGICTIC TIGLSÍZA 7333172332 TCTTIUJJ ”

C73C333WG GÄ3Z? GG3 3233Γ33ΑΓ GIGH5ÄS 772323333 73333V23ľ iCnt? ÄSc sQPCHc iGDmsa: <373333232 attttagz 022233333

G27G33333 333333333i. 77232237 GIKETSOA C23333 £ A G333322F7>.

033323333 703733332 73332277 (33337722 3372332Γ 03333 ^ 723

Gffinoňd ftnsaa (332133332 «2372322 77333721333 22332333) 3333333337 g ^ otozt ttcstctstt 72102237 ewwäet ccsíms

03723233 G3233337T 73X37I2A 77702203 ÄM237333 GL32333

02332223 (3333332337 C732333333 203333333 03332332 =, G333333337 Clinic 32233733: 07332223 022333 = 23 G3323332 <233332333

323 333 = 70th 33233033 G32327G3 703327733 723323333 (322232332

77233K2IG HJĽIUllll <33332332 CJGSJ: 3332 = 7032 332733333

0IG3IQLLA <33322335 033333232 ΊΤ32ϋτΟ3 233332335 02 = 02333

C23IULL23 33321LLD5 <2323 = 012 <323333332 <3333022 (352333255

G323IU12C ICTHIDÄ. <323237777 0332333IG 7COT235 0123003 <32322235 cnrooo G33330335 tiuzess 722333333 032223332

TOTcascA oarmriT 03023333 03330233 702223327 G5222322

702117722 (I-203330: 03233023 03323033 72 = 017233 022332333

900

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ClcllllUj (jCOIxjtOC Οΐ-ΛΛ-Τ-ibj iu r rtujlun. 00733232 CttZľu, ..,

Ú.A i i.iaQTZ 20233203 <37707322 AlCeiOtClä 27330270. G333273373

CAJTiOILu Äxii.t_ril_LA G733333333 G-u.i.'iUOO. CC2ilJJjO>. C77O73333

Q1-07 07 07 07_IGIGIGIGG-A (íí.3í.j.j.j.j.j.j.j), .j C C C C C C.IG G G G G G G G G G G G G G G G G G:: G:::.

332203333 gtsoaso ottococ czotoas 073333332 033333333

Oll i .1.113,7 03033333 (JUdIGC-A GHOCOTZ 20P730CT 3233-22222

Gi i hi.iáój 2dÍČt33j Olom-lG OcľtOíCij ilii i-i''JJ lliiiriCOC

CLáiiijiILä 733737333 Li, i (.. il.i; I? C 273333370. 733µftlU22 C7377O7

273333Γ733 333777333 CO273773 00233322. C3C2737333 (3.133333.1 i. '

10730113 207277332 C727O7272 00732272 ΊΟ7332233 O23Z7372

A22G722733 C7G3O733 073000. 0073370 2C ~ 73OC2A 0777072

GOOCT332 2G002227 07377027 2273733737 233737732 33037773

C3O3372T 0733700. O2OG37A 0070732 733377337 (3333332773)

G3O3C2733 27OS7373 037727127 01370277 270330727 (37737377)

0013720 GTOTO 7333333773 0370277 20733330 73772073 ”7732272733 2LiyftJiC'iG 033133333 OG3337O7 TC233OG® 7717333733

0733.113 ^ 1207377723 033307373 OO273333 737773377 C37727CC

133213777 G72O7372 Cit x 11.3313 733773073 33077302. 7333327333 <2332270®. 7337373OG 0730207 (337Ώ33373 277233072 Ο737Ώ3Ο

072733333 OG333377 (337337027 277773333 G722G3373 2273) (73333

73373

2230

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2700

27602820

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3420

3425 (2) INFORMATION ON SEQUENCE IDENTIFICATION NUMBER 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3432 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (genomic) (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vii) DIRECT SOURCE :

(B) CLONE: rTHDKS-IRES-bDBH (ix)

(A) NAME / BRAND: 5'UTR (B) POSITION: 1..13 (ix) BRAND:

(A) NAME / BRAND: exon (B) LOCATION: 14..1024 (ix) BRAND:

(A) NAME / BRAND: intron (B) LOCATION: 1025..1624 (ix) BRAND:

(A) NAME / BRAND: exon (B) LOCATION: 1625..3418 (ix) BRAND:

(A) NAME / BRAND: 3'UTR (B) POSITION: 3419..3432 (ix) BRAND:

(A) NAME / BRAND: miscellaneous characters (B) LOCATION: 1032..1624 (D) ADDITIONAL INFORMATION: product is IRES region (xi) SEQUENCE DESCRIPTION: IDENTIFICATION NO. SEQUENCE: 19:

ASSYTlCT ÍCľTJGälGľ (ΠΕΖΊΠΖ íàffiňPSGIG ARAGIGICA 60 eoaznssiii x & fcrnG Juncacr gscoigsc ccxsszr iuictscľa 120

G3IGKIC33 CkLGlUxA ÄdJlUATIGi: ΑλΞτΠΟΟΣ THIKUDi ÄSľALGKÄ

180

A_i. i.-i (.ii-^ ^ ST ASSZtGZT rCZZujAjj AOS —-— “2d 240

CTClj-Hj-SG GjZZIdr.3j G? C3Zr2GZ blink-i-OrG CA32TG?? Gj?

