EP0833935A1 - 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

Cell line producing analgesic compounds for treating pain

Field of the Invention

The present invention relates to a cell line useful for the treatment of pain. More particularly, the cell line of this invention has been genetically engineered to produce at least one analgesic compound from each of the groups consisting of endorphins, enkephalins, and catecholamines.

Background of the Invention

Pain is a common symptom of disease. The superficial dorsal horn of the spinal cord, where primary afferent fibers carrying nociceptive information terminate, contains enkephalinergic interneurons and high densities of opiate receptors. In addition, there is a dense concentration of noradrenergic fibers in the superficial laminae of the spinal cord.

Acute pain arises in response to acute noxious stimuli. Chronic pain is predominantly due to neuropathies of central or peripheral origin. This neuropathic pain is the result of aberrant somatosensory processing that can result in increased sensitivity to a painful stimulus (hyperalgesia) and pain associated with a stimulus that does not usually provoke pain (allodynia) .

Intrathecal injection of morphine into the spinal subarachnoid space produces potent analgesia. Similarly, intrathecal administration of norepinephrine or noradrenergic agonists also produces analgesia. See, e.g., Sagen et al., Proc. Natl. Acad. Sci. USA. 83, pp. 7522-26 (1986) .

Co-administration of subeffective doses of opiates, such as enkephalins, and catecholamines, such as norepinephrine, may synergize to produce analgesia. Ibid. Chromaffin cells in the adrenal medulla produce and release several neuroactive substances including norepinephrine, epinephrine, met-enkephalin, leu- enkephalin, neuropeptide Y, vasoactive intestinal polypeptide, so atostatin, neurotensin, cholecystokinin and calcitonin gene-related peptide. See, e.g., Sagen et al., Proc. Natl. Acad. Sci. USA, 83, pp. 7522-26 (1986); Sagen et al., Jour. Neurochem., 56, pp. 623-27 (1991) .

Because chromaffin cells produce both opioid peptides and catecholamines, one approach to reduction of nociceptive response or pain sensitivity has investigated transplanting adrenal medullary tissue, as well as isolated adrenal chromaffin cells, directly into CNS pain modulatory regions, in attempts to provide analgesia. See, e.g., Sagen et al., Brain Research. 384, pp. 189-94 (1986); Vaguero et al., Neuroreport. 2, pp. 149-51 (1991); Ginzberg and

SUBSTrrUTE SHEET RULE26 Seltzer, Brain Research. 523, pp. 147-50 (1990); Sagen et al., Eain, 42. pp. 69-79 (1990).

Attempts to produce analgesic have been made using both allogeneic and xenogeneic chromaffin tissue or cells transplants. Allograft tissue is in limited supply, and is not readily available, particularly for in human pain treatment programs. In addition, allogeneic human tissue carries the risk of pathogenic contamination. .See e.g., Hama and Sagen, Brain Research. 651, pp. 183-93 (1994) .

Xenogeneic donors may provide large quantities of material that can be readily obtained. For this reason, bovine adrenal tissue has been used. See, e.g., Hama and Sagen, Brajn Research, 651, pp. 183-93 (1994) .

However, potentially serious host consequences, as well as ultimate graft rejection, are inherent problems in transplantation between disparate species. Complete graft rejection of whole or dissociated tissue may occur even in the CNS, normally thought to be immunologically privileged, due to presence of highly antigenic cells in the xenografts, particularly endothelial cells. In addition, the donor tissue must be carefully screened to avoid introduction of viral contaminants, or other pathogens, to the host. To overcome graft rejection, immunosuppression is required typically using cyclosporine A.

Some reduction in pain sensitivity has been reported resulting from these transplants, particularly for the reduction of low intensity chronic pain. In most reports, significant differences between control and transplanted animals were noted only after nicotine

SUBSTITUT administration to stimulate opioid peptide production. However, there have been some reports that analgesia has been observed in a rat chronic pain model from basal level activity of chromaffin tissue allografts. See, e.g., Vaquero et al., NeuroReport. 2 , pp. 149-51 (1991) and Hama and Sagen, Brain Research. 651, pp. 183-93 (1994) .

Bovine adrenal chromaffin cells have been encapsulated to form a bioartificial organ ("BAO") for implantation into rats for the- treatment of acute and chronic pain. See, e.g., Sagen et al., J. Neurosci.. 13, pp. 2415-23 (1993) and Hama et al. , 7th World Congress Pain. Abstract 982, Paris France (1993) . Initial trials in human subject have been conducted using encapsulated bovine chromaffin cells. See, Aebischer et al.. Transplantation. 58, pp. 1275-77 (1994) .

There have also been attempts to induce antinociception using other cells, e.g., AtT-20 cells. AtT-20 cells were originally derived from a mouse anterior pituitary tumor. These cells synthesize and secrete β-endorphin. See, e.g., Wu et al., J. Neural Transol. & Plasticity. 5, pp. 15-26 (1993). AtT-20/hENK cells are AtT-20 cells that have been genetically engineered to carry the entire human pro- enkephalin A gene (i.e. containing 6 met-enkephalin sequences and one leu-enkephalin sequence) with 200 bases of 5'-flanking sequence and 2.66 kilobases of 3'- flanking sequence. See Wu et al., supra. Comb et al. EMBO J.. 4, pp. 3115-22 (1985).

Wu et al., J. Neural Transpl. & Plasticity. 5, pp. 15-26 (1993) refers to rat hosts transplanted with AtT-20 or AtT-20/hENK cells. Unstimulated AtT- 20/hENK cells produced more antinociception (tail flick test) than produced by AtT-20 implants. In contrast, isoproterenol stimulation produced more antinociception with AtT-20 cells than with AtT-20/hENK cells. Ibid.

In mice hosts, AtT-20 or AtT-20/hENK implants did not affect basal response to thermal nociceptive stimuli. Mice receiving AtT-20 implants developed tolerance to β-endorphin and a μ-opioid agonist (DAMGO) . Mice receiving AtT-2Q/hENK implants developed tolerance to an δ-opioid agonist (DPDPE) . In response to repeated doses of an μ opiate agonist, mice receiving AtT-20/hENK implants developed less tolerance compared to mice receiving AtT-20 cells or controls. The antinociceptive effect of isoproterenol treatment appeared equal in mice receiving AtT-20 or AtT-20/hENK cell implants. See, Wu et al., J. Neuroscienπs. 14, pp. 4806-14 (1994) . Wu et al. speculated that one reason for the absence of additional antinociception in mice implanted with enkephalin producing AtT-20/hENK cells may be due to lack of sensitivity of the behavioral assays. Another possible reason was that met-enkephalin's known antagonist effect on morphine induced antinociception offset the potentiating effect of the single leu-enkephalin, particularly since there are 6 met- enkephalin sequences for each leu-enkephalin sequence in pro-enkephalin A.

RULE 26 Summary of the invention

The present invention provides a cell line that has been genetically engineered to produce at least one analgesic compound from each of the groups consisting of endorphins, enkephalins, and catecholamines. The cell line may be used in the treatment of pain.

There are advantages to using a cell line over the use of primary cells. Expensive and time consuming testing to ensure safety and performance criteria for cells must be performed for individual isolations of primary cells. Less testing is required . of a cell bank. There is no need to isolate primary cells. Output of the desired analgesics may be more stable since the performance of primary cells may be dependent on the age, sex, health or hormonal status of the donor animal. It is also possible to achieve higher output of the desired products, as well as to engineer specifically modified peptides into the cell line. This permits delivery of multiple analgesics simultaneously. Expression of one or more of the analgesics can be regulated (by using a regulatable promoter to drive expression) . In addition, for safety, a "suicide" gene can be incorporated into the cell line. Further, for encapsulation purposes proliferating cells have the advantage that they divide to replace dying or dead cells. Brief Description of the Drawing

Figure 1 is a plasmid map of vector pBS- hPOMC-027, pBS-IgSP-hPOMC-028 and pBS-IgSP-hPOMC-ΔACTH- 029. Figure 2 is a plasmid map of vectors pCEP4- hPOMC-030, pCEP4-hPOMC-031, pcDNA3-hPOMC-034 and pcDNA3-hPOMC-035.

Figure 3 is a plasmid map of vectors pCEP4- hPOMC-ΔACTH-032, pCEP4-hPOMC-ΔACTH-033, pcDNA3-hPOMO ΔACTH-36 and pcDNA3-hPOMC-ΔACTH-037.

Figure 4 is a plasmid map of vectors pcDNA3- rTH-044, pcDNA3-rTHΔ-045, and pcDNA3-rTHDKS-075 (also represented as pcDNA3-rTHΔKS-075) .

Figure 5 is a plasmid map of vectors pcDNA3- rTHΔ-IRES-bDBH-088 and pcDNA3-rTHΔKS-IRES-bDBH-076.

Figure 6 is a plasmid map of vector pZeo- Pcmv-rTHΔKS-IRES-bDBH-088.

Figure 7 is a plasmid map of vector pBS-Pcmv- rTHΔIRES-bDBH-067. Figure 8 is a plasmid map of vector pBS- hPOMC-ΔACTH-IRES-rTHΔIRES-bDBH-068.

Figure 9 is a plasmid map of vector pcDNA3- hPOMC-ΔACTH-IRES-rTHΔ-IRES-bDBH-069.

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

Figure 11 is a plasmid map of vector pcDNA3- hPOMC-ΔACTH-IRES-rTHΔ-IRES-bDBH-IRES-Zeocin-073.

Figure 12 is a plasmid map of vector pcDNA3- hPROA+KS-091. Detailed Dps ription of the Invention

In order that this invention may be more fully understood, the following detailed description is set forth. Any suitable cell may be transformed with the recombinant DNA molecules of this invention. Among the contemplated cells are chromaffin cells, including conditionally immortalized chromaffin cells such as those described in WO 96/02646, Neuro-2A, PC12, PC12a, SK-N-MC, AtT-20, and RIN cells including RINa and RINb. Preferably the cell has endogenous prohormone convertases and/or dopa decarboxylases .

SK-N-MC cells, a neuroepithelioma cell line, co-expresses several neuropeptides, including enkephalin, cholecystokinin and gastrin-releasing peptide. See, e.g., Verbeeck et al., J. Biol. Chem. , 265, pp. 18087-090 (1990) . The pro-enkephalin A gene has been expressed in SK-N-MC cells. See, e.g., Folkesson et al., Mol . Brain Res . , 3, pp. 147-54 (1988) . We prefer AtT-20 and RIN cells, most preferably RIN cells.

RIN cells are a pancreatic endocrine cell line derived from rat. See, e.g., Horellou et al., J. Phvsiol.. 85, pp. 158-70 (1991) . RIN cells are known to endogenously produce GABA and β-endorphin. Some of the characteristics of various contemplated cells are shown in Table 1. Table 1

Cells Analgesic Substances Other Components

Chromaffin NE, met-enkephalin TH, DDC, DβH, PC

PC12, PC12a low NE & met-enkephalin DDC, DβH, PC

AtT-20 β-endorphin DDC, PC

RINa β-endorphin, GABA DDC, PC

RINb β-endorphin DDC, PC

Neuro 2A DDC, DβH, PC

TH = Tyrosine hydroxy lase converts tyrosine - l-dopa DDC = Dopamine decarboxylase converts l-dopa - dopamine (DA) DβH = Dopamine β-Hydroxyiase converts DA - norepinephrine (NE) PC = Prohormone Convertases process POMC to β-endorphin and Pro- enkephalin A (ProA) to met-enkephalin.

AtT20 = Mouse pituitary corticotroph cell line that endogenously secretes β-endorphin via expression of Pro-opiomelanocortin (POMC).

RIN = Rat insulinoma Neuro 2A ^■• Mouse neuroblastoma

The primary delivery products include at least one each of an endorphin, an enkephalin and a catecholamine.

Enkephalins and endorphins are endogenous opioid peptides in humans. These opioid peptides comprise approximately 15 compounds ranging from 5 to 31 amino acids. These compounds bind to and act at least in part via the same μ opioid receptor as morphine, but are chemically unrelated to morphine. In addition, these compounds stimulate other opiate receptors. Yaksh and Malmberg, Textbook of Pain. 3rd Ed. (Eds. P. Wall and R. Melzack) , "Central Pharmacology of Nociceptive Transmission," pp. 165-200, 1994 (New York) .

The opioid peptides have common chemical properties, but are synthesized in different pathways.

SHEET 1'J E 26) β-endorphin, the most abundant endorphin, is synthesized as part of a larger precursor molecule, pro-opiomelanocortin ("POMC") . The POMC molecule contains the full sequence of adrenocorticotrophic hormone ("ACTH"), α-melanocyte-stimulating hormone

(" -MSH"), β-MSH, and β-lipotropin. The POMC precursor molecule also has the potential to generate other endorphins, including α-endorphin and gamma-endorphin. Processing of the POMC precursor occurs differently within various tissues according to the localization of cleavage enzymes, such as prohormone convertases, within those tissues.

In the pituitary, POMC is cleaved to produce ACTH and β-endorphin, and the ACTH is not further processed. In contrast, in the hypothalamus, ACTH is converted to β-MSH. While different cell types may synthesize the same primary gene product, the final profile of hormone secretion may differ widely.

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

SUBSTITUTE SHEET PLE26) Dayhoff in Atlas of Protein Sequence and Structure. 5, (1978) and by Argos, Embo J.. 3, pp. 779-85 (1989) .

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

This invention contemplates using a DNA sequence encoding the entire POMC precursor molecule, This embodiment takes advantage of the host cell's cleavage enzymes (i.e., Prohormone convertase 2) to - generate a suite of endorphins, some or all of which may have analgesic properties.

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

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

We prefer a DNA sequence encoding POMC in which the ACTH coding region has been deleted. The preferred endorphin encoded by this construct is β-endorphin. Some enkephalins are synthesized in the adrenal glands as part of a large protein, pro- enkephalin A, that 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 have significant antinociceptive activity. See, e.g., Sagen et al., Brain Res.. 502, pp. 1-10 (1989) . Other enkephalins, i.e., dynorphins and neo- endorphins are derived from a distinct molecule, pro- enkephalin B. Additional "cryptic" peptides are also encoded within the structure of these precursor proteins, and may be released by "pro-hormone-type" cleavage. See, e.g., Harrison's "Principles Of Internal Medicine", 12th Edition, pp. 1168-69 (1991) .

This invention contemplates use of a DNA sequence encoding any suitable enkephalin that has analgesic activity. Analogs and active fragments that have analgesic properties are also contemplated. Such analogs or fragments may thus have amino acid insertions, deletions, substitutions at one or more sites in the naturally occurring amino acid sequence. Such variants may be generated as described above. This invention contemplates use of a DNA sequence encoding a desired enkephalin in its "mature" form. In addition, this invention contemplates using a DNA sequence encoding the entire pro-enkephalin A precursor, or the entire pro-enkephalin B precursor. Further, we also contemplate using DNA encoding a fusion, or fragment of these sequences, that upon expression yields one or more enkephalin-like molecules that have analgesic properties. We prefer use of a DNA sequence encoding the entire pro-enkephalin A precursor molecule. The DNA and amino acid sequence of pro-enkephalin A are well known. Folkesson. supra. This embodiment takes advantage of the host cell's cleavage enzymes, such as prohormone convertase, to generate a suite of enkephalins, some or all of which may have analgesic properties. The preferred enkephalin encoded by this construct is Met-enkephalin.

There are three naturally occurring catecholamines which function as neurotransmitters in the central nervous system; norepinephrine ("NE"), epinephrine ("E"), and dopamine. NE is associated with postganglionic sympathetic nerve endings. NE exerts its effects locally in the immediate vicinity of its release. Catecholamines are synthesized from the amino acid tyrosine, which is sequentially hydroxylated to form dihydroxyphenylalanine (dopa) , decarboxylated to form dopamine, and then hydroxylated on the beta position of the side chain by dopamine beta hydroxylase to form NE. Harrison's, supra, pp. 380. NE is

N-methylated to E by phenylethanolamine-N methyltransferase ("PNMT") .

Hydroxylation of tyrosine by tyrosine hydroxylase ("TH") is the rate limiting step in NE synthesis. Regulation of dopa and NE synthesis in the adrenal medulla may be accomplished by changes in the amount and the activity of TH.

In addition, regulation of synthesis of E from NE may occur by changes in the amount and the activity of phenylethanolamine-N-methyltransferase

("PNMT") . PNMT is inducible by glucocorticoids from the adrenal cortex. Ibid.

Catecholamines are maintained in high concentration in adrenal medullary chromaffin tissue, mostly as E. Opioid peptides are also stored in the adrenal gland. NE and E have similar affinities at ₂ receptors and therefore both potentially contribute to analgesia. Bylund, FASEB J.. 6, PP. 832-39 (1992) . The enkephalin peptides that predominantly include met- enkephalin selectively activate delta (δ) opioid receptors. Reisine and Bell, Trends Neurosci.. 16, pp. 506-10 (1993) . Activation of α₂ adrenergic and δ opioid receptors in the spinal cord each result in antinociception and are potentially synergistic. Yaksh and Malmberg, Progress in Pain Research and Management. Vol. 1, Ed. Fields and Lisbeskind, IASP Press, • Seattle, pp. 141-71 (1994) . Activation of δ versus (μ) opioid receptors in experimental animals results in fewer adverse side effects including constipation and addiction liability (Lee et al., J. Pharmacol. Exp. Ther. , 267, pp. 883-87 (1993) . The combined delivery of different opioidergic and adrenergic agents may decrease the magnitude of tolerance that develops to a single agent and lead to sustained pain relief. Yaksh and Reddy, Anesthesiol.. 54, pp. 451-67 (1981) .

This invention contemplates use of a DNA sequence encoding catecholamine biosynthetic enzymes or analogs or fragments thereof to obtain catecholamines that have analgesic properties. The preferred catecholamines in this invention are NE and E. In one embodiment, the host cell is transformed with the genes necessary to accomplish production of NE or E, as desired. The selection of heterologous gene sequences required depends upon the complement of catecholamine synthesizing enzymes normally occurring in the host cell. For example, RIN cells, and AtT-20 cells lack tyrosine hydroxylase ("TH") and dopamine beta hydroxylase ("DBH") . However, RIN and AtT-20 cells contain endogenous dopa decarboxylase ("DDC") . If the desired catecholamine is E, then the gene encoding PNMT is also required. The gene encoding PNMT is known. Baetge et al. , Proc. Nat'l Acad. Sci.. 83, pp. 5455-58 (1986) .

