KR19990022414A - Cell line producing pain compounds for pain treatment - Google Patents

Abstract

The present invention relates to genetically engineered cell lines that produce one or more catecholamines, one or more endorphins, and one or more enkephalins for the treatment of pain. The cells can be administered to patients in need of pain relief and can be encapsulated to form living artificial organs.

Description

Cell line producing pain compounds for pain treatment

Pain is a common sign of disease. Surface dorsal projections, which are primary afferent fibers that hold information terminals in response to painful stimulation of the spinal cord, contain enkephalin-functional interneurons and high-density analgesic receptors. In addition, there is a high concentration of noradren functional fiber in the surface thin layer of the spinal cord.

Acute pain occurs in response to a sudden toxic stimulus. Chronic pain is predominantly due to neurological disorders of the central or peripheral medulla. These neuropathic pains are the result of non-ideal body perception that can result in painful stimuli (excessive pain) and increased sensitivity to pain-free stimuli (allodynia).

Intracapsular injection of morphine into the spinal cord subarachnoid space produces potent analgesia. Similarly, intracapsular administration of norepinephrine or noradren agonist also produces analgesia. See, eg, Sagen et al., Proc. Natl. Acad. Sci. USA 83, pp. 7522-26 (1986).

Simultaneous administration of analgesic agents such as enkephalin and catecholamines such as norepinephrine can have a synergistic effect on analgesia production (same reference above). Chromapine cells in the adrenal medulla include norepinephrine, epinephrine, methionine-enkephalin, leucine-enkephalin, neuropeptide Y, intestinal polypeptides acting on blood vessels, somatostatin, neurotensin, cholecystokinin and calcitonin gene-related peptides Produces and releases several neuroactive substances. See, eg, Sagen et al., Proc. Natl. Acad. Sci. USA, 83, pp. 7522-26 (1986); Sagan et al., Jour. Neurochem., 56, pp. 623-27 (1991).

Since cropapin cells produce both opioid peptides and catecholamines, studies that have been studied as a way to reduce trauma soluble response or pain sensitivity have been found to provide pain relief in the control of CNS pain in adrenal bone marrow tissue as well as in isolated adrenal chromaffin cells. Direct implantation into regions [Sagan et al., Brain Research, 384, pp. 189-94 (1986); Vaguero et al., Neuroreport, 2, pp. 149-51 (1991); Ginzberg and Seltzer, Brain Research, 523, pp. 147-50 (1990); Sagen et al., Pain, 42, pp. 69-79 (1990).

Attempts to produce analgesics have been made using both heterologous and heterologous chromaffin tissue or cell implants. Xenograft tissues are limited in supply and are not readily available, especially in human pain treatment programs. In addition, xenotype human tissues present a risk of pathogenic contamination. Hama and Sagen, Brain Research, 651, pp. 183-93 (1994).

Heterologous donors can provide large amounts of material that can be readily obtained. Hama and Sagen, Brain Research, 651, pp. 183-93 (1994).

However, potentially severe host effects as well as ultimate transplant rejection are inherent problems in transplantation between different species. Complete transplant rejection of whole or isolated tissues occurs even within the CNS and is considered immunologically privileged due to the presence of highly antigenic cells in xenografts, especially endothelial cells. In addition, the donor tissue should be screened carefully to avoid introducing viral contaminants or other pathogens into the host. To avoid transplant rejection, immunosuppression with cyclosporin A is typically required.

Some reduction in pain sensitivity, particularly chronic pain with low pain intensity, has been reported to be due to these grafts. In most reports, significant differences between control and transplanted animals were noted only after administration of nicotine to stimulate opioid peptide production. However, there have also been some reports that analgesics are observed from the basic level of activity of chromatin tissue xenografts in a rat chronic pain model. See, eg, 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 in vivo artificial organs (“BAOs”) for implantation into mice for the treatment of acute and chronic pain. See, eg, Sagen et al., J. Neurosci., 13 , pp. 2415-23 (1993) and Hama et al., 7th World Congress Pain, Abstract 982, Paris, France (1993). Initial experiments on human specimens have been performed using encapsulated bovine chromatin cells. See Aebischer et al., Transplantation, 58, pp. 1275-77 (1994).

In addition, other cells, such as AtT-20 cells, have been attempted to induce antinociception. AtT-20 cells were originally derived from mouse pituitary anterior lobe tumors. These cells synthesize and secrete β-endorphins. See, eg, Wu et al., J. Neural Transpl. & Plasticity, 5, pp. 15-26 (1993). AtT-20 / hENK cells display the entire human proenkephalin A gene (ie, six methionine-enkephalin sequences and one leucine-enkephalin) having a 200 base 5'-adjacent sequence and a 2.66 kb 3'-adjacent sequence. AtT-20 sequence genetically engineered to retain. See Wu et al., Supra, Comb et al., EMBO J., 4, pp. 3115-22 (1985).

See Wu et al., J. Neural Transpl. & Plasticity, 5, pp. 15-26 (1993), refer to rat hosts transformed with AtT-20 or AtT-20 / hENK cells. Unstimulated AtT-20 / hENK cells produce more anti-traumatic (tail flick tests) than those produced by AtT-20 implants. In contrast, isoproterenol stimulation produces more anti-trauma uptake by AtT-20 cells than by AtT-20 / hENK cells.