Ή i i-j TA ilniii i í i jtfijA CTĹLžvLÍ. ..Λ CAä-ililrGj AjjljjiL.J. -: JCXJ

Ci'iL.'Í'llaŤ? G i->'- ¹ i <-'- i-th b_í'ÍU-T RII-a-ÍJÍJ ~ bjiij G2322ICZ? Iblt C-b., .. t s 420

KSUTlĽib CZDCHZE CnGZ332ľ GTTK3KGZ ÄTOSMA IĽJSX ^ GZ 480

CIG3D3QZT iZG4TZCC C'jjjjC-jji. CIG2IG23T SGZIGTIGj ffCCGIAZC 540

CTCTIGZT Gtirasr ΤΠΖΠΤΰΙΤ CKH7G7C ÄTIG? CľIG CTCICZGS 600

GZZHGfflT G ¥ G Ä IG IG C C C CA C C CA CA C C C C C C C C C C C C 33 33 33 33 33 660 660 660 660 660 660 660 660 660 660 660 660 660 660 660

A3-íä_j. Lbib (AZTCSOľG ?GG-taj-11 ľ.

Ä3ZIGIG3G CZZAZTAS ÄIGWCZA GTGZľGTG TAŽNCTSľ GZTffiZľT 840 σΚΒ3Ώ3Χ íäZATäjZ lOGGÄZA TUľľŽOL JCCTJm: GTľľOIGT 900

GPAZTnGC GOOlAazAľ IGZTAľlGk GOľAľlGGKľ A3XZI0XA dATZATS 960

ClI.i'ii33G GjjjICZ? Gj äDäjJííáa C? C3ZIGj33 C? Lii? Liji GIG3?? Z?? 1020? □ΑΕΞΑ? ΓSCCZjX?

1Gj3MGIG Ä-i-ij.i, t-i AMZTjjOZZ TľjľCnCTľG AOjAäJu'H- CIAZíjjj’ÍCÍ 1200

HUJJĽ1U1C GXASAjSA TG3SGZCT CľľPPAľGľC GIG = A33A2 CTGITCZľľ 1260

GSSGZUCT T5A5GWA CTGHCIUľ ATSXľľ 7ΤΑ333Ώ2 G3 ¥ mHZ: 1320

AXIGZSC AasIGZZCT CTZD ŕ GA GZKZTZA T? A? 27GC CIGZTcSGjO 1380

G33OAZZ (3GIG33CG TIGUSGľG CTSbľlbib CHWGPGO. MTCCľlCIC 1440

CTOAZEIA TTC7AZ = A33 GGllTAO'A TCXESffiftS GAUDCT GIKSKSC 1500 iraiciLuja aziasnzA aitzľnx: αοογπαο ίοαιιιίμ arzweioa isóo

CTn 111.LA ΑΙΤΓπΖΓΑ CZIGjľmC CľľTSŕfPA CTCjmSZAÄSZTIG3Z7C 1620 ί ···· ϊ * 114 ί i i. Ϊ 33 33 33 33 (3373373733 3733733373 S-ltj-Axu (3733337333 d »ĹJ7T-lĹĹt) ĹĹ. C3337G3333 Ajjitl—.

int -t 'n. i 11. Tlu ^ rCTutA (j — jAHXíaJí. (□ jSú ^ I c7 £ 3773-3 £ 33 7337337333

Gjts-Jlu-tG (33713373733 ilAi'illxSZ biUixi33ä) G333-I33733 TO-TilU-II-.

<3773373373 <773333 ^ 73 Ä3G33C23 <33333 ^ 3777 <3333 ^ 73337 G3 ^ 37? C3 ?.

ffiPG333Z3G G733-C3733 AJILLLGZA GZT3C3G (7777733333 C3 £ žSG? C io3ffi3z: CTzmľľir ισισκτΰ σζπτισζ: atzustzcz c & pczcz ·.

azctLĽLrG GmzxzG 733037337 GiTOGGmc ctszgzc: <3377333377

ÚJltxx <JiLU 7373SC7CSX 007033733 ÍGJiljJTSr ·.

tiU.ťljJJC here AS4'i. 1.44 (jjJIUjjJJJ GjQTåJjJj- ÄJU-ĹÍxxriO. 7333333333

C33CGIUĽ1 ÄIC λΤΟΖχαλΞΖ: Ä3OSC3C OTC333733 00373 = 033 Ä37733333A (3333773333 (3333C3KO. ΊΠΠΟΟΖΆ) (30033303 CTOC33O3 (33 = 703 = 037 <277337300 00013303 777733073 <333333333333333333333333333333333333333333333333333333333333333333333 037337 ii- »i i-i mjj <3337333333 Q3flG3337T ΤΟΟ7ί333λ C? G3-GGG-0 (3377333377

Ú i-s 3i.x G33733737A (niTlCllXU CTTGSfOTT GuiAj-O. 70300133?

GOWOOSZ 033333307 007331130 <333301300 00033013 0301333333

00133030 <32303033 70002330 G3330003 033370023 037033333.

<2307003 0377733733 73033330. (00370370 770733033 037333377. <3333337734. <33307301 3377333377 73037330 70330373 0000037.

G3733733 (3730333334. (3330030 (330 = 000 7O3373A3) G7GOWO34.

<73003334 OCT03O3 Ά3Ο3323Ο GT73ÍO7G <3733737373 77DO33333

7ΘΟ373373 ÄIUOOIOT G3COOA <3O33A3C 703033733 030337333

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3000 SIGIGZjT GrCZrCGlG CA-C27i-thi-Ar-Ari-ll. 'ft rt i: i iGjZZTCZZG! <* .- »-

GäAjZTIC APCGC ^ GZ Ä = £ 3¾Z7C GGZCZAj GZľľCľZľľ CZGw-Aiľi

-i i n i; -, i-11 ^ t STT QUlLt-jJGGGGGZZC Aríiá.L ~ .'ÍGI AJjjJÍ’L-í.í

ÄXEÄICICZ AIGTOZA Ä7GZCTZ G333ZC33ľ ΤΤΖλ3ΞΖ3 ASľľZTAľCZ gz = gzczig azHrfPic: igxtgzt g ^ äjgzc A.i, ... r ^ -cr ghtgzjg