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

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

The sequence of the DBH gene is also well known. See, e.g., Lamoroux et al., EMBO J.. 6, pp. 3931-37 (1987) . It will be appreciated that in addition to the preferred DNA sequences described herein, there will be many degenerate DNA sequences that code for the desired analgesics.

Secondary compounds with potential analgesic action may also be produced by the cells of this invention. Such compounds include galanin and somatostatin. In addition, neuropeptide Y, neurotensin and cholecystokinin may be produced by the transformed cells of this invention. The cells of this invention may normally produce some or all of these compounds, or may be genetically engineered to do so using standard techniques. Standard methods may be used to obtain or synthesize the genes encoding the analgesic compounds to be produced by the cells of this invention.

For example, the complete amino acid sequence of the desired compound may be used to construct a back-translated gene. A DNA oligomer containing a nucleotide sequence coding for the desired analgesic compound may be synthesized. For example, several small oligonucleotides coding for portions of each desired polypeptide may be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for assembly.

The DNA sequence encoding each desired analgesic compound, may or may not also include DNA sequences that encode a signal sequence. Such signal sequence, if present, should be one recognized by the cell chosen for expression of the analgesic compound. It may be prokaryotic, eukaryotic or a combination of the two. It may also be the signal sequence of the native compound. It generally is preferred that a signal sequence be encoded and most preferably that the native signal sequence be used.

Once assembled, the DNA sequences encoding the desired compounds will be inserted into one or more expression vectors and operatively linked to expression control sequences appropriate for expression in the desired transformed cell.

Proper assembly may be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in the transformed cell. As is well known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene must be operatively linked to transcriptional and translational expression control sequences that are functional in the chosen expression cell. The choice of expression control sequence and expression vector will depend upon the choice of cell. A wide variety of expression host/vector combinations may be employed. Useful expression vectors for eukaryotic hosts, include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus.

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

Any of a wide variety of expression control sequences may be used in these vectors. Such useful expression control sequences include the expression control sequences associated with structural genes of the foregoing expression vectors. Examples of useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast -mating system and other sequences known to control the expression of genes of eukaryotic cells or their viruses, and various combinations thereof.

It should of course be understood that not all vectors and expression control sequences will function equally well to express the DNA sequences described herein. Neither will all cells function equally well with the same expression system. However, one of skill in the art may make a selection among these vectors, expression control sequences and cells without undue experimentation. For example, in selecting a vector, the host cell must be considered because the vector must replicate in it. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.

In selecting an expression control sequence, a variety of factors should also be considered. These include, for example, the relative -strength of the sequence, its controllability, and its compatibility with the actual DNA sequence encoding the desired analgesic compounds, particularly as regards potential secondary structures. Host cells should be selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded for by the DNA sequences, their secretion characteristics, their ability to fold the polypeptides correctly, and their culture requirements. If the host cell is to be encapsulated, cell viability when encapsulated and implanted in a recipient should also be considered.

Within these parameters, one of skill in the art may select various vector/expression control sequence/host combinations that will express the desired DNA sequences in 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, amplification of copy number of the heterologous genes is more difficult to achieve. Thus use of fewer expression vectors is preferred. Most preferably, a single expression vector, containing all 4 heterologous genes, is used.

In a particular 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, having the ACTH coding region deleted. The second vector contains the pro-enkephalin A gene operably linked to the CMV promoter. Preferably the proA construct contains the Kozak sequence immediately upstream of the start codon. The third vector contains both the TH gene (preferably truncated and having the Kozak consensus sequence immediately upstream of the start codon) and the DBH gene. In this embodiment, the TH gene is operably linked to the CMV promoter. The DBH gene is operably linked to an internal ribosome entry site promoter sequence. RIN cells are then transformed sequentially with each expression vector according to known protocols.

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

Multiple gene expression from a single transcript is preferred over expression from multiple transcription units. One approach for achieving expression of multiple genes from a single eukaryotic transcript takes advantage of sequences in picorna viral mRNAs known as internal ribosome entry sites ("IRES") . These sites function to facilitate protein translation from sequences located downstream from the first AUG of the RNA.

Macejak and Sarnow reported that the 5' untranslated sequence of the immunoglobulin heavy chain binding protein (BiP, also known as CRP 78, the glucose-regulated protein of molecular weight 78,000) mRNA can directly confer internal ribosome binding to an mRNA in mammalian cells, in a 5'-cap independent manner, indicating that translation initiation by an internal ribosome binding mechanism is used by this cellular mRNA. Nature 353, pp. 90-94 (1991) .

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

This invention also contemplates use of a "suicide" gene in the transformed cells. Most preferably, the cell carries the TK (thymidine kinase) gene as a safety measure, permitting the host cell to be killed in vivo by treatment with gancyclovir.

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 recipient's own immune system provides a first level of protection from adverse reactions to the implanted cells. If encapsulated, the polymer capsule itself may be immuno-isolatory. The presence of the TK gene (or other suicide gene) in the expression construct adds an additional level of safety to the recipient of the implanted cells.

Preferred vectors for use in this invention include those that allow the DNA encoding the analgesic compounds to be amplified in copy number. Such amplifiable vectors are well known in the art. They include, for example, vectors able to be amplified by DHFR amplification (see, e.g., Kaufman, United States Patent 4,470,461, Kaufman and Sharp, "Construction Of A Modular Dihydrafolate Reductase cDNA Gene: Analysis Of Signals Utilized For Efficient Expression", Mol. Cell. Biol.. 2, pp. 1304-19 (1982)) or glutamine synthetase ("GS") amplification (see, e.g., United States patent 5,122,464 and European published application 338,841) . Such amplification can be used to increase output of the desired analgesic compounds.

Other techniques for increasing the output of the desired analgesic compounds are contemplated. For example, subcloning existing polyclonal cell lines is contemplated. Cells are cloned by limiting dilution to a single cell in each well. Cell clones are cultures, and the clones are tested to select the clone with the highest output of analgesic substances. Another technique for increasing the output of the desired analgesic compounds involves cloning altered forms of biosynthetic enzymes with higher activity than the wild type form (i.e., the truncated TH 1-155) . Some truncated forms of TH have 4-6 times increased activity over the wild type form of TH. See, e.g., Daubner et al., "Expression and characterization of catalytic and regulatory domains of rat tyrosine hydroxylase" Protein Science. 2, pp. 1452-60 (1993) . In addition, use of tyrosine-free media to select to increase tetrahydrobiopterin cofactor levels may potentially increase tyrosine hydroxylase activity. See, e.g., Horellou et al., "Retroviral transfer of a human tyrosine hydroxylase cDNA in various cell lines; regulated release of dopamine in mouse anterior pituitary AtT-20 cells", Proc. Natl. Acad. Sci. USA. 86, pp. 7233-37 (1989) . Preferably, the output of β-endorphin ranges between 1 and 10,000 pg/10⁶ cells/hr. Preferably, the output of met-enkephalin ranges between 1 and 10,000 pg/10⁶ cells/hr. Preferably, the output of catecholamines ranges between 1 and 1,000 pmoles/10⁶ cells/hr.

The cells- of this invention may be implanted into a mammal, including a- human, for the treatment, of pain. If implanted unencapsulated, any suitable implantation protocol may be used, including those outlined by Sagen et al., United States patent 4,753,635, incorporated herein by reference.

It may be desirable to encapsulate the genetically modified cells of this invention before implantation. Such encapsulated cells form a bioartificial organ ("BAO") . BAOs may be designed for implantation in a recipient or can be made to function extra-corporeally. The BAOs useful in this invention typically have at least one semipermeable outer surface membrane or jacket surrounding a cell-containing core. The jacket permits the diffusion of nutrients, biologically active molecules and other selected products through the BAO. The BAO is biocompatible.

In some cases, the membrane may serve to also immunoisolate the cells by blocking the cellular and molecular effectors of immunological rejection. The use of immunoisolatory membranes allows for the implantation of allo and xenogeneic cells into an individual without the use of immunosuppression. If biologically active molecules are released from the isolated cells, they pass through the surrounding semipermeable membrane into the recipient's body. If metabolic functions are provided by the isolated cells, the substances to be metabolized enter the BAO from the recipient's body through the membrane to be acted on by the cells.

A variety of types of membranes have been used in the construction of BAOs. Generally, the membranes used in BAOs are either microporous or ultrafiltration grade membranes. A variety of membrane materials have been suggested for use in BAOs, including PAN/PVC, polyurethanes, polysufones, polyvinylidienes, and polystyrenes. Typical membrane geometries include flat sheets, which may be fabricated into "sandwich" type constructions, having a layer of living cells positioned between two essentially planar membranes with seals formed around the perimeter of the device. Alternatively, hollow fiber devices may be used, where the living cells are located in the interior of a tubular membrane. Hollow fiber BAOs may be formed step-wise by loading living cells in the lumen of the hollow fiber and providing seals on the ends of the fiber. Hollow fiber BAOs may also be formed by a coextrusion process, where living cells are coextruded with a polymeric solution which forms a membrane around the cells.

BAOs have been described, for example, in United States patent Nos. 4,892,538, 5,106,627, 5,156,844, 5,158,881, and 5,182,111, and PCT Application Nos. PCT/US/94/07015, WO 92/19195, WO 93/03901, and WO 91/00119, all of which are incorporated herein by reference.

BAOs may contain other components that promote long term survival of the encapsulated cells. For example, WO 92/19195 refers to implantable immunoisolatory biocompatible vehicles having a hydrogel matrix for enhancing cell viability.

The encapsulating membrane of the BAO may be made of a material which is the same as that of the core, or it may be made of a different material. In either case, a surrounding or peripheral membrane region of the BAO which is permselective and biocompatible will be formed. The membrane may also be constructed to be immunoisolatory, if desired. The core contains isolated cells, either suspended in a liquid medium or immobilized within a hydrogel matrix.

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 polymer blends can be used to manufacture the capsule jacket. Polymeric membranes forming the BAO and the growth surfaces therein may include polyacrylates (including acrylic copolymers), polyvinylidenes, polyvinyl chloride copolymers, polyurethanes, polystyrenes, polyamides, cellulose acetates, cellulose nitrates, polysulfones, polyphosphazenes, polyacrylonitriles, poly(acrylonitrile/covinyl chloride), as well as derivatives, copolymers and mixtures thereof. BAOs may be formed by any suitable method known in the art. One such method involves coextrusion of a polymeric casting solution and a coagulant which can include biological tissue fragments, organelles, or suspensions of cells and/or other therapeutic agents, as described in Dionne, WO 92/19195 and United States Patents 5,158,881, 5,283,187 and 5,284,761, incorporated herein by reference.

The jacket may have a single skin or a double skin. A single-skinned hollow fiber may be produced by quenching only one of the surfaces of the polymer solution as it is co-extruded. A double-skinned hollow fiber may be produced by quenching both surfaces of the polymer solution as it is co-extruded.

Numerous capsule configurations, such as cylindrical, disk-shaped or spherical are possible.

The jacket of the BAO will have a pore size that determines the nominal molecular weight cut off (riMWCO) of the permselective membrane. Molecules larger than the nMWCO are physically impeded from traversing the membrane. Nominal molecular weight cut off is defined as 90% rejection under convective conditions. In situations where it is desirable that the BAO is immunoisolatory, the membrane pore size is chosen to permit the particular factors being produced by the cells to diffuse out of the vehicle, but to exclude the entry of host immune response factors into the BAO. Typically the nMWCO ranges between 50 and 200 kD, preferably between 90 and 150 kD. The most suitable membrane composition will also minimize reactivity between host immune effector molecules known to be present at the selected implantation site, and the BAO's outer membrane components.

The core of the BAO is constructed to provide a suitable local environment for the particular cells isolated therein. The core can comprise a liquid medium sufficient to maintain cell growth. Liquid cores are particularly suitable for maintaining transformed cell lines like PC12 cells. Alternatively, the core can comprise a gel matrix. The gel matrix may be composed of hydrogel (alginate, "Vitrogen™", etc.) or extracellular matrix components. See, e.g., Dionne WO 92/19195.

Compositions that form hydrogels fall into three general classes. The first class carries a net negative charge (e.g., alginate). The second class carries a net positive charge (e.g., collagen and laminin) . Examples of commercially available extracellular matrix components include Matrigel™ and Vitrogen™. The third class is net neutral in charge (e.g., highly crosslinked polyethylene oxide, or polyvinylalcohol) .

Any suitable method of sealing the BAO may be used, including the employment of polymer adhesives and/or crimping, knotting and heat sealing. These sealing techniques are known in the art. In addition, any suitable "dry" sealing method can also be used. In such methods, a substantially non-porous fitting is provided through which the cell-containing solution is introduced. Subsequent to filling, the BAO is sealed. Such a .method is described in copending United States application Serial No. 08/082,407, herein incorporated by reference.

One or more in vitro assays are preferably used to establish functionality of the BAO prior to implantation in vivo. Assays or diagnostic tests well known in the art can be used for these purposes. See, e.g., Methods Tn Enzvmoloσv. Abelson [Ed], Academic Press, 1993. For example, an ELISA (enzyme-linked immunosorbent assay) , chromatographic or enzymatic assay, or bioassay specific for the secreted product can be used. If desired, secretory function of an implant can be monitored over time by collecting appropriate samples (e.g., serum) from the recipient and assaying them. If the recipient is a primate, microdialysis may be used. The number of BAOs and BAO size should be sufficient to produce a therapeutic effect upon implantation is determined by the amount of biological activity required for the particular application. In the case of secretory cells releasing therapeutic substances, standard dosage considerations and criteria known to the art are used to determine the amount of secretory substance required. Factors to be considered are discussed in Dionne, WO 92/19195.

Implantation of the BAO is performed under sterile conditions. Generally, the BAO is implanted at a site in the host which will allow appropriate delivery of the secreted product or function to the host and of nutrients to the encapsulated cells or tissue, and will also allow access to the BAO for retrieval and/or replacement. The preferred host is a primate, most preferably a human.

A number of different implantation sites are contemplated. These implantation sites include the central nervous system, including the brain, spinal cord, and aqueous and vitreous humors of the eye.

Preferred sites in the brain include the striatum, the cerebral cortex, subthalamic nuclei and nucleus Basalis of Meynert. Other preferred sites are the cerebrospinal fluid, most preferably the subarachnoid space and the lateral ventricles. This invention also contemplates implantation into the kidney subcapsular site, and intraperitoneal and subcutaneous sites, or any other therapeutically beneficial site.

In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only, and are not to be construed as limiting the scope of this invention in any manner.

Examples

Construction of Polvcistronic Expression Vectors Construction of IgSP-POMC Fusion The Smal-Sall fragment containing the human

POMC exon 3 was subcloned into pBS cloning vector (Stratagene) . See Takahashi. supra; Cochet. supra. The resulting plasmid was named as pBS-hPOMC-027. See Fig. 1. A PCR fragment was generated using two oligonucleotide primers, termed oCNTF-003 (SEQ ID NO: 1) and oIgSP-018, (SEQ ID NO: 2) and the pNUT plasmid containing the human CNTF gene. See Baetge et al., Proc. Natl. Acad. Sci. USA. 83, pp. 5454-58 (1986) . Both primers oCNTF-003 and oIgSP-018, contain synthetic Ba HI and S al restriction sites, respectively, at the 5' ends.

The 196 base pair (bp) PCR fragment was digested with restriction endonucleases BamHI and the Smal-isoschizomer Xmal, and electrophoresed through an 1% SeaPlaque agarose. The 193 bp HindiII/Xmal DNA fragment was excised and purified using the FMC SpinBind DNA purification kit (FMC BioProducts, Rockland, ME) . pBS-hPOMc-027 was also digested with BamHI and Xmal and purified from 1% SeaPlaque agarose using the FMC SpinBind DNA purification kit (FMC BioProducts, Rockland, ME) . The ligation mixture was transformed into E. coli DH5α (Gibco BRL, Gaithersburg, MD) . Positive sub-clones were initially identified by the cracking gel procedure (Promega Protocols and Applications Guide, 1991) . Minilysate DNA was then prepared using the FMC SpinBind DNA purification kit (FMC BioProducts, Rockland, ME) and subject to BamHI and Smal restriction digestions. The positive sub- clone was named as pBS-IgSP-hPOMC-028. See Fig. 1. The nucleotide sequence of the fusion junction in pBS- IgSP-hPOMC-028 was determined by the dideoxynucleotide sequence determination using the Sequenase kit (USBC, Cleveland) . The sequence of the IgSP-hPOMC fusion is shown in SEQ ID NO: 3.

Construction of IgSP-POMC Expression Vectors

The IgSP-hPOMC DNA fragment in pBS-IgSP- hPOMC-028 was subcloned into pcDNA3 (Invitrogen Corp., San Diego, CA) and pCEP4 (Invitrogen Corp., San Diego, CA) in sense and anti-sense orientations.

The Notl-Sall IgSP-hPOMC fragment from pBS- IgSP-hPOMC-028 was ligated with the Notl-Xhol digested pCEP4 resulting in the sense orientation clone named as pCEP4-hPOMC-030. Fig. 2. The BamHI-Sall IgSP-hPOMC fragment from pBS-IgSP-hPOMC-028 was ligated with the BamHI-XhoI digested pCEP4 resulting in the anti-sense orientation clone named as pCEP4-hPOMC-031. Fig. 2. The insert orientation in pCEP4-hPOMC-030 and -031 was confirmed by BamHI, NotI, Sail and Notl/Sall restriction digestions as well as by dideoxynucleotide sequence determination using the Sequenase kit (USBC, Cleveland) .

The Ba HI-Sall IgSP-hPOMC fragment from pBS- IgSP-hPOMC-028 was ligated with the BamHI-XhoI digested pcDNA3 resulting in the sense orientation clone named as pcDNA3-hPOMC-034. Fig. 2. The Notl-Hindlll IgSP- hPOMC fragment from pBS-IgSP-hPOMC-028 was ligated with the Notl-Hindlll digested pcDNA3 resulting in the anti¬ sense orientation clone named as pcDNA3-hPOMC-035. Fig. 2. Restriction digestion using Smal, BamHI,

EcoRI, and BamHI/EcoRI was used to confirm the insert orientation in pcDNA3-hPOMC-034, whereas Hindlll, NotI and Sail were used for pcDNA3-hPOMC-035.