ΙΏ332-3--:-J J (J J J J J J J J J J J j j j j j j j j j

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3432 (2) INFORMATION 0 SEQUENCE WITH IDENTIFICATION NUMBER 20 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULA TYPE: cDNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vii) DIRECT SOURCE:

(B) CLONE: ohPOMC-IRES-070 (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION NO. SEQUENCE: 20:

AGGGCACAGC GGGCCCCTCT CCCTCCCCCC (2) IDENTIFICATION NUMBER SEQUENCE INFORMATION 21

(I) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (Ii) MOLECULA TYPE: cDNA (Iii) HYPOTHETICAL: no (Iv) (Vii) ANTI-SENSE: no DIRECT SOURCE: (B) CLONE: o-IRES-rTHD-071 (Xi) SEQUENCE DESCRIPTION: IDENTIFICATION NO. SEQUENCE: 21 GAACCAGGGG ACCATGGTTG TGGCAAGCTT

2) IDENTIFICATION NUMBER SEQUENCE INFORMATION

(I) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (Ii) and (Iii) MOLECULA TYPE: cDNA HYPOTHETICAL: no (Iv) ANTI-SENSE: no (Vii) DIRECT SOURCE: (Xi) (B) CLONE: o-IRES-rTHD-072 SEQUENCE DESCRIPTION: IDENTIFICATION NO. SEQUENCE: 22 CTTGCCACAA CCATGGTCCC CTGGTTCCCA

2) INFORMATION ON SEQUENCE WITH IDENTIFICATION NUMBER 23: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4499 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULA TYPE: DNA (genomic) (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vii) DIRECT SOURCE :

(B) CLONE: with pomc-th-dbh (ix) fusion gene

(ix) (ix) (ix) (ix) (ix) (ix) (A) NAME / BRAND: 5'UTR (B) POSITION: 1..43 BRAND:

(A) NAME / BRAND: exon (B) POSITION: 44..89 BRAND:

(A) NAME / BRAND: intron (B) POSITION: 90..168 BRAND:

(A) NAME / BRAND: exon (B) LOCATION: 169..482 BRAND:

(A) NAME / BRAND: intron (B) LOCATION: 483..1080 BRAND:

(A) NAME / BRAND: exon (B) LOCATION: 1081..2091 BRAND:

(A) NAME / BRAND: intron (B) LOCATION: 2092..2691

SIGN:

(A) NAME / BRAND: exon (B) LOCATION: 2692..4485 (ix) (ix)

(A) NAME / BRAND: 3'UTR (B) POSITION: 4486..4499 (xi) SEQUENCE DESCRIPTION: IDENTIFICATION NO. SEQUENCE: 23:

— Í Í ^L l ¹ C ¹ CľZZZZZG GGľlCrS. 240ΕΖΏΞΞΞλ u .'— 240 i; t 'ϊ · .-::: ti d 11 i 111. G ^ GSZHrtl · * - jCO

GZZGZGJľ GZHZ2S2 JAíRríIFd ΑΞΞΖΖΣ2Ά CSS3GSG GCľlCdľ 253

1.7 .-. ¾ (jj.J.-rjrLďC Jfta.xa.i.ŕCj GZ22ľľId S-Iu-IG 5ESís_i_rS. 420 ^ 22ZdET221 SG3Z32HC ÄrrrfCjjZA ILrtILfŕSA G22Z3Cnrsj ffUáaújrGT 480 grarasG dwzzzz: iczzczz: azzzzsAzs ^ .2 122 022 222 22222222 540 gtakoo < 2222222212 ίεςεεξζπ ramiu-g gzsozz <222212222 soo

AAíLjÍíjí2La3 <2222? ÀC2 2222222222 ΓΙΙΙιγϊΙλ GFIľGZŕu (3333222222 oOO

0222222237 Ä33A2237 # 322233333 ÍK332222S. # Ξ3 # Ξ3 · .23 3233Z3327A 720

G22IC2IGÄ GdÄdVC (7102237222 ÄÍÚTiL-λ <2237233: # # CXC37O2 780 <3323731333 G222222222 QJirrftjU-G G333I? ĹEPG # 37302332 -i -i ·; -í-840 Σ22 (2? 333333Ξ? # & 21UWí22 900

AIiIhí'íla = £ 3 # 332223 GÄ3272222 <2 = £ = Α233-2 C23732332 223 ^ 3223 ^ 3 950

C222222222 (223237333 CTEGSGl (332372223 # GZTSàKSA C22Z372232 1020 axn =? M ¢ 2232272222 2221222333 gwspctg:? 33 # 3 # 23 igzpcäz ioso

AJ.i7i \ .í_lu1 (33ITC2 ^? G? Fr? 222222 GĹIIC33ijijijG33IC7237 Q22221CC2 1140 ffltjxXlUrCZ ClLrlCil *. C2332rC2 ?72 U xiňa-i'm 'U-LriUlaii2i?

G21C23 # C32 333IE37?>. Si'liái.rii. (3i7ir73:? 722 .PC333EPC2 ^ Ax'íujI ^ Z 1260

GIliŤÄIECÄ CrtjJájSS. <332223722 IG3H33G2 UTlÄilälGC G223-r72222 1220

ClUirtIGilL · ³ . G2Ĺ3322222 uJjj-ťíaj-C C233O2233 IC2? G2I223 GSSúLisirC

IS0

TG3333ZA30 ΕλΟλλ: GlCÚoCu CíixuSZj 7G0033330T user; 1440

^.λ.-1-l · Ju · ^ 10 ^ 003 (333 A-i; r G333Z3333O3) »íj íj G G G 30G333 15 = G? Z = 333 G332G3333 <33303303 1560 nt * i.-irdb Ο-τ-ι-τΡΙτΟ CibiiLuTC Al'Ji.-S_C_-3 G3333333FC ISO