Construction of ACTH Deleted IgSP-POMC The ACTH coding region in the POMC gene in pBS-IgSP-hPOMC-028 was deleted. pBS-IgSP-hPOMC-028 was first digested with Xmal restriction enzyme and treated with pfu DNA polymerase (Promega, Madison, WI) . The Xmal-pfu DNA polymerase treated pBS-IgSP-hPOMC-028 was then digested with StuI restriction enzyme and purified from 1% SeaPlaque agarose using the FMC SpinBind DNA purification kit (FMC BioProducts, Rockland, ME) . The self-ligation mixture was transformed into E. coli DH5α (Gibco BRL, Gaithersburg, MD) . Positive sub-clones were identified by BamHI/Hindlll restriction digestion and named as pBS-IgSP-hPOMCΔACTH-029. See Fig. 1. The nucleotide sequence of the ACTH deletion region in pBS- IgSP-hPOMC-ΔACTH-029 was confirmed by the dideoxynucleotide sequence determination. The sequence of the IgSP-hPOMC-ΔACTH fusion is shown in SEQ ID NO: 4.

Construction of ACTH Deleted IgSP-POMC Expression Vectors

The IgSP-hPOMC-ΔACTH DNA fragment in pBS- IgSP-hPOMC-ΔACTH-029 was subcloned into pdDNA3 (Invitrogen Corp., San Diego, CA) and pCEP4 (Invitrogen Corp., San Diego, CA) in sense and anti-sense orientations. The Notl-Sall IgSP-hPOMC-ΔACTH fragment from pBS-IgSP-hPOMC-ΔACTH-029 was ligated with the Notl-Xhol digested pCEP4 resulting in the sense orientation clone named as pCEP4-hPOMC-ΔACTH-032

(Fig. 3) . The BamHI-Sall IgSP-hPOMC-ΔACTH fragment from pBS-IgSP-hPOMC-ΔACTH-029 was ligated with the BamHI-XhoI digested pCEP4 resulting in the anti-sense orientation clone named as pCEP4-hPOMC-ΔACTH-033 (Fig. 3) . The insert orientation in pCEP4-hPOMC-ΔACTH- 032 and -033 was confirmed by BamHI and EcoRI restriction digestions as well as by dideoxynucleotide sequence determination using the Sequenase kit (USBC, Cleveland) . The BamHI-Sall IgSP-hPOMC-ΔACTH fragment from pBS-IgSP-hPOMC-ΔACTH-029 was ligated with the BamHI- XhoI digested pcDNA3 resulting in the sense orientation clone named as pcDNA3-hPOMΔACTH-036 (Fig. 3) . The Notl-Hindlll IgSP-hPOMC-ΔACTH fragment from pBS-IgSP- hPOMC-ΔACTH-029 was ligated with the Notl-Hindlll digested pcDNA3 resulting in the anti-sense orientation clone named as pcDNA3-hPOMC-ΔACTH-037 (Fig. 3) .

Restriction digestion using PvuII and EcoRI was used to confirm the insert orientation in pcDNA3- hPOMC-ΔACTH-036, whereas Sail and EcoRI were used for pcDNA3-hPOMC-ΔACTH-037.

Cloning of Full Length and Truncated TH cDNA

Total RNA from PC12 cells was prepared using the guanidinium thiocyanate-based TRI reagent (Molecular Research Center, Inc., Cincinnati, OH) . ^' Five hundred ng of PC12 total RNA was reverse transcribed at 42°C for 30 minutes in a 20μl reaction volume containing 10 mM Tris.HCI (pH 8.3), 50 mM KC1, 4 mM of each dNTP, 5 mM MgCl₂, 1.25 μM oligo (dT) 15- mer, 1.25 μM random hexamers, 31 units of RNase Guard RNase Inhibitor (Pharmacia, Sweden) and 200 units of Superscript II reverse transcriptase (Gibco BRL, Gaithersburg, MD) . Two micro-liters of the above reverse transcribed cDNA was added to a 25 μl PCR reaction mixture containing 10 mM Tris.HCI (pH 8.3), 50 mM KC1, 800 of each nM dNTP, 2 mM MgC12, 400 nM of primers #1 and #2, and 2.5 units of Thermus aquaticus (Taq) DNA polymerase (Boehringer Mannheim, Germany) . To generate the full length TH cDNA, oligonucleotide primers orTH-052 (SEQ ID NO: 5) and orTH-053 (SEQ ID NO: 6) were used. For the truncated TH, primers orTH-054 (SEQ ID NO: 7) and orTH-053 (SEQ ID NO: 6) were used instead. These oligonucleotides were constructed based on published TH sequence information in Grima et al . , Nature. 326, pp. 707-11 (1987); US patent 5,300,436, and Daubner. supra. Primers orTH-052 (SEQ ID NO: 5) and orTH-054 (SEQ ID NO: 7) have synthetic Hindlll restriction site at the 5' end where orTH-053 has BamHI at the 5' end. The PCR reaction mixtures were subject to 30 amplification cycles consisted of: denaturation, 94°C 30 seconds (first cycle 2 minutes) ; annealing, 50°C 1 minute; and extension, 72°C 3.5 minutes (last cycle 5 minutes) . The 1537 bp full length and 1087 bp truncated rat TH PCR fragments were digested with restriction endonucleases BamHI and HindiII and resolved on an 1% SeaPlaque agarose gel. The 1531-bp and 1081-bp Hindlll/BamHI DNA fragments were excised and purified using the FMC SpinBind DNA purification kit (FMC BioProducts, Rockland, ME) . pcDNA3 expression vector was also digested with BamHI and Hindlll and purified from 1% SeaPlaque agarose using the FMC SpinBind DNA purification kit (FMC BioProducts, Rockland, ME) . The ligation mixture was transformed into E.coli DH5 (Gibco BRL, Gaithersburg, MD) .

Cracking gel procedure (Promega Protocols and Applications Gμide, 1991) was used to screen out the positive sub-clones. The identity of the correct clones was further verified by BamHI/Hindlll double digestion.

The positive sub-clones for the full-length and truncated rat TH in pcDNA3 were named as pcDNA3- rTH-044 (Fig. 4) and pcDNA3-rTHΔ-045 (Fig. 4), respectively. The nucleotide sequence of both full- length and truncated rat TH PCR clones was determined by the dideoxynucleotide sequence determination using the Sequenase kit (USBC, Cleveland) . The sequence of the rTHΔ construct is shown in SEQ ID NO: 16.

To optimize the translation efficiency of the truncated rat TH, oligonucleotide primer orTH-078 (SEQ ID NO: 8) was designed so that the consensus Kozak sequence is immediate up stream to the start codon ATG. pcDNA3-rTHΔ-45 was used as the template in a 50 μl PCR reaction mixture with reagent composition identical to the one described above with the exception that the oligonucleotide primers were replaced with orTH-078 (SEQ ID NO: 8) and orTH-053 (SEQ ID NO: 6) . The 1097 bp PCR product was cloned into pcDNA3 in the same manner as described above. The resulting sub-clone was named pcDNA3-rTHΔKS-75 (Fig 4) . The sequence of the rTHΔKS construct is shown in SEQ ID NO: 17.

Construction of rTH-IRES-bDBH Fusion Gene

Recombinant PCR methodology was used to generate the rTH-IRES-bDBH fusion gene. Oligonucleotides oIRES-057 (SEQ ID NO: 9) and obDBH-065 (SEQ ID NO: 10) are specific for IRES and bDBH gene sequences, respectively, and contain synthetic BamHI and NotI restriction sites at the 5' end, respectively. Oligonucleotides oIRES-bDBH-064 (SEQ ID NO: 11) and oIRES-bDBH-066 (SEQ ID NO: 12) are complementary to each other. Furthermore, oligonucleotide primer oIRES- bDBH-064 (SEQ ID NO: 11) has its 5' 16 nucleotides identical to the IRES sequence and its 3' 18 nucleotides identical to the bDBH sequence; and vice versa for oIRES-bDBH-066 (SEQ ID NO: 12) . Two first PCR reactions were carried out using oligonucleotide pairs oIRES-057/oIRES-bDBH-066 and oIRES-bDBH-064/obDBH-065 on templates pCTI-001 (with an insert containing the IRES sequence shown in SEQ ID NO: 30) and pBS-bDBH-006 (containing the bovine DBH gene cloned from bovine adrenal chromaffin cells, Lamoroux et al., EMBO J.. 6, pp. 3931-37 (1987)) plasmids, respectively. One hundred ng of template DNA was added to a 50 μl PCR reaction mixture containing 10 mM Tris.HCI (pH 8.3), 50 mM KC1, 800 of each nM dNTP, 2 mM MgCl2, 400 nM of primers #1 and #2, and 2.5 units of Thermus aquaticus (Taq) DNA polymerase (Boehringer Mannheim, German) .

The PCR reaction mixtures were subject to 30 amplification cycles consisted of: denaturation, 94 °C for 30 seconds (first cycle 2 minutes) ; annealing, 50 °C 1 minute; and extension, 72 °C 30 seconds (last cycle 5 minutes) . The PCR products were resolved on 1% TrivieGel 500 (TrivieGen) . Two agarose plugs containing each one of the first PCR products were transfer to a tube containing 50 μl of PCR reaction mixtures identical to the one described above with the exception that the oligonucleotides oIRES-057 and obDBH-065 were used.

The second PCR reaction was subject to 30 amplification cycles consisted of: denaturation, 94 °C for 30 seconds (first cycle 2 minutes) ; annealing, 60 °C 30 seconds (second to fourth cycles 37 °C 2 minutes) ; and extension, 72 °C 30 seconds (last cycle 2 minutes) . The 2407 bp IRES-bDBH fusion PCR product and the cloning vector pcDNA3-rTHΔ- 5 were digested with BamHI and NotI restriction enzymes and subsequently purified from 1 % SeaPlaque agarose gel using the FMC SpinBind DNA purification kit (FMC BioProducts, Rockland, ME) .

The ligation of IRES-bDBH/BamHI/Notl and pcDNA3-rTHΔ-045/BamHI/NotI would generate a rTHΔ-IRES- bDBH expression vector named as pcDNA3-rTHΔ-IRES-bDBH- 066 (Fig. 5) whereas that of IRES-bDBH/BamHI/Notl and pcDNA3-rTHΔKS-075/BamHI/NotI would generate a rTHΔKS- IRES-bDBH expression vector, named as pcDNA3-rTHΔKS- IRES-bDBH-076 (Fig. 5), where the start codon ATG in rTHΔ is preceded with a consensus Kozak sequence. The sequence of the rTHΔ-IRES-bDBH construct is shown in SEQ ID NO: 18. The sequence of the rTHΔKS-IRES-bDBH construct is shown in SEQ ID NO: 19. The ligation mixture was transformed into DH5α (Gibco BRL, Gaithersburg, MD) . The positive clones were identified by the cracking gel procedure (Promega, Madison, WI) and restriction digestions using Hindlll, BamHI, HindiII/BamHI, Smal and NotI.

The 4114 bp Nrul-Xhol fragment containing the CMV promoter-rTHΔKS-IRES-bDBH was excised out of pcDNA3-rTHΔKS-IRES-bDBH-076 and subcloned into pZeoSV cloning vector (Invitrogen Corp., San Diego, CA) digested with Seal and Xhol in the multiple cloning site. The resulting expression vector was named as pZeo-Pcmv-rTHΔKS-IRES-bDBH-088 (Fig. 6) .

Construction of IgSP-hPOMC ACTH- rTHD-IRES-bDBH Fusion Gene

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

The resulting plasmid pBS-Pcmv-rTHΔ-IRES- bDBH-067 (Fig. 7) was used as the intermediary construct to which the recombinant PCR IgSP-hPOMCDACTH- IRES fragment would be inserted.

Oligonucleotide oIgSP-068 (SEQ ID NO: 13), containing a synthetic EcoRV restriction site, is specific for the IgSP sequence. Oligonucleotide primer orTHΔ-073 (SEQ ID_..

NO: 14) is specific for the rTHΔ sequence and contains an endogenous Smal restriction site.

Oligonucleotide primers ohPOMC-IRES-069 (SEQ ID NO: 15) and ohPOMC-IRES-070 (SEQ ID NO: 20) are complementary to each other. Furthermore, oligonucleotide primer ohPOMC-IRES-069 has its 5', 18 nucleotides identical to the hPOMC sequence and its 3' 12 nucleotides identical to the IRES sequence; and vice versa for ohPOMC-IRES-070. Oligonucleotide primers oIRES-rTHΔ-071 (SEQ

ID NO: 21) and oRIRES-rTHΔ-072 (SEQ ID NO: 22) are complementary to each other. In addition, oligonucleotide primer oIRES-rTHΔ-071 has its 5' 15 nucleotides identical to the rTHΔ sequence and its 3' 18 nucleotide identical to the IRES sequence; and vice versa for oRIRES-rTHΔ-072.

Three sets of first PCR reactions were carried out.

PCR reaction A: template pBS-IgSP-hPOMCDACTH-029, oligonucleotides oTgSP-068/ohPOMC-IRES-069; PCR reaction B: template pCTI-001, oligonucleotides ohPOMC-IRES-070/oIRES-rTHΔ-071; and PCR reaction C: template pcDNA3-rTHΔ-045, oligonucleotides orIRES-rTHΔ-072/orTHΔ-073.

The three sets of first PCR reactions were carried in 50 μl PCR reaction mixture containing 100 ng of template DNA, 10 mM Tris. HCI (pH 8.3), 50 mM KCl, 800 of each nM dNTP, 2 mM MgC123, 400nM of primers #1 and #2, and 2.5 units of Thermus aquaticus (Taq) DNA polymerase (Boehringer Mannheim, Germany) .

The PCR reaction mixtures were subject to 30 amplification cycles consisted of: denaturation, 94 °C for 30 seconds (first cycle 2 minutes) ; annealing, 50 °C 1 minute; and extension, 72 ^CC 30 seconds (last cycle 5 minutes) .

The PCR products were resolved on 1% TrivieGel 500 (TrivieGen) . Two agarose plugs containing each one of the PCR products from PCR reactions B and C were transferred to a tube containing 50 μl of PCR reaction mixtures identical to the one described above with the exception that the oligonucleotides ohPOMC-IRES-070 and orTHΔ-073 were used.

The second PCR reaction was subject to 30 amplification cycles consisted of: denaturation, 94 °C for 30 seconds (first cycle 2 minutes) ; annealing, 60 °C 30 seconds (second to fourth cycles 37 °C 2 minutes) ; and extension, 72 °C 30 seconds (last cycle 2 minutes) .

The PCR products were treated as described above. Agarose plugs containing the PCR products from the second PCR reaction and the PCR reaction A were combined and subjected to a third PCR amplification using oIgSP-068/rTHΔ-073. The 1203 bp IgSP-hPOMC-IRES- rTHΔ fusion PCR product and the cloning vector pBS- Pcmv-rTHΔ-IRES-bDBH-067 were digested with EcoRV and Xmal restriction enzymes and subsequently purified from 1% SeaPlaque agarose gel using the FMC SpinBind DNA purification kit (FMC BioProducts, Rockland, ME) . The ligation mixture was transformed into DH5α (Gibco BRL, Gaithersburg, MD) .

The positive clones were identified by the cracking gel procedure (Promega, Madison, WI) and restriction digestions using EcoRI, Kpnl and NotI. The resulting clone was named as pBS-IgSP-hPOMCΔACTH-IRES- rTHΔ-IRES-bDBH-068. Fig. 8. The sequence of this construct is shown in SEQ ID NO: 23.

Construction of IgSP-hPOMCACTH-IRES- rTHΔ-IRES-bDBH Expression Vectors

The 4491 bp NotI fragment containing the IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES-bDBH gene was excised out of the pBS-IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES-bDBH-068 and subcloned into the pcDNA3 (Invitrogen Corp., San Diego, CA) at the NotI site in the multiple cloning site. Restriction digestion using NotI and Smal confirmed that the IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES-bDBH gene was inserted in the sense orientation resulting in pcDNA3-IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES-bDBH-069. See Fig. 9.

Construction of IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES- bDBH-IRES-Zeocine Expression Vector

Recombinant PCR methodology was used to generate the IRES-Zeocine fusion gene. Oligonucleotides oIRES-074 (SEQ ID NO: 24) and oZeocin- 077 (SEQ ID NO: 25) are specific for IRES and Zeocin gene sequences, respectively, and contain synthetic NotI and Xhol restriction sites at the 5' end, respectively. Oligonucleotides oIRES-Zeocin-075 (SEQ ID NO: 26) and oIRES-Zeocin-076 (SEQ ID NO: 27) are complementary to each other. Furthermore, oligonucleotide oIRES-Zeocin-075 has its 5'15 nucleotides identical to the Zeocin sequence and its 3' 18 nucleotides identical to the IRES sequence; and vice versa for oIRES-Zeocin-076.

Two first PCR reactions were carried out .^' using oligonucleotide pairs oIRES-074/oIRES-Zeocin-075 and oIRES-Zeocin-076/oZeocin-075 on templates pCTI-001 and pZeoSV (Invitrogen Corp., San Diego, CA) plasmids, respectively.

One hundred ng of template DNA was added to a 50 μl PCR reaction mixture containing lOmM Tris.HCI (pH 8.3), 50 mM KC1, 800 of each nM dNTP, 2 mM MgC12, 400 nM of primers #1 and #2, and .2.5 units of Thermus aquaticus (Taq) DNA polymerase (Boehringer Mannheim, Germany) .

The PCR reaction mixtures were subject to 30 amplification cycles consisted of: denaturation, 94 °C for 30 seconds (first cycle 2 minutes) ; annealing, 50 °C 1 minute; and extension, 72 °C 30 seconds (last cycle 5 minutes) .

The PCR products were resolved on 1% TrivieGel 500 (TrivieGen) . Two agarose plugs containing each one of the first PCR products were transfer to a tube containing 50 μl of PCR reaction mixtures identical to the one described above with the exception that the oligonucleotides oIRES-074 and oZeocin-077 were used.