G33CA3T33 033033000 <j —- ujCAIT (33033333 0333333333 CIC3SIEA 1630 fIGSIGSSA AA — ICILI —- C (333330333 T33O33333 JAl'li ii-t, ¹ ' ¹ £ ui? £ 3O 1740'i 0333303'fS 0333303'i) “- 3333303033 C33G333O33 ullA,-t * - (333003300 1800

TtXĽlbJCTO ÄiàiijJiLr ·. G33333? O23 ľlii3-tj_i_ “G ££ 3 ^ O_àOG lGioĹ3iil— 1860

G3333333C 3333333003 £ ffO33IC £ A 1 (j-0gJ1 ££ O 1920)

GCffiCG <33 ^ 03330 033333000 COOOjOOOtI 3333333ΞΑ (331330333 1980)

3700033 O333ffi33ľ Α3333ΏΞ3 CTDOWi 033033333 (333330333 2040

G33C3G3rĹD3 Äj-itx / ĹSC (030333330 GÄJlGiíaKa 33Α3Ι3Ο3Ί3 · - AS33GK3O3 2100

ÄRTm'i lt cioľGro cooooospc goizoojll: θ ^ οοοτογο gsstopoo 2160

CO3IG3ZI33 3IG333O33 C333AT33IO3 Α33ΙΚΓ333 0130317333 C = £ 333? O3 2220 (i i.i iaj ^ PSC C333333333 (3003333-03) G333033CIC C333030330 2280 £ £33 333333333333333333333

Α3ΟΓΓΟΑ. (3333333303 «ΰΟΟΟΟΓΰΟ £ 033033« ΑΊΓΟΤΤΟΟ 33333OG3 2400

0333303333 answGz: xsľGuroA «κοοοιο« «333333 äskeccg 2460

3333A33ľ33 TCGTIGSZ Α3Γ33333ΡΑ AGGICTAI 0333330300 JWZGEJlC 2520 ££ «£ 03330 TGÄJSUil C7ffl £ G3IA azmriGIA TGSOCBS. ^ ------ ^ FT 2580

033333ΏΙ G0T3UG133 33ΓΠΡΟΙΌΞ £ Ο3Π ££££ Α £ 03333030 (ΦΟΖΠΣΕΦΟΖ 2640 £ 03333033.G ľ l ľ 33 33 33 £ £ T 333333Ο0 T0330A3 £ C 33 3330330 2700 £ 0333333333333333333333333333333 CTC3033S.G333333333 2820 / • ΑΑΊι Α-ί, ¹ Alt ·· it ENG.ffLL.-iLi_.iiC TľCffGľľC TGSIGSjjff. GOILAjj—

Fusion-f-i-fiff. .AZGľffCľľ GZZCCZGZľC

TGffdffCÄ C4 If - - i i -i ~ CffCľTľGjj ffCGľľľTGff. GZffCffffA jjl-Ci / L AjÍjIC ffCZZGffC GffCffGff. TffCffdlT Llti iá.iaffC AffCffClCO £ ff £ kuJLiL ·

TffftffGZE TTTIGSffCl IGIffCEZA. ÄľffCffCľT CCOffGffC

GSffGľjjľQZ AjLíuJíľiA Itinl'iUJÍb ffGffCSľC ID3G3ICG3ľ GffCIGfflC

AfCÄffTZE CTGffffC Gimt53G ÄZZHGffGl TCTffAZC ffCTCZi:

ÍAZCG3ZC IGZnZGff ffCZlTTCZ ÄIGfffflCľ GZZnXDff CffGľffCľ

CD.T-. I G ľ ff C C C C C ZA C C C C C ľ ľ ľ Gl 3 3 3 3 3 3 3 3 3 3 3 3 3

ÄiGffsncr wciffs: echo ffffcffia: ancncc ctzuhc ffCIQffPffi. lla-ftjjjj-A. GĽjjZIffrC TTCUZUjIC AjJIUJÍLu. Uj_ĽIGjJ-L□□ειτπα Äjjx'ľra. CTcaffffc © αξαξζζ igzcticzs Gszomz lOHCIffffC TICICZZz GffAClffC TCffffàCO ffCICTffC A = ffú! ZC33

CGľffdCľT (ίί-i.t AíllL CClGZPCffC ΊΠ333332Γ? GffdG3G33

AILILIA-GI LI.I.I.> GIffffCGOuO GTffuCSjffi H.I.I.I. GffffΟ333Σ ticgioľíiä anrffcro ffcsffffàc ίεςαπτο: ighctzt 033010033

ÄTiaCSICr TDZľlCD. OTXTOCG ffC01ffC33 33333 ^ 300 GOIffiOSGIG

CIG333A333 ΑΙιΏ 1.4¾¾ ffffffffOC GIffňffG33 AffàCffCIA C3333¾ C7C imffffi 103300017 fffSàGOIC GIGICILÍO3 A33333ffff. 031001000

ÄZicmzA ffmffscc Gfffffcxss αζπεξςά gczgzzh; 00 = 0010333

CIOffGffíff. IGUSlsIlA CffOGIGffC TCDCOOOO ΑΞΏΞΤΰΖΓ GffGľlCIGO

JgffCTnT, 1GffG33IG3 CIGCOIGOC ΑΏΑΟΠΟΟ GZOOOIffA ffGSTOSC

2380

2940

3000

3060

3120

3180

3240

3300

3360

3420

3480

3540

3600

3660

3720

3780

3840

3900

3960

4020

4080

4140

4200

Aiii.TXajň3 iCiti-n ľG C ľľGG 02.