The second PCR reaction was subject to 30 amplification cycles consisted of: denaturation, 94 °C for 30 seconds (first cycle 2 minutes) ; annealing, 50 °C 30 seconds (second to fourth cycles 37 °C 2 minutes) ; and extension, 72 °C 30 seconds (last cycle 2 minutes) .

The 974 bp IRES-Zeocin fusion PCR product and the cloning vector pcDNA3 were digested with NotI and Xhol restriction enzymes and subsequently purified from 1% SeaPlaque agarose gel using the FMC SpinBind DNA purification kit (FMC BioProducts, Rockland, ME) .

The ligation of IRES-Zeocin/Notl/XhoI and pcDNA3/NotI/XhoI would generate an intermediate cloning vector named as pcDNA3-IRES-Zeocin-072. Fig. 10.

The positive clones were identified by the cracking gel procedure (Pro ega, Madison, WI) and restriction digestions using Hindlll, Smal, Xhol, NotI and Notl/Xhol.

To generate the final IgSP-hPOMCDACTH-IRES- rTHD-IRES-bDBH-IRES-Zeocine Expression Vector, a 4491 bp NotI fragment containing the IgSP-hPOMCΔACTH-IRES- rTHΔ-IRES-bDBH gene was excised out of the pBS-IgSP- hPOMCΔACTH-IRES-rTHΔ-IRES-bDBH-068 (Fig. 8; SEQ ID

NO: 23) and subcloned in to the pcDNA3-IRES-Zeocin-072 (Fig. 10) at the NotI site in the multiple cloning site.

Restriction digestion using NotI and Smal confirmed that the IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES-bDBH gene was inserted in the sense orientation resulting in pcDNA3-IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES-bDBH-IRES-Zeocin- 073. The sequence of this construct is shown in SEQ ID NO: 28. Fig. 11.

Construction of ProA+KS Fusion

A construct containing the coding region of the human pro-enkephalin A gene with the consensus

Kozak sequence immediately upstream to the start codon ATG. The sequence of this construct is shown in SEQ ID NO: 29.

Construction of hProA+KS Expression Vector The Hindlll/BamHI fragment containing the hProA+KS fusion was ligated into BamHI and Hind III digested pcDNA3 expression vector substantially as described above. After screening as described above, a positive sub-clone was named pcDNA3-hProA+KS-091. Fig. 12. Construction of the pBS-CMV Pro A vector is detailed in Mothis, J. and Lindberg, I., Endocrinology.

131, pp. 2287-96 (1992) .

Transformation of Cells

RIN and AtT-20 cells were transformed as follows.

The RINa and AtT-20 based cell lines were grown in DMEM (Gibco) with 10% fetal bovine serum and pen-strep-fungizone (Gibco) base media. The cells were plated out in PlOO petri dishes (750,000 cells/dish) in 10 ml of base media. 18-24 hours later, the cells were transfected using calcium phosphate method with a kit made by Stratagene (San Diego, CA) . A 10 μg amount of the plasmid vector DNA was diluted in 450 μl of deionized sterile water. Then, 50 μl of a lOx buffer (solution #1) was added to the plasmid DNA. A 500 μl amount of solution #2 was immediately added to the DNA containing solution and mixed gently. This was incubated at room temperature for 20 minutes and then the 1.0 ml solution was added to the cells in the petri dish. The cells were incubated overnight and 18-24 hours later the cells were washed 2x with Hanks balanced salt solution without calcium and magnesium. Then, the cells were cultured in base media + selection drugs. The cells were selected in either 600 μg/ml geneticin (Gibco) or 400 μg/ml hygromycin (Boehringer Mannheim) or 500 μg/ml Zeocin (In Vitrogen, San Diego, CA) . Cells were sequentially transfected and selected to obtain the final cell line. The RINa cells were transfected with plasmid pCEP4-hPOMC-030 containing the POMC gene. This is a hygromycin resistant vector. The cells were also transformed with plasmid pcDNA3-hProA+KS-091. This is a geneticin resistant vector. Finally, the cells were transfected with plasmid pZeo-PCMV-rTHΔKS-IRES-bDBH-088 which conferred Zeocin resistance.

The AtT-20 cells were transfected with plasmid pBS-CMV-ProA and pCEP4-POMC-ΔACTH-32 which conferred geneticin and hygromycin resistance, respectively. Finally, the cells were transfected with plasmid pZeo-Pcmv-rTHΔKS-IRES-bDBH-088.

We have tested a number of media for cell growth. Surprisingly we have found that in certain serum-free medias, the above cell lines have enhanced neurotransmitter output, compared to serum-containing media. We prefer CHO-Ultra (Biowhitaker) for the growth of AtT-20 cells, and Ultra-Culture (Biowhitaker) for the growth of RINa cells.

Output of various analgesics from one transformed RINa cell line (RINa/ProA/P030/P088) is shown in Table 2. All values represent unstimulated cells. Output of β-endorphin and met-enkephalin is in pg/10 cells/hr. β-endorphin and met-enkephalin were measured by radioimmunoassay using Incstar kits (Stillwater, Minnesota) . Catecholamine output is in pmoles/10 cells/hr. The numbers in parentheses represent values from cells that were preincubated 18^' hours with 100 μM tetrahydrobiopterin. Catecholamines were measured by high performance liquid chromatography as described in Lavoie et al., "Two PC12 pheochromocytoma lines sealed in hollow fiber-based capsules tonically release l-dopa in vitro". Cell transplantation. 2, pp. 163-73 (1993) . GABA output from these RINa cells was 28 ng/10 cells/hrs.

Met-enk DA E

17 3 0 (6) (2)

There are encrypted enkephalin fragments which are not fully processed from the pro-enkephalin precursor molecule. These encrypted enkephalins have opioid receptor binding activity. We digested these encrypted enkephalins to measure opioid activity. The trypsin digest protocol is as follows. A 2 μg/ml trypsin (Worthington #34E470) solution is added to media samples on ice. Samples are vortexed, then incubated for 20 minutes in a 37°C waterbath. After the 20 minute digest, samples are returned to ice and 100 ng/ml carboxypeptidase B (Sigma #C-7011) is added. Samples are mixed by vortexing, and returned to the 37°C waterbath for 15 minutes. Samples are placed on ice once more and 10 ug/ l trypsin inhibitor is added. At this stage, samples are either extracted for met- enkephalin or immediately frozen for future extraction. This results in the full enzymatic cleavage to free all met-enkaphalin from^' the longer encrypted fragments^'. A met-enkaphalin radioimmunoassay of the digested sample gives total met-enkaphalin from the supermatant. The transformed RINa cells appear to have greater than 5 fold more encrypted enkaphalins compared to fully processed met-enkaphalin.

Fiber capsule formation and characteristics

Hollow fibers are spun from a 12.5-13.5% poly(acrylonitrile vinylchloride) solution by a wet spinning technique. Cabasso, Hollow Fiber Membranes, vol. 12, Kirk-Othmer Encyclopedia of Chemical Technology. Wiley, New York, 3rd Ed. pp. 492-517 (1980), Unites States patent 5,158,881, incorporated herein by reference. The resulting membrane fibers may either be double skinned or single skinned PAN/PVC fibers. In order to make implantable capsules, lengths of fiber are first cut into 5 cm long segments and the distal extremity of each segment sealed with an acrylic glue. Encapsulation hub assemblies are prepared by providing lengths of the membrane described above, sealing one end of the fiber with a single drop of LCM 24 (Light curable acrylate glue, available from ICI), curing the glue with blue light, and repeating the step with a second drop. The opposite end is previously attached to a frangible necked hub assembly, having a silicone septum through which the cell solution may be introduced. The fiber is glued to the hub assembly by applying LCM 22 to the outer diameter of the hub assembly, pulling the fiber up over it, and curing with blue light. The hub/fiber assemblies are placed in sterilization bags and are ETO sterilized.

Following sterilization with ethylene oxide and outgassing, the fibers are deglycerinated by ultrafiltering first 70% EtOH, and then HEPES buffered saline solution through the walls of the fiber under vacuum.

Preparation and Encapsulation of Transformed Cells

The transformed cells are prepared and encapsulated as follows: A matrix solution is prepared using a commercially available alginate, collagen or other suitable matrix material. The cell solution was diluted in the ratio of two parts matrix solution to one part cell solution containing the transformed cells described above. We prefer Vitrogen (Celtix, Santa Clara) as a matrix for AtT-20 cells.

We prefer Organogen (Organogenesis, Canton, MA) as a matrix for RINa cells. The RINa based cells are prepared for encapsulation by the following method. The cells are grown in base media of DMEM + 10% fetal bovine serum during the proliferation phase. These cells can be removed from the tissue culture flasks by two washes in Hanks balanced salt solution without calcium and magnesium. Then the cells are incubated in 0.25% trypsin + EDTA for 1 minute. This is removed and the cells are rinsed free of the flask using Hanks balanced salt solution without calcium and magnesium solution. The cells are placed in 10 is of base media and centrifuged at 100 x g for 2 minutes. The cells are resuspended in 10 is of the preferred serum free media (Ultra culture, Biowhitaker, Walkersville, MD) . Surprisingly, the RINa cells secrete more analgesic substances when cultured in this serum free media ■ relative to serum continuing base media.

The cells are centrifuged at 100 g twice in the preferred serum free media before the cells are concentrated 1:1 with the preferred Organogen matrix. Organogen is a 1% bovine tendon collagen obtained 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 attained (approximately 250 μls) .

The final concentration of the cell + matrix solution used for encapsulation can range from 20,000 - 50,000 cells/μl. The cells are counted in a standard manner on a hemocytometer.

The cell/matrix suspension is placed in a 1 ml syringe. A Hamilton 1800 Series 50 microliter syringe is set for a 15 microliter air bubble, is inserted into a 1 ml syringe containing the cell solution and 30 microliters are drawn up. The cell solution is injected through the silicone seal of the hub/fiber assembly into the lumen of a modacrylic hollow fiber membrane with a molecular weight cutoff of approximately 50,000-100,000 daltons. Ultrafiltration should be observed along the entire length of the fiber. After one minute, the hub is snapped off the sub-hub, exposing a fresh surface, unwet by cell solution. A single drop of LCM 24 is applied and the adhesive cured with blue light. The device is placed first in HEPES buffered NaCl solution and then in CaCl₂ solution for five minutes to cross-link the alginate. Each implant is about 5 cm long, 1 mm in diameter, and contained approximately 2.5 million cells.

After the devices are filled and sealed,^' a silicone tether (Speciality Silcone Fabrication, Paso Robles, CA) (ID: 0.69, OD: 1.25) is then placed over the proximal end of the fiber. A radiopaque titanium plug is inserted in the lumen of the silicone tether to act as a radiographic marker. The devices are then placed in 100 mm tissue culture dishes in 1.5 ml PC-1 medium, and stored at 37°C, in a 5% CO_: incubator for in vi tro analysis and for storage until implantation. The encapsulated cells are then implanted into the human sub-arachnoid space as follows:

Surgical Procedure

After establishing IV access and administering prophylactic antibiotics (cefazolin sodium, 1 gram IV) , the patient is positioned on the operating table, generally in either the lateral decubitus or genu-pectoral position, with the lumbar spine flexed anteriorly. The operative field is sterily prepared and draped exposing the midline dorsal lumbar region from the levels of S-l to L-l, and allowing for intraoperative imaging of the lumbar spine with C-arm fluoroscopy. Local infiltration with 1.0% lidocaine is used to establish anesthesia of the skin as well as the periosteum and other deep connective tissue structures down to and including the ligamentum flavum.

A 3-5 cm skin incision is made in the parasagital plane 1-2 cm to the right or left of the midline and is continued down to the lumbodorsal fascia using electrocautery for hemostasis. Using traditional bony landmarks including the iliac crests and the lumbar spinous processes, as well as fluoroscopic guidance, and 18 gauge Touhy needle is introduced into the subarachnoid space between L-3 and L-4 via an oblique paramedian approach. The needle is directed so that it enters the space at a shallow, superiorly directed angle that is no greater than 30- 35° with respect to the spinal cord in either the sagittal or transverse plane. Appropriate position of the tip of the needle is confirmed by withdrawal of several ml of cerebrospinal fluid (CSF) for preimplantation catecholamine, enkephalin, glucose, and protein levels and cell counts.

The Touhy needle hub is reexamined to confirm that the opening at the tip is oriented superiorly

(opening direction is marked by the indexing notch for the obturator on the needle hub) , and the guide wire is passed down the lumen of the needle until it extends 4- 5 cm into the subarachnoid space (determined by premeasuring) . Care is taken during passage of the wire that there is not resistance to advancement of the wire out of the needle and that the patient does not complain of significant neurogenic symptoms, either of which observations might indicate misdirection of the guide wire and possible impending nerve root or spinal cord injury. After the guide wire appears to be appropriately placed in the subarachnoid space, the Touhy needle is separately withdrawn and removed from the wire. The position of the wire in the midline of the spinal canal, anterior to the expected location of the caud equina, and without kinks or unexplainable bends is then confirmed with fluoroscopy. After removal of the Touhy needle the guide wire should be able to be moved freely into and out of the space with only very slight resistance due to the rough surface of the wire running through the dense and fibrous ligamentum flavum.

The 7 French dilator is then placed over the guide wire and the wire is used to direct the dilator as it is gently but firmly pushed through the fascia, paraspinous muscle, and ligamentum flavum, following the track of the wire toward the subarachnoid space. Advancement of the 7 French dilator is stopped and the dilator removed from the wire as soon as a loss of resistance is detected after passing the ligamentum flavum. This is done in order to avoid advancing and manipulating this relatively rigid dilator within the subarachnoid space to any significant degree.

After the wire track is "overdilated" by the 7 French dilator, the 6 French dilator and cannula sheath are assembled and placed over the guide wire.

The 6 French dilator and cannula are advanced carefully into the subarachnoid space until the opening tip of the cannula is positioned 7 cm within the space. As with the 7 French dilator, the assembled 6 French dilator and cannula are directed by the wire within the lumen of the dilator. Position within the subarachnoid space is determined by premeasuring the device and is grossly confirmed by fluoroscopy. Great care is taken with manipulation of the dilators and cannula within the subarachnoid space to avoid misdirection and possible neurologic injury. When appropriate positioning of the cannula^' is assured, the guide wire and the 6 French dilator are gently removed from the lumen of the cannula in sequence. Depending on the patient's position on the operating table, CSF flow through the cannula at this point should be noticeable and may be very brisk, requiring capping the cannula or very prompt placement of the capsule implant in order to prevent excessive CSF.

The encapsulated (transformed cells) is provided in a sterile, double envelope container, bathed in transport medium, and fully assembled including a tubular silicone tether. Prior to implantation through the cannula and into the subarachnoid space, the capsule is transferred to the insertion kit tray where it is positioned in a location that allowed the capsule to be maintained in transport medium while it is grossly examined for damage or major defects, and while the silicone tether is trimmed, adjusting its length to the pusher and removing the hemaclip™ that plugs its external end.

The tether portion of the capsule is mounted onto the stainless steel pusher by inserting the small diameter wire portion of the pusher as the membrane portion of the device is carefully introduced into the cannula. The capsule is advanced until the tip of the membrane reaches a point that is 2-10 mm within the cranial tip of the cannula in the subarachnoid space. This placement is achieved by premeasuring the cannula and the capsule-tether-pusher assembly, and it assures that the membrane portion of the capsule is protected by the cannula for the entire time that it is being advanced into position.

After the capsule is positioned within the cannula, the pusher is used to hold the capsule in position (without advancing or withdrawing) in the subarachnoid space while the cannula is completely withdrawn from over the capsule and pusher. The pusher is then removed from the capsule by sliding its wire portion out of the silicone tether. Using this method the final placement of the capsule is such that the 5 cm long membrane portion of the device lay entirely within the CSF containing subarachnoid space ventral to the cauda equina. It is anchored at its caudal end by a roughly 1-2 cm length of silicone tether that runs within the subarachnoid space before the tether exits through the dura and ligamentum flavum. The tether continues externally from this level through the paraspinous muscle and emerges from the lumbodorsal fascia leaving generally 10-12 cm of free tether material that is available for securing the device.

CSF leakage is minimized by injecting fibrin glue (Tissel®) into the track occupied by the tether in the paraspinous muscle, and by firmly closing the superficial fascial opening of the track with a purse- string suture. The free end of the tether is then anchored with non-absorbable suture and completely covered with a 2 layer closure of the skin and subcutaneous tissue. The patient is then transferred to the neurosurgical recovery area and kept at strict bed rest, recumbent, for 24 hours postoperatively. Antibiotic prophylaxis is also continued for 24 hours following the implantation procedure.

Sequences

The following is a summary of the sequences set forth in the Sequence Listing: SEQ ID NO:l — DNA sequence of oligo oCNTF-003 SEQ ID NO:2 — DNA sequence of oligo oIgSP-018 SEQ ID NO: 3 — DNA sequence of IgSP-hPOMC fusion

SEQ ID NO: 4 — DNA sequence of IgSP-hPOMC-ΔACTH fusion SEQ ID NO: 5 — DNA sequence of oligo orTH-052 SEQ ID NO: 6 — DNA sequence of oligo orTH-053 SEQ ID NO: 7 — DNA sequence of oligo orTH-054 SEQ ID NO: 8 — DNA sequence of oligo orTH-078 SEQ ID NO: 9 — DNA sequence of oligo oIRES-057 SEQ ID NO: 10 — DNA sequence of oligo obDBH-065 SEQ ID NO: 11 — DNA sequence of oligo oIRES-bDBH-064 SEQ ID NO: 12 ~ DNA sequence of oligo oIRES-bDBH-066 SEQ ID NO: 13 — DNA sequence of oligo oIRE-068 SEQ ID NO: 14 — DNA sequence of oligo orTHΔ-073 SEQ ID NO:15 ~ DNA sequence of oligo ohPOMC-IRES-069 SEQ ID NO: 16 — DNA sequence of rTHΔl-155 SEQ ID NO:17 ~ DNA sequence of rTHΔ+KS SEQ ID NO: 18 — DNA sequence of rTHΔ-IRES-bDBH SEQ ID NO:19 — DNA sequence of rTHΔKS-IRES-bDBH SEQ ID NO:20 — DNA sequence of oligo ohPOMC-IRES-070 SEQ ID NO:21 ~ DNA sequence of oligo oIRES-rTHΔ-071 SEQ ID NO:22 — DNA sequence of oligo orIRES-rTHΔ-072 SEQ ID NO:23 — DNA sequence of IgSP-hPOMCΔACTH-IRES- rTHΔ-IRES-bDBH-068 fusion

SEQ ID NO:24 — DNA sequence oIRES-074 SEQ ID NO:25 — DNA sequence of oligo oZeocin-077 SEQ ID NO:26 — DNA sequence of oligo oIRES-Zeocin-075 SEQ ID NO:27 — DNA sequence of oligo oIRES-Zeocin-076 SEQ ID NO:28 ~ DNA sequence IgSP-hPOMCΔACTH-IRES-rTKΔ

-IRES-bDBH-IRES-Zeocin-073 SEQ ID NO:29 — DNA sequence of proA+KS SEQ ID NO:30 — DNA sequence of IRES fragment

Deposits RINa/ProA/POMC/TH-IRES-DBH cells, transformed to produce a catecholamine, an enkephalin and an endorphin, as described above in the example (and in Table 2) , named RINa/ProA/P030/P088, have been deposited. The deposit was made in accordance with the Budapest Treaty and was deposited at the American Type Culture Collection, Rockville, Maryland, U.S.A. on June 7, 1995. The deposit received accession number CRL 11921.