4260

TGZÄZCZT TGSdZSi GZGZCŕu (ΖΠΖΠΞΤΟΞ GľľľCGľľCZ CSZ3--2G 4220 CTCH33SCÄ. GSICZZCjjZ GSIC222ZT2 GGjjGSGT (ΧτΑΙΙϋΞΆ G2222ZGG2ľ 4j80 GSZGHGľ GDAZľľľGGi ASjZZTCG GZICOZZ (4440 3ιΖΣλΖΖ ?. UuJilXFuZ π-ιητ; Ί · Γ 'CCYZGľGľľ GÄSTCGľ GGGZxASfG GDICSfCGIG G222S Z3ľ-4459 (2) INFORMATION 0 SEQUENCE WITH IDENTIFICATION NUMBER 24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULA TYPE: cDNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vii) DIRECT SOURCE:

(B) CLONE: o-IRES-074 (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION NO. SEQUENCE: 24

AAAGCGGCCG CCCCTCTCCC TCCCCCCCCC (2) INFORMATION ON SEQUENCE WITH IDENTIFICATION NUMBER 25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULA TYPE: cDNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vii) DIRECT SOURCE:

(B) CLONE: oZeocin-077 (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION NO. SEQUENCE:

AAACTCGAGT CAGTCCTGCT CCTCGGCCAC (2) INFORMATION ON SEQUENCE WITH IDENTIFICATION NUMBER 26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULA TYPE: cDNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vii) DIRECT SOURCE:

(B) CLONE: 0IRES-Zeocin-075 (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION NO. SEQUENCE: 26

GGTCAACTTG GCCATGGTTG TGGCAAGCTT (2) INFORMATION ON SEQUENCE IDENTIFICATION NUMBER 27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULA TYPE: cDNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vii) DIRECT SOURCE:

(B) CLONE: o-IRES-rTHD-076 (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION NO. SEQUENCE: 27:

CTTGCCACAA CCATGGCCAA GTTGACCAGT

2) INFORMATION ON SEQUENCE WITH IDENTIFICATION NUMBER 28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5540 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (genomic) (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vii) DIRECT SOURCE :

(B) CLONE: POMCDACTH-IRES-THD — IRES-DBH-IRES-Zeocin (ix)

(A) NAME / BRAND: 5'UTR (B) POSITION: 1..118 (ix) BRAND:

(A) NAME / BRAND: exon (B) LOCATION: 119..164 (ix) BRAND:

(A) NAME / BRAND: intron (B) POSITION: 165..243 (ix) BRAND:

(A) NAME / BRAND: exon (B) LOCATION: 244..557 (Ix) SIGN: (A) NAME / BRAND: intron (B) LOCATION: 558..1155 (Ix) SIGN: (A) NAME / BRAND: exon (B) LOCATION: 1156..2166 (Ix) SIGN: (A) NAME / BRAND: intron (B) LOCATION: 2167..2766 (Ix) SIGN: (A) NAME / BRAND: exon (B) LOCATION: 2767..4560 (Ix) SIGN: (A) NAME / BRAND: intron (B) LOCATION: 4561..5159 (Ix) SIGN: (A) NAME / BRAND: exon (B) LOCATION: 5160..5534 (Ix) SIGN: (A) NAME / BRAND: 3'UTR (B) LOCATION: 5535..5540 (Xi) SEQUENCE DESCRIPTION: IDENTIFICATION NO. SEQUENCE

PfGľľGCTA (ZSGZGSS ÄICľAľUGr Ä3IG23ZIG3 AKTICISTG 60

ALWILUÄLU (2CIUC-jj ~ OjJjICAíI. CÍCTLLGjj IGľllADVxľ 120

GAAICZPGľ ibjjľlíSľľ xLilU-ikjiľ GZTGIGZľAAnnCHňACC G3ZIO3ľ? 1803 180 lOSAPàCľT (SÍwSaÄT ÄSčCICIGIG JOGIG33A TGYľľlGľZ ΤΠΟΤΣΊΠΆ 240

CSG33HCÄ Timrmc 11 ktetiu anajcrr cigcigzľ 300

A333CTOJS GrGZ4FlT t-U 1111 ^ 3 GZnSXSi IglHT1-1JA. O23j3Z? GC 360

033QZIGÄ G3QGSZIG QGjIGjZGj Q33GS®A G50jG333 11- 420

Τ33Ώ3ΟΤ UJCTĽGX Α3ΖΠ3ΊΣΑ Ä33OW333 070321321 TIOTCCLT 480 dSG ^ GrG GTffGLL CIGZCCjZ IGLlCWàA

A3à®SG33 GTGTCTGSS GOtTRIT CU1IC1QJ2I αΤΠΊΠΤΤ ΊΑ ^ ΣΠΚΞΓ 600

Citi AL. GZHG3ÍS. ΞΞΖ ΞΤ Γ ľ ľ 660 660 660 660 660 660 660 660 660 660 660 660 660 660 660 660 660 660 660 660 660 660 660 660 · · · · · · · · · · · · · · · · - ~ j ~ i ~ 11; {..! ' G λ ľABňjjA AlLLAruJlu ΊΕΓΣΕΡΣ <2Γ GZZ ^ rGjà. i o C

G3GTICZZC IGSPGZZiC x’IWSGCA AZ? «_ XjÍ2 íj Ľ + + ľ ΐΞ ^ χ-Κϋ- · ΐΞ S40

GjjAjjjj- QlLLLuZZA GGSIGZľlU ΞΞ2ΞΞ33? Α? G3Z? CjIGT.-SrG-CAZ-- SCO ozzíhäs cľr-c + c ctgigztc Gra? ST G ^ msncr Gsspffizc seo

APnlGZlCr aZG ¥ £ ÄlDÄHC GZZHTAZ ÄIG2Z7GÄ CTCH ^ 1020 'iLsxÄiliia-T Clúnitlltl-G GZZICjjIGZ Ä_Äita — ΙΤΙΑ CAib'Uji'i'xÄ (ILUjrúull

1140.AJ.IJJ.iiiτ žBlUĽItíh A.->> x1 x1 x1 x1 x1 x1 11 ¹¹ 40 40 40 40 1140

AFtjJl'HÁJUA CSSCZSIGjT G222IG2ITC CĽArfflftòPG ‘IGICjSSZT (IxíTäjíuI 1200 σαταπΕ icmøcľ 73 = cnc? I brick chg3 jmams cthchtc 1260

CYSIGEIC Q23G3ZG3 SÄZIGtľ G3GSHG GľKlTGIA CMZ? G22ľ 1320

SPCCÄnC CDZ = 3ZGGk ÄIÄ7OGľ3 (PŕSSTIG dHZIGPA GSGZÄKT 13Θ0

GK3CEHGIΑ23ΖΖ7ΣΆ 1CTCCT G22ItmS2 Ä33CT3Ä G33TICCC 1440

CHOGCPPC. GjíJj'UÍU- 1500

GGlTlL'ľiW. XSGTHTC 1G2 ľ IQ7G ZZ? C C2S U2S TGUHľXľT äHGIUGáí. 1560

CXiXTiC TI · t · '^; Gι '!: ^{| L} G2221T2222

GXILĽIUC CTĽĽATX ÄZH3GZC GkľlGľKDl ÄHSGZIGTT G2? C? 2GIA 1680 aCTGTIGZ CIGC2ZľC ÄTTEZUTG TICHmE ÍCOSCT 'lliAZdOG 1740

G3ZmC? G ÄIGWSMT IGSSPAPCIC 1Q? C2SIGl JOGZICrC IGICCPAlC 1800 (μαΠΚΠΞΙΆ ÄOSKIGS G3GZISPG CLT1ÄIUJ1G CTtiájJlUJr GICIICZUC 1860

CTGSGZIQZ T33CIG2ZT GP3SG5G CTTGZCZ GW2IT1LA 0X? FfĹ3CA 1920

G3GZGIG2 Ä3ZC3CA APTOPAZ TSCDSnG CTCTOTIGT CTIffGG: 1980

TIOKOCu OTàGSGA (3'K3G3CC TSIGZZCIC G72O3G33 ΟΧΚΓΟΖΓ 2040

CTSSdTIG S ľ--i · at (((g g g g g g g g 2

Cii JlL.'I. Crt-Ujljjj CJL-lj-tCT

ÄZSàÄIGZ ΑΏΞΞΕΟΖ (llT'iĽLUJL. TC2ZHZ2Z C27VC2ZgC TGZnSňZľ

GjZHGSäT äGjZZjjIG IG-GUTUjIC (KÄibx'iftT ITIC ^ CZŕZ AZIGCZjICľ

X'X'ltxiJflIIj ILt-t λ-. i. ί I ät GrrAj — ILu.- GdGZďľCT ΊΐΛχχϋΑΙ aLĽZAííjj.

CmLLUJlL HNnTSAS £ MGgÄ33ľ OGľiGÄÍG IGSTGAZÄ ÄZgSTZľľ

C2G3SGZIT øGÄĽICľ G27G2? C32 TľľTGZA G3Gj? * OZZ

CCCZUiu A3GGH3XT CTZKa. 1Ά Aft333CZG EOMffiI = C Α2ΖΠ33 ¥ «3

Q3333OSC G2ZA2'G23k GG'lGTGCT IGSHTGTxG lG3rG £ T GSMGZZC

IUUilAŕGZ TCTCÄTA GGGG1'GSrG CTGZITffl. ΑΞΖΚΣΠΑ THľglGGGt

IľlWiUli, GZrZOZHG σΟΟΖΣΤΓ A33GIGm ΑΠΌλμΓΓ Ä ^ CGIC

ΊΆΤΠΤ1Ί!. ' G ¥ CTC33G SCGIGGITľ TXTTnSfA AATQAIIP. TľítľľľlGC:

Ä3Aj_AíIjá 'AJJaAXíJC GGÄGGGOGaG ICGZOGIGGG IGZàCÄGgG

Q3ZiQiĽi iraTnsffG GzzľiQZĽ TľUT-ccu: cxaz c? Cgi? C:

CIG3GZIGT C18GJAĹTATE CGlgJGL, GSTGKZA. TľXľTIUľk GľľCCIGGTG

Clx.X J-Li-XLA. A.i.3. .iiäjAijA U_Hji'I'iijjj ÄÁ1ja \ jjjCZ ffÁ ii Ť Í Í-T Gg £? KGZľ

GCTIGGÄGG IG2ÄCIG3C 1GOGH? C GZSZxzCT ΊΤ3ΞΞΏ32 CTSSGICC

C3ffiK3333Z ΑΞΗϋΞΏΖΓ G3CIOI7G CTGGtľUQľ AjJľlUiljuG GZTCSffGG

JO23GÄ3 GXICTClr G3C2IGx®. aZCWOSC imcts and ^ cht-c azasrac gigstost txigsgs. ollľ-mľs itmGSGT ctoätc äictztig ogrcnrr tztgszz czsg ^ shg ίΑ IΑΖΑ ICHOTCC ΠΤΠΤΠΧΙΧ GGGjQCGG

Cl l.tjrUjiLU. ILnlQ.1,1, l, ii ul-fiigffGZ AjjlA-lLuT GlffQGIGil Q3G2ID33G

CT33ZKZA GQQHC7-IG ΈΏΞΏΖΕΑ 'RĽICAZXA G333ÄZ5G

OC

2160

2220

2280

2340

2400

2460

2520

2530

2640

2700

2760

2Θ20

28Θ0

2940

3000

3060

3120

3180

3240

3300

3360

3420

3480

-L T.

ÄJTAJATjjI (333377130 Ii.xiS.i.ij.J. ^ .3

L.I. ιΊΊΊ LC LC LC-C7r0n73? G XaO-äJjX.