The foregoing description has been for the purpose of illustration and description only. This description is not intended to limit the invention to the precise form exemplified. It is intended that the scope of the invention be defined by the claims appended hereto. SEQUENCE LISTING

(1) CΪNEESL INEOMΪΠCN:

(i) APPLIONT: Cytol erapeutics, Inc. (Ebr purposes of all designated states exoφt IB)

Shou Wong (For purposes of US cnly) Joel Saydbf f (For purposes of IB cnly)

(ii) TITLE OF INVENΠCN: PAIN CKTL LINE

(iii) NCM6ER. OF SEQUENCES: 30 (iv) CCRRESPCNDΞ E ALTEESS:

Elrifi

(v) CCMEUER READABLE ECFM: (A) MEDILM TΪEE: Elcppy disk

(B) CCMEUIER: IBM PC cαipatible

(C) CEERATING SYSEM: PC-DC6 M3-DCS

(D) SOFTWARE: Batentln Release #1.0, Version #1.30 (vi) CUPRENΓ APPLICATION DATA:

(A) APPLICKITCN LΗffiR:

(B) FTLINS EKIE:

(C) OASSIΠ AΠCN:

(vii) PRIOR APPLICATION EKEA: (A) APPLICATION NLMBER: IB 08/481, 917

(B) FTLINΞ EKE: 07-JUNE-1995

(viϋ) ATT PNEY/PQENΓ INECPMKITCN: (A) IΨ .: Elrifi, Ivor R (B) REGISIRAΠCN NLMEER: 39, 529

(C) EEEEREN-E/DOCKEπ' N MER: CIT-29 ZIP PCT

(ix) TFTFIXMtMCATICN MFCR RTICN: (A) TEEEHCNE: 212 596-9000 (B) IELEEPX: 212 596-9090 (2) INEOMOTCN ECR SEQ ID ND:1:

(i) SEQ ENΕ CHARSCIERISΠCS:

(A) LEN3IH: 33 base pairs

(B) TYEE: πxieic acid

(C) STRPNLΕD ESS: single

(D) TOPOLOGY: linear

(ii) MXECULE TYPE: c∑tφ. (iii) HYPOTHETICAL: N3 (iv) JMT-SE1BE: NO

(vϋ) MEDIATE SORE:

(B) CLONE: OCNΓF-003

(xi) SEQUENCE EESCRIFTTCN: SEQ 3D ND:1: 033330033 03I 3Ω333r ASGIO3Ω2 TGT 33

(2) INEΠMATICN KE SEQ ID N0:2:

(i) SEQLUCE CHRRPCIERISπCS: (A) IEN3IH: 23 base pairs (B) TYEE: nucleic acid

(C) SMMEENESS: single

(D) TCPdCGY: linear

(ii) MTEEIIF, TΪEE: cQ^.

(iϋ) HYPOTHETICAL: ro

(iv) 2NTT-SENΞE: ND

(vii) IMMEDIATE SCURCE:

(B) CLOE: oIgSP-018

(xi) SEL EKΣ EESCRIPITCN: SEQ ID N0:2:

TΠU UU A33 aκrτ σc 23 (2) πNECRβTICN ECR SEQ ID N3:3:

(i) SEQUENCE OϊrøCIERISTTCS: (A) LENSIH: 849 base pairs

(B) TYEE: nucleic acid

(C) STBflNfFTNESS: single

(D) TCKICGY: linear (ii) MTFΠJLF. TYEE: EISA (gesxniic)

(ϋi) HYPOTHETICAL: O (iv) J^SNΠ-SEΪBE: O

(vii) M4EDIKIE SOURCE:

(B) CLCNE: IgSP-hPClC (ix) FEATURE:

(A) N?ME/KEY: 5'UIR

(B) LOCAπCN: 1. .43

(ix) EEAI FE: (A) JSFME/KEY: excn

(B) LOCKΠCN: 44. .89

(ix) FEATCRE:

(A) NPME/KEY: intrcn (B) LOCATION: 90. .168

(ix) EEKTLRE:

(A) NPME/KEY: 3'UIR

(B) LOCAITCN: 807. .849

(ix) EEA3LRE:

(A) WVE/VEX: π sc_feature

(B) IOCATICN: 43. .186

(D) CTHER INFCHYKITCN: /products "IgS > regicn"

(ix) EE¥TϋRE:

(A) NK KEY: πύ≤c_feature

(B) LOCKLTCN: 187. .806

(D) OTHER TNKRΦOICN: /products "hPCMC regicn"

(xi) SEQUENCE EESCRIPπCN: SEQ ID N0:3: αsααuπr CKI OΣPGF. Giαsαcicr fflα33iαcττ A3KD3« T αsociossr 60

TAICπ πC Ol_HKϊX_flG TG3TEOΩ3 TrøG3333IC CIAPGKrrA AACm3Ω33 120

^■TCEZflARSCT CTGB30GT Q33»ICA3r TlU 'mCT TTCB G33 GH3AΠD33 180

CITI033333 2ftAIG333C CΪG3G33IC TOCOSfflA 0333333A&G TA33ItA103 240 03301033 CIQ33033V TTCU lTAr G3A3Q3G CA 3G333C A33G33333 300

C2G3330Α G3333G3C GICICA3333 G33Φ003 03333333IG CCIOG3333 360

033333033 0333Ω33ΪΓ Q3IQ33ΦGC C333333333 Q3Q333SG 033IO33Cr . 420

03IG3G3. CTTCDXTCG Q33AA33333 TUΪ3 Z¥ A 0333333331 GT&PΩGΪGΪ ^' 480

PΩLU PΩ3G 03333G3C (3GI033333 A33XTΛJX 03IG3GITC 2&3G3 GC 540 T3CI0333A GOS UAi OG33ΩOG 033333033 OXTOUGM¹ (3033J33G 600

0 Q.U. 3 a3C3IQ3G σ OC T33ia333X 03C3A3SG (303Ω33X 660

033-3G3T Q303CπC O CI033GCA 03333333A 03003333 TZπ33333IT 720

TCAΠPCCIC σs zooc CASΏSHΓ TO H3Ω3CΓ GΓKAAAAA G acac 780

A3A3333IA CAA3W333C OGT3Ω33C A3033333C 03Ω33CISC 031033330 840 <3G3I03C 849

(2) INECFMAITCN ECR SEQ ID N3:4:

(i) SEQUENCE OffiROERISITCS: (A) IENSIH: 525 base pai s

(B) TYEE: rax±eic acid

(C) SIRPNEEDNESS: single

(D) TCEOLCGY: linear (ii) MXECULE TYEE: INA (genαnic)

(iii) HYPOIHEΠCAL: NO (iv) JΦVTΠ-SENSE: NO

(vii) I EDIKIE SCU€E: (B) CX£NE: IgSP-hPCMCCPCIH

(ix) EEAIURE:

(B) LOCATION: 1..43

(ix) E5AIIFE:

(A) m& X: exon

(B) I03TICN: 44..89

(ix) EEAIURE:

(A) IW4E/KEY: intrcn

(B) IOCKTICN: 90..168 (ix) FEATURE:

(A) rø*C/KEY: exen

(B) ICCATICN: 169..482

(ix) EEAKEE: (A) NPME/KEY: 3'UIR

(B) LOCATICN: 483. .525

(ix) EEAIURE:

(A) NPM EY: misc_feature (B) IOCAITCN: 44..188

(D) OIHER INE MOTCN: /products "IgSP regicn"

(ix) EEAIURE:

(A) ISPME/KEY: misc_feature (B) X3ITCN: 189. .482

(D) OTHER INFOFMAITCN: /products "hEO regicn"

(xi) SEQUENCE EESCRIPπCN: SEQ ID ND:4:

0310 3331 (3LUXTK3 GI0303IGr <3033I03IT J AK3AKT 03Q3I033T 60

TAILT1LT1C IGMHJ3G T03ITft3G3 TAPG3333IC α3SGI033A AACIT3Q33 120 TCCAD " CIGT30GT Q33KB3CT TlUJJπTLT TTCB 033 GIt3ATT033 180

CπTC03333 LT1U3CJX'1G 3GTK3A3 G33G3K3C TQ333033A CI03333G3 240

3GOQ33X 030333XT G333S303 033303333 03G3333C CIQ303 300

Q33IG3IQ3T 03333333G AA3A3303 A333D333IA 3Q3TG3G O TO333T 360 03333Ω333 Q333S03C ΛA3333303 0333ITKAT GAX1LU3G AP0G33O. 420

(3333331031 (3033IGITC AAZWΩ333A TCAT3A3A 0333I7OA3 AA33333GT 480 (30333 & CQ333333G Q33m333IC 03330303 T03C 525

(2) iLNEOMOTC FOR SEQ ID N0:5:

(i) SEQϋE E OErøCIERISTTCS: (A) LENGIH: 30 base pairs

(B) TYPE: nucleic acid

(C) SIK TTNESS: single

(D) TCEOLCGY: linear (ii) MIFllJLE, TYEE: EN .

(iii) HYP3IHETTCAL: NO (iv) -^ONπ-SEtsEE: ND

(vii) IMYEDIATE SOURCE:

(B) CLONE: orTH-052

(xi) SEQUENCE INSCRIPTION: SEQ ID N0:5: 033A33TIG C2CIAIG33C AD3333033 30 (2) INECHΦTTCN FOR SEQ ID D: 6:

(i) SEQUENCE CHARZOERISTTCS:

(A) I£N3IH: 30 base pairs

(B) TYPE: nucleic acid (C) STRSNEETNESS: single

(D) TOPOLOGY: linear

(ii) M3LB3UIE TYEE: cENA. (iϋ) HYPOIHEΠCAL: NO

(iv) ANTI-SENSE: ND

(vii) IMMEDIATE SOURCE:

(B) CLONE: orTH-053

SUBSTTTUTESHEET RULE2β (xi) SEQLHCE EEΞCRIPΠCN: SEQ ID MD: 6:

OJJGCW-LU Γ AlU Ai lAS dAAK333C 30

(2) INFCFMffilCN FOR SEQ ID ND:7:

(i) SEQUEME CHARACTERISTICS: (A) IEN3IH: 30 base pairs (B) TYEE: nucleic acid

(C) STRANTETNESS: single

(D) TCEOEOGY: linear

(ii) MlFπ.ITF. TYEE: cΩ<S

(iϋ) HYPOIHEΠCAL: ND

(iv) JΦvπ-SElvEE: NO

(vii) IMMEDIATE SOURCE:

(B) CUNE: orTH-054

(xi) SEQUENΕ EESCRIPπCN: SEQ ID ND:7:

OCCAAGCΠΆ TCOIUXCIG GΠCCEKO 30

(2) INEO ΦΑTCN ECR SEQ ID NO: 8 :

(i) SEQUENCE CHARACTERISTICS:

(A) ENGIH: 33 base pairs

(B) TΪΪE: nucleic acid

(C) SIRANrFrNESS: single (D) TCP3LOGY: linear

(ii) MJFπiF. TYEE: cENA

(iii) HYPOTHETICAL: NO

(iv) ^NTT-SENSE: NO

(vii) MEDIATE SOURCE: (B) CLCNE: orTH-078 (xi) SEQUENCE EESCRIPπCN: SEQ ID NO: 8: 33AG3TIC 03303203 T0333IQ3IT OX 33 (2) JNEOttKTICN ECR SEQ ID N0:9:

(i) SEQ ENΕ OfflRACEERISπCS:

(A) IEN3TH: 30 base pairs

(B) TYPE: nucleic acid (C) STRA CEDESS: single

(D) TCEΌDDGY: linear

(ii) M3LB3ULE TYEE: dOSA (i i) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vii) MEDIATE SOURCE:

(B) CLONE: oIRES-057

(Xi) SEQUENCE EESCRIPπCN: SEQ ID ND: 9: AAG33D33 UJXTLTUJC TUllilOX 30

(2) I ECR KπC EOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS:

(A) LENJEH: 30 base pairs

(B) TYPE: nirleic acid

(C) SIRANEEENESS: single

(D) TCECLOGY: linear

(ii) XEEULE TYEE: dNA

(iϋ) HYPOΓHEΠCAL: NO (iv) .ANTI-SENSE: NO

(vii) MEDIATE SCtFCE:

(B) CL tE: ctIEH-065

(xi) SEQ3ENΕ IESCRIPΠCN: SEQ ID NO: 10: ΛAAΩ333333 03303πCA G33πTQ333 30

(2) TN OMαiαN ECR SEQ ID ND:11:

(i) SEQUENT CHARACTERISTICS:

(A) LEN3IH: 30 base pairs

(B) TYEE: nucleic acid

(C) STRANEEHESS: single (D) TCBXOGY: linear

(ii) M3LE3UIE TYEE: cDSA

(iii) HYPOIHETICAL: NO

(iv) JANET-SENSE: NO

(vii) MEDIATE SORE: (B) CUNE: oIRES-iEEH-064

(xi) SEQUENT EESCRIPπCN: SEQ ID N0:11: CπυJ30A 03003033 (3033333IG 30

(2) Γ EOMOTCN ECR SEQ ID N0:12:

(i) SEQUENCE CHARACTERISTICS: (A) IEN3IH: 30 base pairs

(B) TYEE: nucleic acid

(C) SIRANEEENESS: single

(D) TCEOmr: linear (ii) MTFΠTTF, TYEE: cDNA

(iii) HYPOTHETICAL: ND

(iv) ANTI-SENSE: ND

(vii) MEDIATE SCLKE:

(B) CLCNE: oIRES-tfEH-066

(xi) SEQESCE DESCRIPΠCN: SEQ ID ND:12: 03333IQ333 T3 T03ΠG T033AA03IT 30

(2) INEOMΪΠCN ECR SEQ ID M :13: (i) SEQUENCE CHARACTERISTICS:

(A) LEN3IH: 30 base pai rs

(B) TYEE: nucleic acid

(C) STRANEEHSESS: single

(D) TCEOOGY: linear

(ii) TFCUIE TYEE: cQΦA.

(iii) HYPOTHETICAL: NO (iv) TI-SEΪEE: O

(vii) MEDIATE SOURCE:

(B) CLONE: oIgSP-068

(xi) SEQUENX EESCRIPπCN: SEQ ID NO: 13: iAAAO-TATOS 03333333IC M333C G 30

(2) INEOMAΠCN ECR SEQ 3D D: 14 :

(i) SEQUENCE CHARACTERISTICS: (A) IIN3IH: 25 base pairs (B) TYEE: nucleic acid

(C) STRANEEENESS: single

(D) TCEOOGY: linear

(ϋ) M EΠTΓF, TYEE: CDMA.

(iii) HYPOIHEΠCAL: NO (iv) ANTI-SENSE: NO

(vii) MEDIATE SOURCE:

(B) CLONE: orTHD-073

(xi) SEQUENCE EESCRIETTCN: SEQ ID ND: 14:

ADOCCiαS T3 SAA03 03333 25 (2) INKHffiπCN ECR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS: (A) IEN lH: 30 base pairs

(B) TYEE: nucleic acid

(C) SIRANEEENESS: single

(D) TOPOLOGY: linear (ii) MTFQ1F, TYPE: cQΦA.

(iii) HYPOIHEΠCAL: ND (iv) _ONΠ-SEΪEE: ND

(vii) MEDIAE SOURCE:

(B) CLONE: chPCM IBES-069

(xi) SEQUENCE EESCRIPπCN: SEQ ID ND:15f 033333033 A30333333 O IGT03XT 30 (2) INECRΦAπCN FOR SEQ ID N0: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) IEN3IH: 1030 base pairs

(B) TYEE: nucleic acid (C) STRANEETNESS: single

(D) TC DLOGY: linear

(ii) M3EB3ULE TYEE: ENA (genariic) (iii) HYPOTHETICAL: ND

(iv) ANIT-SEΪEE: NO

(vii) MEDIATE SOURCE:

(B) CLONE: rTHD

(ix) EEAKRE:

(A) N*ME EY: 5'UIR (B) I03TICN: 1. .6

(ix) EEAIURE:

B TTTUTE HEET RULE 26 (A) E*E KEY: ex n

(B) LOCATICN: 7. .1017

(ix) EEATCPE:

(A) N3E/KEY: 3'UIR

(B) LOCATICN: 1018..1030

(xi) SEQUENCE EESCRIPHCN: SEQ ID ND:16:

AAUJrJALU3 T03XTG3IT 033AGAAAA GTGID33AKr T03 AGTG T30303IG 60

GE303A?Cr TK3033K3. TCIG303IG 30303333 03πCICH3. 03 331031 120 C033Ω33IC Q3AA03B3T T03 A1T 033Trα3GT A3AG3033 T3AAOC2AATT 180

033310103 AATAOOG3 Q3AAG IT 033031031 A0303IATΑ 101303310 240

ΛAG3330TCT AIQJfAOXA 1033103333 0*3303103 Aϋ l lULΛ GCITCIQSAA ^■ 300

033IZ 3IG 03320330. 03 03TC 03O03IQ3 7Ω303IGIC 033311010 360

AAG3G333A. CT0XTT03. 031033033 GHX-U-OJTC TACIGI033C 033B3πTT 420 CI0333GIC lUAJ TIUJG CGIGΠTOAA T03 3GT ATAiα3333A TG33103ICΑ 480

03EAia3ATT σ03IGA03C 03003103 CAD303TGT T033 IGT A333ATGITG 540

03130333. CAITTOIXA GT1UIUJ3G (3CATI03C TTO3AICICT Q333333TCA 600

C3T3AA£3AA TT3AAAAACT CI03033IG TZ 33IT0A CIGIQ3A.T 03333IKD3r 660

AAACAGAAIG 03303P3A Q33ITAD33T O30333IO3 TGICπOCTA 033GA03IC 720 CI03 333 TGB3 03A. Q33H3Q3IC 03Q33πTG JA033OOC A33G3IGIG 780

CAG333303 AAGAKAAAC C3C3Ω33T GIG3CITTG TGI033OG CIT3AAH3C 840

033AAG3O. A33T3Q3A CIAI033ICT CGIAD33Q3 ODCEATICTC TGK3AGITT 900

(3G333BO. <3CI0333T T3033CIG (3O0333IC A303T03A. 0333103110 960

(303333103 A33n30 T 03 033IG 0333033C T3GI033T TA333AATG 1020 CA3Q3AI03 1030

(2) INECEMATICN ECR SEQ ID N0:17: (i) SEQUENCE CHARACTERISTICS:

(A) LEN3IH: 1037 base pai rs

(B) TYEE: nucleic acid (C) STRANEEENESS: single

(D) TOPOLOGY: linear

(ii) M Fπi.F, TYEE: ENA (gerrrnic) (iii) IiYPOTHETICAL: ND

(iv) .ANTI-SENSE: ND

(vii) MEDIATE SOURCE:

(B) CLONE: rTHEKS

(ix) EEAIURE:

(A) NPME/KEY: 5'UTR (B) LOCATICN: 1. .13

(ix) EEAIURE:

(B) LOCATICN: 14. .1024

(ix) EEAIURE:

(A) NJΪE/KEY: 3'UIR

(B) I03AΪTCN: 1025. .1037

(xi) SEQUENCE TESCRIPΠCN: SEQ ID ND: 17 :

JΦUJΠUJJ ; j Aiosiαc UJIUJΠUJ : AAGAAAAOIG TCOGAAΠGG TOAGIOEA 60 0303IQ3IC JΩ3AAGTTTG J0 33CT 0303IG3C (30333333T TCICT303A. 120

⁽331031033 020331033. A33II3--TG3 A3SATIG33 TT03A03CA A30AO33IGA 180 iΩ3ATTCD3 CAD3I03AKr J OG333A. AGAGKΠOCT A33IG3AG3 J*33DATAIGT 240

CA333TGAAG G3XTCIAIG C3033AT03 CIQ33333G 303IQ3A03 GΠTO3Q3T 300

T3IG3AA033 T3 0IUXT 2033 G3A. CAQ3AΪ03CΑ (303T0303 A33IGI0333 360 CπCITGAAG 30333 0 TKOΏCT 033O 3IG Q3333ICrAC TOI0333333 420

T3-i πL'lG 033 CIQG OJiTUJ333T GITTCAKrOC A333GTAIA 103333003 480 CTLLTU ' Aiα3Tfl3C CI303333A. CIQ3I033T 303101103 (3 1G303 540

C IOΠU Γ GAOCOCACAΓ TTQOOCAGIT CΓO3303C AΠO3CΠG CAΓCTCTO33 6OO

03XT3 T SAAGAAATIG AAAAACICIC 3CG3IG3C TG3TICACIG T03ATIO33 660

G3IATGTAAA (3SAKr0333 A33K3AG33 TrAT03I03A. OSXTXTOT CTT33TA033 720 A3G3IC3IG C3O033IGr CA3G3G33 T3G3I033A. GJ3ITH303 0A3 OΩ3 780

A33IGI03G α33rA03AG AT3AAAC0IA 03033IGIG TTOTTGIGr 033 03IT 840

OAK30333 ΛAG3 A03 T3G3AACIA TU TCIU3T AIO3033X CAπCTCIGT 900

SAAGTTD3C 033IZ CAC T0333ATK3A. 033CIG3C A3333ICA3A. 03AT03033 -960

C1UJ1TU30 033310303 AD3G3IG3A. 303310333 30J3CKA GT033TIAG 1020 CIAAKiαCAr 2Q3AT03 1037

(2) INEOMATICN ECR SEQ ID ND:18:

(i) SEQUEME CHARACTERISTICS: (A) IEN3IH: 3425 base pairs

(B) TYEE: nucleic acid

(C) SIRANΓEENESS: single

(D) TC IOGY: linear (ii) M1FY1JLE TYEE: EfcA (genαnic)

(iii) HYPOIHEΠCAL: NO

(iv) JANTI-SEIEE: ND

(vii) MEDIATE SOURCE:

(B) CLONE: rTH-IRES-tCHT (ix) EEAIURE:

(B) IOCAΠCN: 1. .6

(ix) EEAIUFE: (A) KPME KEY: exen

(B) LOCKΠCN: 7. .1017 (ix) EEAIURE:

(A) NAtE KEY: intrcn

(B) LOCATICN: 1018..1617 (ix) EEAKFE:

(B) LOCATICN: 1618..3411

(ix) EEAIURE: (A) N»E KEY: 3'UIR

(B) LOCATICN: 3412..3425

(ix) EEK JRE:

(A) NMl KEY: ιrάsc_feature (B) LOCATICN: 1025..1617

(D) OTHER INEO ATICN: /products "IRES sequence"

(xi) SEQUENCE DESCRIPTION: SEQ ID ND:18:

2AUJl'iAlU3 lUJJ 'lUΪLT 033AAOAAA GTGI033AAT TQ3OA0IG T303A03IG 60

GKA33AAGT TT3033IO. TCIQ3D3IG GA0303333 OCπCICTO 03031031 120 0333G33IC G3AAΩ3H3T T03 OTT UU TΛIBGT A A03033 T3AA03ATT 180

033310103 AA300Q3 Q3AS TT Q33CCIQ3A. A33G3TATA TGICA033IG 240

AAQ3333ICT AD33K033L T033I03333 3G303I03 A033πT03A. Q3πCIG3A 300

03330010 G33C03O. G3 00A 3 03003103 A3303IGIC 033311010 360

AAG3G333A CT i IOA G3IQ330X GI033333TC 3CIGI0333 CDGIGATΠT 420 CE33330C 1033311033 UJIUI ICAA 1Q3033GT AEAI03333A. 1G33I03KA 480

03DAT03ATT C3O3H3G33 03O03I03 CAT3Q3IGT ϊ033OIGr A333ATGTIG 540

Q3IGA0333A. CAiTiUJJ3A GT1LIUJ3G (3 TI03C TT03ICICT Q333333ICA 600

3CT3AGAAA TIGAAAAACT CI03033IG 3003110 CIGIQ3AATT 03333IAIGT 660

ΛAAOGAALG Q33G3IOA QX IA1U3T 030333103 IGICTI03IΑ 033OQ3IC 720 CT03CI0X TGP3O03A. Q33IGPG3IC 030331110 AD33AGPOC A33Q3IGIG 780

30333303 2AGAP3AAC C303A033T GIGTAOTTG 1GT333 0 CTTCAAT3C 840 Q33AG30 A33ICA33A αAiα33ICr 03310303 G3330TCTC TGIGAAGTTT 900

3033330 C2O03D3T T3Ω33CIG 3O0333IC A303AI03A. G333IO3ITG 960

(303333103 2Q33GAG3T G3O033IG 0333033C T3 033AT 3Q3IAAAIG 1020

020203103 (JJJJ 'IL'IUJ CI03333333 adAA03TIA CIQ3333AAG 0 TIU3AA 1080 3 rrro33τ GIUJ3ΓΓI T OAIKIGΠA TΓHU AOO TKΠΠXGΓC TI IUOAT ιi40

GK3Q33333 Q3AA03I03 UXTGTCTIC TTO03G3. T1CCI20333 1CITIO333T 1200

QUJJCAAAG OAIQCAAQS^'TCiσπGAAT GIOJiOAAOS AA33 103 1CI03AA03r 1260

TCTH3AGAC JAAA AC3IC TG303303 OTT03G33 G333AA033 0330O033 1320

(30031UJ3 TU3U33CA 2AAG33D3T GIA3A IA C2O3iα02AA 033333OA 1380 03330033 203I1GD3G TTGSATAGIT GIG3AAAOG T3AAAIQ33T CI03IOAA03 1440

G3ATICAA Xmi.lXZA 03003330 2AQ33Q333 AI GIAI033 AIU10AT 1G 1500

Q3333iα33T 03 I03IT TAOαGIGIT TSGICOOOT T7AAAAAA03T CTAQ3333X 1560

03AA03α33 QGAϋGIU ϋT TKCITIOA 2AAO03AΪG ATAAQCπOC C2OA03ATG 1620

T20333033 CQ3IQ3333T CTIUJlUJlt ATOOICGIOS CI03003A. 0333103331 1680 0333333 Q3333TTOX CTT03OIC 033310303 0330333C 031030310 1740

I03I03AO 13033103 00203003 AP33OT03 A03IO3IQ3T 0333303TC 1800

2AG33I03IG 1031011103 GAIGI033C 03G333O3 T03 AIO3 T3 T03IG 1860

GIQ3ICIG3A. CE3O033A. 0333333I2C TTI0333aG 0310300 03 A0333 1920

3Q3IO303 TG3 033A. Q3Q3AT3C (30311003 0333 OG (3 002OA 1980 0333TGI203 1UJILTJOA 30333TπT 033D3IGIG 2Q333AA03A. C303ICAIC 2040

3Q30333A. OCOTCOOT 03I01ΑT03A. TT03I03Ω3 A33333T033 GIQ33I03G 2100

103ATCAA OI03333IT 0C2O03333 O03O033 10303100 GAAO33303 2160

AI0333A03 0333331033 03333003 CQ3A03IG3 A IC03333 0333303IC 2220 OO.LO3333 033A03 C 3033OQ3 1G3303IO O33G3I033 030330IC 2280

0333333AO3 2OI03I T G3C303X AD33TC2033 2G333AA03A. Q3333I03IG 2340 (3030103 20310100 GIG3333333 (301030 03103033. 01003333 2400

03310330 03A 13A Q3333G333 OOAOIO 03331000 000333333 2460

1033333103 03333APO33 CπTIA02C 03O03AAG O0333IQ33 010333333 2520

033333100 COOTTIO OJGXI03A GTIC2O203 20A0330 03D3AIAAO 2580

0333333333 2O0O0333 CAI03333IG TA 2O033 O0333IG33 033311030 2640 G33333AIO 10301033 03I03333C 2033333IO 1O333AT033 0333303G 2700

2033331103 103100333 C3O0303 3OAO0O 033031033 0310333333 2760

TCTQ33HC 20301033 O O03IC (30030203 130333333 GAA03IG3IC 2820

2000003 03Q33A033 0333300 O 303IO 2O033 AA 03C3 03 2880

03 O103 20301033 OIGTIOAG AAO3I03IGr CIGICOG33 G33 03IG 2940 OOIOOO 01Q30ATA CAAO033AA O G3G33 10333030 0333333110 3000

033ATσθ03 2Q3 I0IG COIOAOKr 003020 203333OC 030O03G 3060

OOG3A 033333IQ3A. O IOXTIC Q02OA 20103330 03IOA003 3120

TT A 033 2Ω3AO G C20O033X C2Q333I G T033I3ΩO GITI03O03 3180

003331002 IU010A 0333303IG CI AG3333 1G3033OT CD3033AIC 3240 T03AI03 G3AAO03TC O033333IC 033H0303 G33 03A IO3302G0D3 3300

CI030O TU31GI03G O103AAOG 0330333IC 2O0333G3 3Q3303O 3360

C2GA033333 03333333C 03I0O AC AD3O0333 03AP030G 2A03I03333 3420

03333 3425

(2) INECFMATICN ECR SEQ ID NO: 19: (i) SEQUENCE CHARAOERISTICS:

(A) LENGIH: 3432 base pairs

(B) TYEE: nucleic acid (C) SIKAMEENESS: single

(D) TCEOEOGY: linear

(ii) MJFΠJTF, TYEE: I2ΦA. (gaxmic) (iii) HYPOTHETICAL: NO (iv) 2NTI-SEΪBE: NO

(vii) MEDIATE SOURCE:

(B) CLCNE: rTHHS-IRES-*£EH

(ix) EEAIURE: (A) IWE/KEY: 5'UTR

(B) I03TICN: 1..13

(ix) EEAIURE: (B) IOCAΠCN: 14. .1024

(ix) EEAIURE:

(A) ΪΦME/KEY: intr n

(B) IOOTICN: 1025. .1624

(ix) EEAIURE:

(A) NPIE/KEY: excn

(B) IOCKTICN: 1625. .3418 (ix) EEAIURE:

(A) røJE/KEY: 3'UIR

(B) X3HCN: 3419. .3432

(ix) EEAItRE: (A) rø*E/KEY: ιnisc_feature

(B) IOCATICN: 1032. .1624 (D) OTHER INFCEMATTCN: /products "IPES sequence"

(xi) SEQUENCE EESCRIPπCN: SEQ ID 0: 19:

AAG3TKJ3X 203303X0 ϋCTCOπ U 2AOAAAOG 1033AAπ03 2OAO0TO 60

0303I03IC 203AAO11G 20 3I I Q30003C C2033333O TOOOOO 120

031033033 C2033T33 203T H03 2O 31033 TT03O2O 2GC2C33I 180 2COA31033 O30T33AAT 2000333A 230TIQO 203IO3ZΩ3 20333210 240

C203O AG 033 OAIG O2033AI03 CIQ33333AG C203I0303 GTITCOQO 300 TCiσ3A033 T2C1U1UXT 2033000 0033030 0O0303 2031010333 360

CπCTTOAG O0333 G QOTKOGO 033033310 Q3333IC3C TGI0333333 420 loamαo QOOOCIQS 031103330 GΓΠOKIOC 20330ATA 103333103 480 ocoσαo 2303310c 0003330 OOOOOOT oooσriαs SOIGIACC 540

03011030 30330203' 0033300 03C2G3AC 23103OTG CAΓO O33 600 Q333TOOT AOAA31G 222AAOOC C2033IGI2C TQ3TI OG lOG ATTCOS 660

GOAI03AA C2 A30333 20OO2Q33 T33Q3IGO 0330000 OT033033 . ^'720 ^■

20000310 OCI0X1GT C2OQ30X 10031030 Q33ITT3CC OOOOGD 780

2GOGI03G 033I203AG 23 AA03IA COOOOOTG TAOTIGIO 033 00T 840

CAAIGAOXC 2A03 AG3 TO03AAOΑ 10331000 2300203333 0231000 900 020TE3C 0333 OC 10333AIT CG3O03C 2Q33000 003002033 960 σα 1Q3G Q333IO02O3 AI3Ω3IG 00300333 303300 033ATTAG 1020

CTAAA303T 203303333 000030C 03333333O 2A03TI2CIG G333AA0333 1080

CTIG3AI2A 0333331010 COTIGIOA TAD3TTAITT TO02O3T23' T0333IOTT 1140

T333AA3GIG 2G3333333A.2A0O033X TOrOTOTG 20303A3TC C30333I 1200 TT0333IOC Q33AAA03AA 10C2AG3I OTOA30IC O A03AAG 30T300 1260

Q3AAGOTO IGAAOOAA (3A03TO 203303OT TQ3Ω33G3 032033333 1320

203IG333C 203I03O 033333AAAA Q3303IGIA TAA0320C CIQ3AA033 1380

G33 A033 C2OG3303 TIGTOGITG O32O10IG OAAOGTCA AATGX1CIC 1440

OOAG33IA 1T AOA03 Q33IGAAO T0333OAG GTA03333T A30333C 1500 T030Q333 OJ1U 3100 C230OTI2C 23GIOTI2G I0303TIAA 2AAAC3IOΑ 1560

Q3333333020303330 03103000 OTIOAAAA C2033O3A A3O1O302C 1620 22O0AIGI2C Q33CD3333103333I T 0O03ICAIC 03IG33I00003G33 1680

CT03X1OIJ 0333 033 ULΥ1UJLU1T 03030333 CIQ303333 A333303 1740

Q3G3IOIO3103AACA3 03330330 Q3O0CAIC 301002031031031033 1800

03AGO AG 0310310103 'lUlTlULOT 0033030 Q3330 O32GAA303I 1860 010310310 CICIG3O02 G330330333300 Q333303O Q3OO0O 1920 A03333G 000200032OC030 Q33T2C3G 01003333 C20003C 1980

103 AQ3300200031CTIOA0303310103320O0IO03 C A03 A 2040

0O 303G GAQ3330331030000 GTAI033TC CI03030303O0333IC 2100

Q Q3 03 AT0200T 033X003. O033333IG OGAG33IO 2GO03I A^' 2160 Q333G30C 033AA033330331033333 Q3 033332002303010333333X 2220

03O3IU3HJ 2303333333203A 03AC G3OQ3I03 T203IO03G 203103330 2280

03331100030333020 '10310303. C 03333C 033A0303A03G33 2340

Q3K33IQC2C C2 303G3101030003333333G TT030001033302OT 2400

OΘ33333X 1Q33O0 2 3 A0X O02O333OC 2AO1O0X OD3I0O 2460 033333003 Q33O03333 C AQ330T OA3303O GA0OA03G 03OG333IT 2520

0333333333 G33IO C C231TO0330O03APGO C2 203A 2033AO0 2580 0AA G330333333 00033302103333IG3C 30033000331033333 2640

C1IU30333033AI 3O320OG333O 0333120030333IOIQ303AIU333X 2700

Q3AQ3GA030X0031031 03333TA CI03033C 2AGIO02OX 2QO0333 2760 03333331003331 1O1O330C TC2O O02C 203303IO 03333333A 2820

G3KO G3I033 03303333303 OG 2303IOAO 033 20 2880

C3 033 (3010203203033021 OTOAOAG O03IGIOG ΪO0AG33333 2940

2003KOC 23O0OCIT 0C2O32 A C20332 C 2Q3033IQ3030331033 3000 0333110333 230310303 2 TGI03 OAC3IGTG OC3O203 03020030 3060

QOG3G3IC T03A Q33 033103033 T03OTα3IG 0OA02 T03333I0 3120 OA G3TIC AAO033Q3 AAOO03C O0333302G 033I O03 CI G02OT 3180

1033103310 CD3IQ3A 03TIOA033 C 03IQ3IC 22033300 203330033 3240

2D33AICI03 A1U3O0 20Q3I03rC 0333310333 TTC 03333 2 Q3AAI03 3300

03033300 OOOOICG 1GICO03 03AAOQ333 2033000 03330330 3360

C 03OOG 2033333333 G3333A03 0O AO3C 2O03333 AA03OO2C 3420 O03333333 03 3432

(2) INECR4A3TCN ECR SEQ ID ND:20:

(i) SB3 NE CHARACTERISTICS: (A) LENGIH: 30 base pairs

(B) TYEE: nucleic acid

(C) STRANTETNESS: single

(D) TCEOEOGY: linear (ii) MXEEULE TYEE: HΦA.