äotx

C3303O333

733073303 aOXj Lai — 30333 77730003 <0300?

2540

3600

3650

Til i - »- i4ň4 ·» '0333333373 (03037333 Ι.ί.τ. ϊ,.' ΙΙϊτγΟ i'ÍLàCi / iULÄ OPÚLOUTu

OitóHMCG C333333333 Alt. in. 1.101 'A030-0330' Lily 'RT 14.-1 uj'1 (j303 02333170-0 Ua (i.íx7äA-A 033333730 i44 13711 11)

03330313 cGzmGSľ crzccciz: ΊΑ3733-33 ät 03 03 = 0273322 3 J 3 3J-T <33331373

ASG373373A (307337333 (3G33G333 G333SGO) AS7337 ^ A C7G33GrC ¢ 002 = 0 = 033 οώζγτςοα Gxffzmz ατττοαξι A337337373 733770333

G3A3O3733 731730333 73330130 ÄOG3SSG # 3030373 (333303373 (-3.T ..- Í3.aJľiUj (jjåsAIUIuj U1l.1Sc.i.-03 7XÄ2L4LXA ÚXU3SG3))

3720

3780

3840

3900

3960

4020

4080

4140

4200

4260 (alla-iSCAjj ΊΟττΟΑΧίτ-. G3ŕO73333 SO37333333 AáäJjTvlUi 'U — ilAj — O 4j20

Ti aOJ-Tujj TIijjJT03A C333733PC G333G3332 KAAXijjJI (□ xpOSSOTTu 4380

OTOZÄílL G31733O3 OSÍ3G3333 7333033102 037333023 03GIG3S3 4440

O332A33222 73337337 O373733O3 7733A3O2 03033731 07333303 4500

A333A3Z73 A3O332232 033337703 G73373A3 73 = 0737373 C3SA23-33A 4560

A337373333 0333333733 003373.1 .: 033ľ3A33ľ 30333333. ÄJXJX.7733 4620

ÄSZASG3333 (3737333177 GT10-TrTCT OTITIILSC (33303333 'I777773331 4680

Ä7373O332 G373fA33T G33337372T 73173030 (37733303 (3373IT7333 4740)

C7223373A A33Ä773A 0373731731ÄO3373A G3A33O7T 03ICT33PG. 4800

O773773 = A3 AOSAOA23 737330331 0X717330 (3303330 03333 = 0233 4860

AftjJLLrfjlkj (J —'iiii.’iUiíJ iiUiluArG rGZCPArJGO Ciulw - '. ICA 4980

C ji U i L L C C C A Z Z Z IG IG IG 2 2 2 2 2 ilt ilt j j j (j

GmArrArC GTZ3GG22Z 5100

C 3 C 3 C 3 - C 3 C 3. TGGruľT GZ7CÄZA 5150

It.iá.tJApi'i 'SJCAj'ÍG-- (1 í í í-::::): ICA-UálaJj

TC CTCZn? £ IGHCSGZ

KľKnC GZSGZSGľ. CIG0GIGZ7C ¾C GjjHCIOOO GjjíľnCľľ GjvSGSD Π0

Itiiii í lájji CdLxCrOZ CIGlOmCt GUjOmOZA. GSrGGIGGIT.iJ..i.i; SCA

AC30OZIGjZ CIGSjIGIGj iqzgíulä: ggczmnG

CljjjjiiiijjjjjjjjCCl.

A33G3G31 CT5CICSG

5280

5340

5400

5460

5520

5540

2) INFORMATION ON SEQUENCE WITH IDENTIFICATION NUMBER 29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 829 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULA TYPE: DNA (genomic) (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vii) DIRECT SOURCE :

(B) CLONE: ProAKS (ix)

(A) NAME / BRAND: 5'UTR (B) POSITION: 1..16 (ix) BRAND:

(A) NAME / BRAND: exon (B) LOCATION: 17..820 (ix) BRAND:

(A) NAME / BRAND: 3'UTR (B) POSITION: 821..829 (xi) SEQUENCE DESCRIPTION: IDENTIFICATION NO. SEQUENCE: 29:

G03PGHTC GXPC ^ IGj U.-I GrfZPClTlGlAlGjZIGZ TGTZGZICjj 60

Ci i ii, -i aIT t. ^J IT LT Tíí.í..í.5.íXíjť I4. ATGiAj_i_Aj iTi'HÁTiiri OJ1110-ii-iA 120

O23ZZSGIG (πζππ, ΊΣΐ íGICÄZľ CLTUrnUJ CľEAZGZrAT GľGVGSTA ISO

ÍCHZZncr CGWÄľľ GSPPPGľľG CTGSZľZC CTZTGľlGT CC ^ VCTGk 240

Gľľ1GľKľPA CTGZ7G3. GTGZZOG PGAPAECO TransS3 SSKSAll 300

GZEGCTA PGZKIG323 GUSILA PPGZATOÄ (JXTIUO®. ÄSPPHnS. 360

IGUJTlXT CXAIGEG: GffiffPÄ G3X? PIG3i ASIS32n: IGSEÄHS 420

GIKIGSHZ: TICKIGA. ASE ^ UľrS. G2GZCZC TCZnmZA ATTOZICGC 480

CT33SWA ΘΞΣΊΕΤΞΞ ŕSGSSR C = A22? FfG GSPG ľľľ 3333G52G3 540

CTGľSZTAT GGGVSAO TGAZPPffG AKHSS.T € 32G3 CTTAPff 600 ffi CĽGSVYTSS PA223APÄ GľZ ^ ffÄS CTSKGZS GnnCAIGAS 660

PPGCTGST azroscol GH13? TC3l CĽPGEGVA CSSTSGSS Gmcngft. 720 aiXiLWJĽ GSGZlOGO CTZffiCÄ Ä5PG3ZPA PGTEOTA PPffPGlOO 780