(iϋ) HYEΌIHEΓICAL: ND

(iv) 2NO-SENEE: ND

(vii) MEDIATE SOURCE:

(B) CLCNE: c PQ -IEES-070

(xi) SEQUENCE IESCRIFΠCN: SEQ ID N0:20: 20333003 033333310 0331033333 30 (2) INECHΦ3ICN ECR SEQ ID ND:21:

(i) SEQ3ENE CHARACTERISTICS:

(A) IEN3IH: 30 base pairs

(B) TYEE: nucleic acid (C) SffJANTETNRSS: single

(D) TCFOOGY: linear (ii) MTR3ULE TYEE: LWtλ (iii) HYP3THEIICAL: ND (iv) ANO-SEKEE: NO

(vii) TMEDIATE SCURCE:

(B) CLOE: oIPES-nHD-071

(xi) SEQUENCE DΞSCRIETICN: SEQ ID ND:21:

OAOC2Q333 203AIG3 G 1033AA03 -30

(2) INECFMAΠCN ECR SEQ ID N0:22:

(i) SEQUENE CHARACTERISTICS: (A) IENGIH: 30 base pairs (B) TYEE: nucleic acid

(C) STRAMEENESS: single

(D) 1CEOL0GY: linear

(ii) MΣE33ULE TYEE: cENA (iii) HYPOHEnOL: NO (iv) ANO-SE^EE: ND

(vii) IMEDIAIE SOURCE:

(B) CICNE: oIRES-rTHD-072

(xi) SEQUENCE EESCRIETICN: SEQ ID ND:22:

C00330A 03AIO3 O3 0030030 30

(2) INFOMAITCN ECR SEQ 3D NO:23:

(i) SEQUENCE CHARAOERISTICS:

(A) LEN3IH: 4499 base pairs

(B) TYEE: nucleic acid

(C) SIRA TETNESS: single (D) IDEOLOGY: linear

(ii) DLE3ULE TYEE: ENA (ga mic)

E 26 (iii) HYEOIHEΠOL: MD (iv) 2NIT-SEJSEE: ND

(vii) -MEDIATE SCCKE:

(B) CECNE: pαrc-th-cfch fusion (ix) FEAItRE:

(A) NPME/KEY: 5'UIR

(B) lOCATICN: 1..43

(ix) EEAIURE: (A) 1ΦME/ EY: excn

(B) lOCATICN: 44..89

(ix) EEAIURE:

(A) ΪWE/KEY: intrcn (B) lOCATICN: 90..168

(ix) EEAIURE:

(A) røME/KEY: excn

(B) IOOOCN: 169..482

(ix) EE23T E:

(A) NHE/KEY: intrcn

(B) IOCA3TCN: 483. .1080 (ix) EEAIURE:

(A) NHE/KEY: ex n

(B) LOCATION: 1081. .2091

(ix) EEAIURE: (A) NHE/KEY: intrcn

(B) lOCATICN: 2092..2691

(ix) EEAIURE:

(A) 1ΦME/KEY: excn (B) lOCATICN: 2692..4485

(ix) EEAIURE:

(A) NPME/ EY: 3'UIR

(B) lOCATICN: 4486. . 499

(xi) SEQUENT EESCRIPπCN: SEQ ID ND: 23: OX-HXOICT 0033330 OCO OGT OOSOOOT 20AI A2T 03GOQ3 60

TATCriLTlC C1OI033G TUJi'120Q3 TAA03333IC 03AAOCD AAC OQ33 120

^■KOOAAACT CIGIOCZO Q33AIOO 1103000 0020033 O A3T033 180 THJJLUJL'. 010333310 GAOIOAO Q330OOC TQ333A03O 003333Ω3 240 IQ3333 03Ω3333O 0333AIGA03 G333AQ3333 COΩ3333AC 003030 300

033IQ3IOO Q3333333G AA A0303 203333333. C203303G 3C 033O 360

Q3333G3X G3332G3C 220333303 0333100 " 3U3TCCOG 2A0G33O 420

0333X100 GAOXTOTT AAAAA033 T3AT A A 0333I2C22G 2A03333 ^• 480

3G3302 G q -mX TC 10X103333 03333T2A03 02003333 22Q3333I1G ^' 540 OATAAG333 031010330 TGIC33A3G TIAlTπUO COlAriQX 0001033 600

AAIGI3G33 03333AAA03 10333OOC O 1 0O Q3A 033G Q33IO1103 660

0000330 AAG3AAIQ 203I 1G AA3GI03I 2032030 10O Q3AA 720

GOTOIOA OOAAOAC 0002033 20330100 Q33G333A 0333330O 780

0333 0 033100333 03AAAA030 03IG33APG 23200003 222033330 840 0203330" G3303ITGT OO103ATA O1GI03AAA OOOAAIG OOOOOO 900

2033331 2OAG3330 OA03AIQX OGAAQ33C 03331031 Q33ATO T 960

CI033333IC G3I002O3G C012O3CT OT3O0 Q3TIAAAAAA COCI203X 1020

03333AA0 033330310 GTTOCOO GAAAAAO03 23 3AA0O 1Q33OA03 1080

230310330 0300X220 AAAAOGI03 OA3IG3AO 2OOO0 COOOOOC 1140 22O O03 CIOICIOO O3IO3O02C 03333O1 OO03Q GTA-OSCOG 1200

Q3I033AG3 TO3103O GA31033ITC C2G3OA03 2033I AC3 AAOCOCOT 1260

0032320 0333AA 03103303 TQ3AAQ303 TA33GIOC QO A0333 1320

CICTAIUJIA 030230300 0333303C CI03G33O T030OT 03AA0333C 1380

SUBSTTTUTE SHEET (RULE 26) Ϊ 033I2CC OOQ3C2G O303C2OG OQ30303 1GIO3330T OTOA03G 1440

0330030 T030O033 20333IQ3X 0310200 0333333IO OTIO03X 1500 2OO033O TU.U.GTG 1OA30303 OOAIADX 03C23O33TC OO033IG 1560

OTIOOOG 203333 G CK333AT G O0 033AC AD33033AT OIGXTOC 1620

0330300 033O1CIC 0020300 Q3O103AT CI Q33333 OOOIOA 1680

OAAOOAA AAOOCOC 03IG3OG3 000003 AA 0333O 23GI2AAOG 1740

2A30333G3 1 A033TIA 103103033 OOOGTOT 0330330 OOOOOCAC 1800 T03OGIC2G 2Q3033T Q3TG330X TII 0302G 1Ω&GS&: 1GIQ303X . 1860

T203AAGATC AAA033CO G33IGI03C T GIGI033 200300A 1O0333AAG 1920

OOAG3I 032C3T03 0003330 OOD333T lOOOOA 0000333 ^' 1980

T2ODO03 CO OOO 2CIQ30G3 0OC2O0O TOCAQOOOC 01030333 2040 0C2G33G 203I002OC 0003X C 03AOOOG COOAOOA 2A3O0232O3 2100 23033333O CI033I03X 03333332C GT3003X OA03333 Q3AA322Q33 2160

C03IGIG3 GICI2TAT GΠATOTX 203AT23103 03100103 CAATOO03 2220

Q33333AAAC CIG3333I C O1 03 2Q33T033. G333ICI C 033I 0333 2280

AAAQ3AAT03 2AG3ICIOT A3GI03IG 2A03A03G OOOCIOO 2001 1 2340

200AAOA 03100203 O03O 03 203303302033330203 1Q333O03 2400 10300033 Q33AAAAQX 203IGI23AA O32O0OG 32AQ33333 2 A0333AG 2460

TGOOCΠΠG TOGΓIQSAT 201GI03AA 2000AAΓ Q3dCiα3IC AA03333IC 2520

220AQ3333 T3AAG3A303 03OA333 0333Ω1 Α 1Q33AIO ΪOQ33330 2580

03310301 03102030 '1OT1201U3 2Q3ITAAAAA 203IC3G33 CDXU3A03 2640

20333300 QSOTKXO lOAAAAOC 3OTAA03 0302OAC 03030333 2700 2D33333IQ3 03310100 Q3IOI03IC GIO33IQ02C 103AQ333IC 03 UXJ3X 2760

0003330 10333000 O303333IG 30333303 0303000 00003103 2820 AACATOGO 2I0333G3A. 033AIC3C ΩO02O3IO3 TG r :03^":A GOOAG30 2880

03IGI0OO OOCOIGIC Q3G33033 O03I03O 2303IOOT Q3IQ3IQ3P3 2940

T03O O 033033333 0200033 (3303000 OO0C2 A G333303IC 3000 03T03 0330303. T3O02O3IT O0333302C 2OQ3O03 2OA0333IG 3060 TAOOOOO T A 0333 00030203 TGIO033 2030200 O303Q3C 3120

0330003 2OJIU3I01Α TG3O0OG GAQ3Q3333 TXOOCQO Q3O002IC 3180

2AO α33 U 1UJ C Q3333I03G 2Q33IO02O3 T03I 2033 C2O0AIO3X 3240

AAO33330D3 TQ3333333A, O03333A03 23Q3GAT03 033333330 03I0OO3C 3300

O3333302Q3 2G20303IΑ OQ3I033C GIO03303 103333033 O10333333 . 3360 0030103 T TG30 GX AIU3IC 2033AQ33 203G333 Q3IQ303C 3420

2303Q3I 10300333 03333O1C OO02I03 03C2O1OG 0333333103 3480

G 003AO 1 A03333A. 0333OOAC 0333IC A03I03IQ33 03XTG33X 3540

003333302033O TA OA033OG AQ02Q333 1Q333O033 0333333333 3600

ICCICOOT 0003330 03AAO1OC T203OA03 C2 03IOT AA0033333 3660 αXGAOUO 033X23033 0OGI2OA3 2033OQ333 103333330 C 0333333 3720

2TOI0303 TO33XT0X: G3O03333 GI 3033O 103333330 03O033X 3780

T CEIOOO 033333OG C2033OAG TOC203C2G3 103333IOX 03300033 3840

AITC20I T3333I O 03X0003 0003033 033332Q 03100010 3900

003302033 203333333. GAOOOIC GIGAAOG33 2OA03OA OQ3302OC 3960 TT03Q30 T03333GIT OAOAG3IC GIGIOOO3 2033333O 03IQOOIC 4020

2000100 CA32020C 03AGA003 2G33IG330 0331033333 C 0333A1C 4080 03A03 IGI033IOA CI23GIO02C T2O2033X 2 03CA3O 03QOOQ3 4140

2A0033333 103033103 LOUJIU3C 22020103 03O03IOA O03 OAC 4200 2Q33G3AAG T 002OO0 033X20333 TOGI03OG 20301103 O033I03X 4260

1Q3AA 0 1C2A0333 Q3I0O AG 033OO203 0300X203 CAIOOOAIG 4320 C2O03AA 0310310333 031033303 0033330 03230330 03330030 4380

GAOiαSIGT 002030002GAQ33303 OOOOOX 30302Q3O Q33IO OC 4440 co:o:r.Q3 002033100 GAAOICZO 033333AAAG OOOAOGIG 033333333 4499

(2) INfOMATICN FOR SEQ ID ND:24:

(i) SEQUENCE CHARAOERISOCS: (A) IEN3IH: 30 base pairs (B) TYEE: nucleic acid

(C) STOANTEΓNESS: single

(D) TCEOOGY: linear

(ii) TFO.TTF. TYEE: cQΦA. (iii) HYPOTHETICAL: ND (iv) ANTI-SENSE: NO

(vii) MDIA3E SOURCE:

(B) CECNE: oIBES-074

(xi) SEQUENCE DΞSCKEPOCN: SEQ ID ND:24:

Pmi Ϊ U (X03I UX T033333C03 30

(2) INECEMOICN ECR SEQ ID ND:25:

(i) SEQUENCE CHARAOERISOCS:

(A) IENGIH: 30 base pai rs

(B) TYEE: nucleic acid

(C) STEANEEENESS: single (D) TCEOLCGY: linear

(ii) 3LEEUIE TYEE: cEMA.

(iii) HYEOIHEOOL: NO

(iv) 2N -SEΪEE: ND (vii) ItøEDIAIE SOURCE:

(B) CLONE: oZeocin-077

(xi) SEQUENCE EE90KEEOCN: SEQ ID ND:25:

AAAOCOO σoαooo σoαsxoc 30 (2) INECFMAπCN ECR SEQ ID ND:26:

(i) SEQUENCE CHARAOERISOCS:

(A) IENOH:. 30 base pairs

(B) TYEE: nucleic acid (C) STRANEEENESS: single

(D) TCKΪCGY: linear

(ii) MDIEEUIE TYEE: cQΦA. (iϋ) HYEΌΠEΓICAL: ND

(iv) 2NIT-SEΪEE: ND

(vii) Η EDIAIE SOURCE:

(B) CXNE: OIRES-Zeocin-075

(xi) SEQUENCE EESCEIPOCN: SEQ ID ND:26:

03I AO1G 033303 G 1033AA03 30

(2) INECKΦAOCN ECR SEQ ID N0:27: (i) SEQUENCE CHARAOERISOCS:

(A) IEN3IH: 30 base pairs

(B) TYEE: nucleic acid

(C) STRANEEENESS: single

(D) TCEOLOGY: linear

(ii) MDTFΠJLE TYEE: cDNA

(iii) HYEΌΠEΠOL: ND (iv) ANO-SENSE: ND (vii) IMEDIA3E SOURCE:

(B) LHE: oIEES-Zeαcin-076

(xi) SEQUENCE EESCRIPOCN: SEQ ID N0:27: C 0J3OA C I0333AA GOO03 30

(2) INEOMOICN ECR SEQ ID ND:28:

(i) SEQUENCE CHARACTERISTICS: ^' (A) IEN3IH: 5540 base pairs

(B) TYEE: nucleic acid

(C) STRANTEΓNESS: single (D) TOB3LOGY: linear

(ii) MJFDUIE TYEE: HA. (gaxitac) (iii) HYEOIHEOCAL: ND (iv) 2NTI-SEieE: ND

(vii) 3MEDIA3E SOURCE: (B) CECNE: EO>030H-IPES-1HL ]I^-IΞ^IPE-3-Zeoc-in

(ix) EEATURE:

(A) NAME/KEY: 5'UIR

(B) LCOOCN: 1. .118

(ix) EEAIURE:

(A) NPtE/KEY: excn

(B) IOOOCN: 119. .164 (ix) EE23UFE:

(A) NAME/KEY: intrcn

(B) lOCATICN: 165. .243

(ix) EE23URE: (A) NPME/KEY: excn

(B) IOOOCN: 244. .557

(ix) EEAItRE:

(A) NA E/KEY: intrcn (B) IOOOCN: 558. .1155

(ix) EEAIURE: (A) IΦfcE KEY: excn

(B) IOOOCN: 1156. .2166

(ix) EEAIURE: (A) 1>P$E/KEY: intrcn

(B) IOOOCN: 2167..2766

(ix) EEAIURE:

(A) ^W /KE : excn (B) IOOOCN: 2767.. 560

(ix) EEAIURE:

(A) ΪΦ^!ME/KEY: intrcn

(B) IOOOCN: 4561..5159

(ix) EEA31EE:

(B) IOOOCN: 5160..5534 (ix) EE23LKE:

(A) røME/KEY: 3'UIR

(B) IOOOCN: 5535. .5540

(xi) SEQUENΕ EESCRIPOCN: SEQ ID ND:28:

2AOJnU31A 033031033 23C002 2A033333X 20GIQOQ3 AAΩOG3G 60

23A303A3 30Q33333 0333ICACD3 020000 03IGT3O33 TOOTAOAT 120

OAA303Q3 'lUJJl'iAIO TOIOOOT Q33AOQ3IT 20Q33AQ3 α33I0332G 180

ΕUAAAOT O033BXAT AAAOOGIG 2OO0XAA TOOOOX TOOOOA 240 σθG3T A 003300C 033330103 03003 T C2A0033G OOO03X 300

2033O033 03G33OT 033333303 03300330 TGA03333 03333X203 360

033 03IQ Q30030G 003103333 C03A AOA 03COQ333 0333OQ 420

103G3OT 0333103333 20333333 203OA033 02033330 003000 480

033OA G 002003003 O03IO033 TGOOAAAA C030AIO3C 2AOA033O 540 20AOAG33 03OO033 OOQ33333 03OO03 03333333X TAACOTAO 600

033332033 001032020J3X GT ULOOGIO 23230ITAIT 0X2030 660 00333100 0033230 (303333333 AAA03iα3X OOOT T GA030CATT 720

Q32ffi-35TC TΓIUXOLT Q3CPAAQP AΓG3AQ3IC 1G0 A3 CO A03A 780

0301000 TQ3A03OC OOAffOAA 2O203IOG TAQ330XT TTG3ΩX2G 840

O33AAO30O3 C203IG33 OQ3I03OC T033333AA 203303I 23AA0320 900 0O03AAAQ3 0333AOA03 03O033C O10IOGO 03AT2OT Q3AAA OC 960

AAAT03O 0OOAG3 ATTOAOAG Q333TOA03 AIQ333A A 03I20333T 1020

IGI2I033AT CK3AICI033 0331033103 2O303I Α 33GIGOTA OCOGSITA 1080

AAA2A03ICT 2G3J33XJCO AAO02O3333 AJJlUb'OIT 0OO AAA 2O03AIOT _.1140

2AQ01G30 OAC 30O 0333IQ3OC C AOAA2G 1GI033AAIT 03O2O 1200 (30020003 lOCOAOT IGAOXTOT CIG303I03 203Ω33333 COC1U3C 1260

CA03IGI23C 03CAO33I03 OA03I Q02O OG OOICOOA C2AO02O3O 1320

OACOAΠC αcoiGiαo A1200033 GAAOOΠG COCCΓOSAA 03033321 1380

0003002G3333ICIA 1Q33033AT 0331033333 20020000 Q33O103G 1440

CΠOOSAAC 0330003 0203300 OOOCAIΌC CAOOOQO GGACOOIOC lsoo CUJi'iOIO.2Q3A0333C 1Q T1U3G O0330333 103333310 2 0IUJJX 1560

03IOOOC T0333 G333O0333 OOOOAT QC2033 A 121033331 1620

Q3O0OOC C3303AOC 2000Q333 OOOOOX AD30O0 Q33O30IA 1680

03330003 OO0333C AiT10JU3G OO03C203 A3AΩ03 IQ023OCIG 1740

Q33333IOG AIOAOAKT TOAAAAOC TX2033I 2O03O C I 03AAOC 1800 03X12303. AA OA303 03QO AG 0OTA1U31G C2Q33O0 G 'IU CRC 1860

03OQ3I03 TU300XT OO 03G 03I Q3I03 3Q330T OOOOOO 1920

03O1GIUJ 2G3333C 2 IOAA03 12O02O3OG 'lUl JlTI T 0033003 1980

OOA3O03 C 203OA 03I 03AAC TATG33ICIC G3IO02G33 033000 2040 GIOAOOG 203333OC 20Q333A 03T2O03 20Ω333IO CA3310C2G 2100

0331031103 2Q3333I0 03AI30OG O033I03 033A033 (30033A 2160 2Q33AAIG3 A32GO3033 OODOOOX 'H,I ..I^'. U.OJ CTAACOOC 103333A03 2220 α33TIQ3AT AAG33333IG T033 1 C IATATOIKΓ TOO3O0AT 2O0333I 2280

OT033AAIG 1 0333333 OAA03I033 COGIOTO 1 0303T T033Q33 2340

C 10333IC 10333AAA03 AA10C2AGO OOIOAIG T03I 20 2G3OT0 2400

OG3A2G30 COOAOO AAOA03I G3033OE '0103030 G333A03X 2460 C .X1U3J3 2 03I0XT O0X03LAA AAQ3303IG TA3AA I2C 2O3IO02AAG 2520

03333AOAC CD3AOOCO COIGIOO T03A32O1G 103AA2O OAA3033IC 2580

1031132033 TAOOAOA G33300AG O303330.203IA03XA O0IAiα3O 2640

1003003 Q333I033IG OOIQOO 20IOGOT 2003030 2222AA03IC 2700

T2Q33333X Aα30333 3000310 ICOOOAA AAO03AI TAA03O0X 2760 20A03I 20333003 (33I03333IC OO Q3IO 100031033 10070030 2820

Q33T0333IC CD3333GAG CD3O103X 00020303 033IO30X 03G333A03 2880

003000 03I03AAOT (30333033 C203OCO 1C3O10 00000310 2940

033300 2G30Q3I 0OOTIG33 210103303 0033300 Q302IG 3000

30103103 T0OO03C TOOG33C 03333330 00333303 CI03OOC 3060 O AQ3333 2G3I002OO Q3O033AG PG3-LTIKZ 20300033 0X20003 3120

2O002 AG 0300300 QOCOOAG 2G3330OG 03AOOGI 0333AA03C 3180

3000303 2Q30333C CO030OG OGIAIQ33' 10310300 0333310333 3240

T033IO3 αCAIOAOC AI03333 G C2O033333 T03 03 G3Q3I03IG 3300

22G333G 1UX AQX Q3333I03X 03330033 00002100 O303333X 3360 0333Ω3I03 IO3033333 CCAQ3AO03 2033O0O 03303IGAC 03000333 3420

0333X20 O303IOIG T2030XO T03ICA03 Q333AA03G 3480 0333103103203 30 Q3TO103G 10333333332O103OC O30333C2C 3540

OOG3333303I033OC OAGA30AG CD302O33X T02010G 033IO03IG 3600 ai^'.^' U:i.-l' ui.-tI.OU33 CD3C2AG3X OO2003 OOG3AAG32G3XIU3X 3660 ππτn:-πτ: oorπαc 033OQ3AAG 000200 OAOOXAOG 3720 003AA G α.I.miU A. CX 03XC 230333O 20200333 TG330Q333 3780

0XTXO0303333AT T Q3GOQ3330Q3333 033333IOT Q3X2I03X 3840

0333AG3 033330000 O03333120002033 A3AGIQ3C 030OQ3X 3900

CI033333O O03331O C23 T03X T O0O0320030200303333333 ^" 3960

2AG3I03IO 3000033 O033A03X C033 OG 2 I03IOA C2033OAC 4020 C202CZ C OOOTOO Q3 3033323G AO 2Q3I03IOC 10030333' 4080

Q3 CO03 TC2IC20OC OQ02 I2C AAO033AAG 2O03Q3 Q333A033IG 4140

(■^■OX-XTIUG GGKTOOQO 0303003 GIOAOAIG T03Aa2OA 033302 03 4200

3GO03G31O002AOG OOXEIOSC 00033003103AOA A 01033000 4260

O A G3T TO2O03 03AAO 032031033X020333IO 03OO03G 4320 0103003310333IO3A CI0O1 AC O30 03I03 ΪOA0333 2033OTC 4380

03033A3 002300200 C22OQ3I03 T033XO030OTC G3303003AAT 4440

033303X0 lOXπOGKT 031000203 OQ3A 0300203300 O03X20X 4500

2G33AG33IC 2 033333303333X203 GIQOOAO 10003333 AAQ300 4560

AXllrϋl -i OLUXOL'ia' OXIOXaX OXOAAOO 003330 0333 0 4620 2A3AA03333 GIGI033OT OOATAIO TATTTKOC C23AO0333 TC00030 4680

230003330333AAA0O 03333IGI 1C O03G 01103303 QOCOIOX 4740

CICIUXOA 203AKD3C2A 0310000 AIGICOrOA O3202OT 0OCI03AG 4800

COCOGAAG 2 A2 A03 TO03GXA O3OTIO02G X20333AAC 033330OG 4860 033A3G3IG 00003333 OAAAQ33C OG332AO T2O0O0O AAO333302C 4920

AA0333OG 0020300102O103A32G OOGGAAAG 2OOAA303 O 0OOA 4980 QCGTAOOA C2A0333OG AAO3A3O0D3 2 AQ3303 03A GIAIG Q3AIOGAIC 5040 l ΩXIJIUJ 03 3Q3 0120300 TT3O03AG OTAAAAAAC OOA333X 5100

0333203C 03330O03 OOOOTIG 2AAAA003. 103AAQ3IT 033OA0 5160

TG333AAOT OOCAOOX 010333103 TOCD33333 C 03IUJX 030333103 5220

2 1 03C 030X0310 0331101033 0330100 G3G30 C 003333310 5280 103103330 0303I 03 001030 033333100 Q3O02G3ΓG O03333O 5340

2 033I033 00331003 GI033333X T03G30T G303333G 103103303 540θ^" lOSIOCOC (32010333 00330033 0333333021 3033AGA3C G333GC2GC: 5460

OLllSmXX. Q3GO03X CI0333303 033333XAA OO33IO02C 003103333 5520

203 2Q CT O03G 5540 (2) INEOMAOCN ECR SEQ ID ND:29:

(i) SEQUENCE CHARAOERISOCS:

(A) IEN3IH: 829 base pairs

(B) TYEE: racleic acid (C) S1RANEEENESS: single

(D) TCECϋXGY: linear

(ii) DEΣEULE TYEE: ENA (gercmic) (iii) HYEOIHEOCAL: NO

(iv) 2N -SENSE: ND

(vii) MEDIATE SOURCE:

(B) CUNE: EccAKS

(ix) EEAIURE:

(A) ΪΦ_E/KEY: 5'UIR (B) lOCATICN: 1. .16

(ix) EE23LRE: (A) 1ΦME/KEY: excn

(B) IOOOCN: 17. .820

(ix) EEAIURE:

(A) røMEVKEY: 3»UIR

(B) IOOOCN: 821..829

(xi) SEQUENT EESCRIPOCN: SEQ ID N0:29:

033AAQ3I1C Q302O02IQ3 033.0TCLT (300003 20TQX1Q3 10100033 60

C03333XTC 0033300 10X133330 AI03OOG SAO0333 0310300 120 C03333OG 0333333333 2030AOT 03I033 Q3 G3Aiα3KT GIGAAG3IAA 180

2O03OT OOAAATO 033AAA0OG OAQ30OC 0002000 C AA0020 240

Q30000A 0303300 0303OOG 2 AAA3203 2220333AAG 2A2033A3O 300

Q33GC AA 203330333 Q30 3GAA AP0333Q 0XO 3 20222300 360

TOQOTΓAΓ αoioso OOAOAO OXOAIQO ASΓOOIΌ: TCQOOAG 420 G3IO33333 TT023 A 2G330C2 Q30303C 1UXTG30 ATTOOOO 480

OOCOAAAA OG3O 03 22200330 OA033OG 033030203 2O3033Q3 540

C20032AT 303AAOAG TOQ3AAOG 2323G333X TTOIOOG 03T3AAOG 600

2AQ33333AA O03AA IG 2A033A22 0O0C2OAG 033210333 QOIOTOG 660

2A3G3Ω 03330200 03I03ATO OAOCAOAA 03333030 GTOOOGAA 720 ϋJX π-lUJJ 0030003 03BXO0O 2 A0333A 2OTA 0O 2AOAOT03 780

TOAA303AA 2AAA 303 033TIAT (333ΩT2A 03ATO3333 829

(2) INEOMAOCN ECR SEQ ID ND:30:

(i) SEQUENCE CHARAOERISπCS:

(A) EN3IH: 598 base pairs

(B) TYEE: nucleic acid

(C) STRANEEENESS: single (D) TCEOLOGY: linear

(ii) DEEΠIE TYEE: QΦA. (genαnic) (iii) HYHJIHLTIOL: ND (iv) 2N -SEN3E: ND

(vii) JMEDI2TE SOURCE:

(B) CLCNE: IRES sequence (ix) EEAIURE:

(A) røME/KEY: intrcn

(B) IOOOCN: 1..598

(xi) SEQUENCE EESCRIFΠCN: SEQ ID ND:30:

OKΠOOOX OOOOXIC αxixπxo AAOEΓSOG QCDOAAGCDS OIGSAAJAA 60

033X0010 αSTOOOA TATGITAOT TCOCOSKT T0333IO T T033AIGIG 120

A.-»:-t:ι:ι:ι:-t:-A AAαoo33x TOICOC O 203020c 02033310 ocoxioc lβo

Q33A2AQ3A 103A03I GπOAIGTC O A03AAG 3O10 Q3AA03O 240 TGAAOOAA OACGIOO 2033033 T03Q33G3 03AAO3XD3 20OQ333C 300

2G3IQ33ICT 033333AAA 033A03IOΑ TAA0320C CIO02AAG33 03020AOX 360

O 03303 OOOOIG O32OT0IG C3AA O 223033100 OOA033IA 420

TT 2 A03 G33I AGO T0333OAG G30333O G33Q333C TGAICIG333 480 αiTCQGKXA O303ITI2C A10101T12G lOGAGOIAA 2AAA03ICIA Q33333330 540 20303330 COOSOTIC OOGAAAAA 003303203O033AC AAO02IQ3 598

90/1

Applicant -or agents file m TQ CIP PCT , International appticaiionNo. reterenee number v- i. ^£. -

INDI_CATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule I3bis)

90/2

Applicant s or agent s t] lc International application No. reterence number CTI /2 9 CI P PCT

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule I3bιs)

A. l^"h indications πuuc n i w rciate to tne microorganism reterreu lo in ibe description on pace 54 •, . line S L 4 — 23

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet | |

Name ol depositary institution

American Type Culture Collection

Address HI depositary institution _lincl tUn_f po at oat end courunrl

12301 Par lav/n Drive Rockville, Marylmd 20852

United States of America cell Line, RINa/ProA/ Identification Reference by Depositor: P030/P088

Dale ol deposit Accession Number

07 June 1995 ( 07 . 06 . 95 ) CRL 11921

ADDITIONAL INDICATIONS tl avcnlankifruHtppiicsbic. This information is continued on an additional sheet { l

In respect of the de?^1'.gnation of Finland, until the application has been laid open to public inspection by the Finnish Patent Office, or has been finally decided upon by the Finnish Patent Office without having been laid open to public inspection, samples of the deposited microorganisms will be made available only to an expert in the art.

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE tif the imm au j, re wt for aUm p βiSlβla)

Finland

E. SEPARATE FURNISHING OF INDICATIONS (l r t blank, fnatppiicmblέl

The indications listed below will be submitted lo the International Bureau lmla(sptaf ihefattrmi auinofiht mmtao mt. , 'Ac NmmbrofD pmn

90/3

_* IniernaiionjiapplicaiionNo. reference number CTI/2 9 CI P PCT

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule \3bis)

A. ITie indications auc ncuiw relate to ine microorganism referred to in the description "n page 54 . line S 14 -23

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additions! sheet ( ]

Name of depositary institution

American Type Culture Collection

Address ol depositary institution unci unt postal coat and ctmrun)

12301 Parklav/n Drive Rockville, Maryland 20852 United States of America Cell Line, RINa/ProA/

Identification Reference bv D posito P030/P088

Dale ol deposit Accession Number

07 June 1995 ( 07 . 06 . 95 ) CRL 11921

C. ADDITIONAL INDICATIONS (leave nlank if not applicable, This information is continued on an additional sheet {"^*"]

ΪΪSi i^S!_u ^{erβby giVe notice} of my/our intention that samples of the above-identified culture shall _be available on^ly to experts in accordance with paragraph 3 of the ^Fourth Schedule to the Patents Rules 1995.

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the i^uaύm* arc ma for mUiamfi ai Sttn,

Singapore

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the International Bureau later (spcafvihegaitraimatMnef ιhemmirmιwm€.g_m 'A NmmOmT of Depot!!-)

Claims

WE CLAIM :

1. A cell stably transformed to produce at least one analgesic compound from each of the groups consisting of endorphins, enkephalins, and

catecholamines.

2. The cell of claim 1, wherein the endorphin is β-endorphin.

3. The cell of claim 1, wherein the enkephalin is met-enkephalin.

4. The cell of claim 1, wherein the catecholamine is norepinephrine or epinephrine.

5. The cell of any one of claims 1-4 wherein the cell is a RIN cell.

6. The cell of any one of claims 1-4 wherein the cell is an AtT-20 cell.

7. The cell of any one of claims 1-6 wherein the cell additionally produces a compound selected from the group consisting of galanin,

somatostatin, neuropeptide Y, neurotensin, or

cholecystokinin.

8. A cell transformed with a DNA encoding POMC, a DNA encoding TH, a DNA encoding DBH, and a DNA encoding ProA, each DNA molecule operably linked to an expression control sequence.

9. The cell of claim 8 wherein the cell is transformed with pCEP4-POMC-030, pcDNA3-hproA+KS-091, and pZeo-pCMV-rTHΔKS-IRES-bDBH-088.

10. The cell of claim 8 wherein the cell is transformed with pCEP4-h POMC-ΔACTH-032, pBS-CMV-proA, and pZeo-pCMV-rTHΔKS-IRES-bDBH-088.

11. The cell of claim 8 wherein the cell is transformed with pcDNA3-hPOMCDACTH-IRES-rTHD-IRES-bDBH- IRES-Zeocin-073 and pcDNA3-proA+KS-091.

12. A transformed cell producing at least one enkephalin, one endorphin and one catecholamine, wherein the cell is transformed with:

a first vector containing a DNA encoding POMC operably linked to an expression control sequence, a second vector containing a DNA

encoding pro-enkephalin A operably linked to an

expression control sequence,

a third vector containing a DNA encoding TH operably linked to an expression control sequence and a DNA encoding dopamine beta hydroxylase operably linked to an expression control sequence.

13. A method for treating pain comprising implanting at an implantation site in a patient a therapeutically effective number of the cells of any of claims 1-12.

14. The method of claim 13 wherein the cells are encapsulated in a semi -permeable membrane to form a bioartificial organ.

15. The method of claim 14 wherein the bioartificial organ is immunoisolatory.

16. The method of any one of claims 13-15 wherein the implantation site is the CNS.

17. The method of any one of claims 13-15 wherein the implantation site is the sub-arachnoid space.

18. A method of producing a cell that secretes at least one enkephalin, one endorphin and one catecholamine, comprising transforming the cell with a DNA encoding POMC operably linked to a first expression control sequence, a DNA encoding pro-enkephalin A operably linked to a second expression control

sequence, and a DNA encoding TH operably linked to a third expression control sequence and a DNA encoding dopamine beta hydroxylase operably linked to a fourth expression control sequence.

19. The method of claim 18 wherein said first, second, third and fourth expression control sequences are identical.

20. The use of the cells of any of claims 1- 12 to manufacture a medicant for treatment of pain.

21. The cells of claim 20 wherein the cells are implanted.

22. The cells of any one of claims 21-22 wherein the cells are encapsulated in a semi-permeable membrane to form a bioartificial organ.

23. The cells of claim 22 wherein the bioartificial organ is immunoisolatory.

24. The cells of any one of claims 21-23 wherein the implantation site is the CNS.

25. The cells of any one of claims 21-23 wherein the implantation site is the sub-arachnoid space.

26. A bioartificial organ comprising:

(a) a biocompatible, permeable jacket surrounding a core; and

(b) said core comprising at least one living cell transformed to produce at least one analgesic compound from each of the groups consisting of endorphins, enkephalins, and catecholamines.

27. The bioartificial organ of claim 26 for use in treating pain.

28. A method of making a bioartificial organ comprising encapsulating a core comprising at least one living cell transformed to produce at least one analgesic compound from each of the groups consisting of endorphins, enkephalins, and catecholamines, with a biocompatible, permeable jacket.

29. The use of a bioartificial organ

comprising the cells of claims 1-12 in manufacture of a medicament for treating of pain.