TCMKDZEÄ PPPPSITCj ffiSTSTIST GG-ľľľťA GSGQjjjj

829

2) INFORMATION ON SEQUENCE WITH IDENTIFICATION NUMBER 30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 598 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) MOLECULA TYPE: DNA (genomic) (iii) HYPOTHETICAL: no

(Iv) ANTI-SENSE: no (Vii) DIRECT SOURCE: (B) CLOSE: IRES sequence (Ix) SIGN: (A) NAME / BRAND: intron (B) LOCATION: 1..598

(xi) SEQUENCE DESCRIPTION: IDENTIFICATION NO. SEQUENCES:

Garaiuu cnrairac crnxmrr τταοπτοτζ G33370333 ctcoaoa ω

G333233232 CĽiTiĽIĽJA 33733ΠΚΠΤ TZAZAKT 321032317 TG31772G32 120 'Α3Ξ333323. 7702311332 IdCnCTIG ÄffiZOTIC 070332323 3333323232 180

G2377G3A IGOftiiiT GT33A3232 G357GG-7G 33002303 (3277022331 240

35 «27Ο77Α <2770232323 702303217 332703003 G2 = 7O33U2 702322330 300

7Q23332333 O33332777A G33G333IA T77333C 33777023 G327C77O33 360

3331702 733330332 33033232 3773033 7733333233 C3237O33L7 420

T3373702 G33337G3. 32333373 & 7,332ΑΓΓ <33333333 333233323 480

033332100. 333233170 73232ΓΠ73 Τ23Ο2Π7Α 7777Ο333ΣΆ (333111110 540

A2OG333C7 Ο3333ΓΓΓ33 C3333777A 3030337 A3333333O 77032333

598

Claims (23)

PATENT CLAIMS A cell stably transformed to produce at least one compound having analgesic effect from each of the groups comprising endorphins, enkephalins and catecholamines. The cell of claim 1, wherein the endorphin is β-endorphin. The cell of claim 1, wherein the enkephalin is met-enkephalin. 4. Cell by of claim 1, wherein the catecholamine is norepinephrine or epinephrine. 5 . cell by any of the claims 1 to 4, which is cell RIN. 6 . cell by any of the claims 1 to 4, which is cell AtT-20th 7 . cell by any one of claims 1 to 6, which in addition produces a compound selected from the group consisting of galanin, somatostatin, neuropeptide Y, neurotensin or cholecystokinin. A cell transformed with POMC-encoding DNA, TH-encoding DNA, DBH-encoding DNA, and ProA-encoding DNA, wherein each DNA molecule is operably linked to an expression control sequence. The cell of claim 8, wherein pCEP4-POMC-030, pcDNA3-hproA + KS-091 and -bDBH-088. is transformed with pZeo-pCMV-rTHAKS-IRES89 10. Cell by claim 8, which is a transformovaná s pCEP4-hPOMC-AACTH-032, pBS-CMV-proA and pZeo-pCMV-rTHÁKS- -IRES-bDBH-088th 11. Cell by claim 8, which is a transformovaná s pcDNA3-hPOMCDACTH-IRES-rTHD-IRES-bDBH-IRES-Zeoc.n-073 and pcDNA3proA + KS-091. 12. A transformed cell producing at least one enkephalin, one endorphin and one catecholamine that is transformed with: a first vector comprising DNA encoding POMC operably linked to an expression control sequence, a second vector comprising DNA encoding pro-enkephalin A operably linked to an expression control sequence, a third vector containing DNA encoding TH operably linked to an expression control sequence, and DNA encoding dopamine beta-hydroxydlase operably linked control expression. A method of treating pain comprising implanting a therapeutically effective amount of cells according to any one of claims 1 to 12 into a patient at the implantation site. 14. The method of claim 13, wherein the cells are encapsulated in a semipermeable membrane to form a biological artificial organ. 15. The method of claim 14, wherein the biological artificial organ is immunologically isolating. Method according to any one of claims 13 to 15, characterized in that the implantation site is a CNS. Method according to any one of claims 13 to 15, characterized in that the implantation site is a subarachnoid space. 18. A method of producing a cell that secretes at least one enkephalin, one endorphine and one catecholamine, comprising transforming a cell with DNA encoding POMC operably linked to a first expression control sequence, with DNA encoding pro-enkephalin A operably linked to a second sequence expression control and DNA encoding TH operably linked to a third expression control sequence and DNA encoding dopamine beta-hydroxylase operably linked to a fourth expression control sequence. 19. The method of claim 18, wherein the first, second, third, and fourth expression control sequences are identical. Use of cells according to any one of claims 1 to 12 for the preparation of a medicament for the treatment of pain. The cells of claim 20, wherein the cells are implanted. Cells according to any one of claims 21 and 22 which are encapsulated in a semipermeable membrane to form an artificial biological organ. Cells according to claim 22, for generating an artificial biological organ that is immunologically isolating. The cells of any one of claims 21 to 23, wherein the implant site is the CNS. The cells of any one of claims 21 to 23, wherein the implant site is a sub-arachnoid space. 26. An artificial biological organ comprising: (a) a biocompatible, permeable sheath surrounding the core; and (b) a core comprising at least one living cell transformed to produce at least one compound having an analgesic effect from each of the groups comprising endorphins, enkephalins, and catecholamines. The artificial biological organ of claim 26 for use in treating pain. 28. A method of forming an artificial biological organ, comprising encapsulating a nucleus comprising at least one living cell transformed to produce at least one compound having an analgesic effect from each of the group consisting of endorphins, enkephalins and catecholamines with a biocompatible permeable sheath. Use of an artificial biological organ comprising cells according to claims 1 to 12 for the preparation of a medicament for the treatment of pain.