In mouse hosts, AtT-20 or AtT-20 / hENK implants do not affect the baseline response to thermal painful stimulation. Mice receiving an AtT-20 implant show resistance to β-endorphin and μ-opioid agonists (DAMGO). Mice receiving an AtT-20 / hMEK implant show resistance to δ-opioid agonists (DPDPE). For repeated administrations of μ analgesic agonists, mice receiving AtT-20 / hENK implants show less resistance than AtT-20 cells or mice receiving control.

The anti-traumatic effect of isoproterenol treatment is the same in mice receiving AtT-20 or AtT-20 / hENK cell implants. Wu et al., Neuroscience, 14, pp. 4806-14 (1994). Wu et al. Considered the lack of sensitivity of behavioral analysis as a cause of the absence of further anti-trauma acceptance in mice transplanted with enkephalin-producing AtT-20 / hENK cells. Another possible reason is that known antagonist effects on morphine-induced anti-trauma uptake of methionine-enkephalin resemble the possible effects of single leucine-enkephalin. This is because there are six methionine-enkephalin sequences for each leucine-enkephalin sequence in pro-enkephalin A.

The present invention relates to cell lines useful for the treatment of pain. In particular, the cell lines of the present invention are genetically engineered to produce one or more analgesic compounds selected from the group consisting of endorphins, enkephalins and catecholamines.

1 is a diagram showing plasmid maps of the vectors pBS-hPOMC-027, pBS-IgSP-hPOMC-028 and pBS-IgSP-hPOMC-ΔACTH-029.

Fig. 2 shows the plasmid maps of the vectors pCEP4-hPOMC-030, pCEP4-hPOMC-031, pcDNA3-hPOMC-034 and pcDNA3-hPOMC-035.

Fig. 3 shows the plasmid map of the vectors pCEP4-hPOMC-ΔACTH-032, pCEP4-hPOMC-ΔACTH-033, pcDNA3-hPOMC-ΔATCH-36 and pcDNA3-hPOMC-ΔACTH-037.

Fig. 4 is a diagram showing plasmid maps of the vectors pcDNA3-rTH-044, pcDNA3-rTHΔ-045 and pcDNA3-rTHDKS-075 (also indicated as pcDNA3-rTHΔKS-075).

Fig. 5 shows the plasmid maps of the vectors pcDNA3-rTHΔ-IRES-bDBH-088 and pcDNA3-rTHΔKS-IRES-bDBH-076.

Fig. 6 shows the plasmid map of the vector pZeo-Pcmv-rTHΔKS-IRES-bDBH-088.

Fig. 7 shows the plasmid map of pBS-Pcmv-rTHΔIRES-bDBH-067.

Fig. 8 shows the plasmid map of the vector pBS-hPOMC-ΔACTH-IRES-rTHΔIRES-bDBH-068.

Fig. 9 shows the plasmid map of the vector pcDNA3-hPOMC-ΔACTH-IRES-rTHΔ-IRES-bDBH-069.

Fig. 10 shows the plasmid map of the vector pcDNA3-IRES-Zeocin-072.

Fig. 11 shows the plasmid map of the vector pcDNA3-hPOMC-ΔACTH-IRES-rTHΔ-IRES-bDBH-IRES-Zeocin-073.

Fig. 12 shows the plasmid map of the vector pcDNA3-hPROA + KS-091.

Detailed description of the invention

In order to more fully understand the present invention, the following detailed description of the invention will be given.

Any suitable cell can be transformed with the recombinant DNA molecule of the invention. Among the cells contemplated are chromatin cells, including the conditionally endogenous chromaffin cell lines described in WO 96/02646, neuro-2A, PC12, PC12a, SK-N-MC, AtT-20 and RIN comprising RINa and RINb. Cells are included. The cells preferably have endogenous prohormone convertases and / or dopa decarboxylase.

The neuronal endothelial cell line, SK-N-MC cells, co-express several neuropeptides, including enkephalin, cholecystokinin and gastrin-releasing peptides. See, eg, Verbeeck et al., J. Biol. Chem., 265, pp. 18087-090 (1990). The pro-enkephalin A gene is expressed in SK-N-MC cells. See, eg, Folkesson et al., Mol. Brain Res., 3, pp. 147-54 (1988). We preferred AtT-20 and RIN cells, with RIN cells being most preferred.

RIN cells are pancreatic cell lines derived from rats. See, eg, Horellou et al., J. Physiol., 85, pp. 158-70 (1991). RIN cells are known to produce GABA and β-endorphins endogenously.

Some properties of the various considered cells are described in Table 1 below.

The initial delivery product includes one or more endorphins, enkephalins and catecholamines.

Enkephalins and endorphins are human endogenous opioid peptides. These opioid peptides comprise about 15 compounds consisting of 5 to 31 amino acids. These compounds are at least partially bound and secreted by the same μ opioid receptor as morphine, but are chemically independent of morphine. In addition, these compounds stimulate other analgesic receptors, see Yaksh and Malmberg, Textbook of Pain, 3rd edition (P. Wall and R. Melzack), "Central Pharmacology of Nociceptive Transmission", pp. 165-200, New York (1994)].

The opioid peptides possess common chemical properties but are synthesized by different pathways.

The most endorphins, β-endorphins, are synthesized as part of the larger precursor molecule, pro-opiomelanocortin ("POMC"). The POMC molecule contains the entire sequence of pituitary hormone (“ACTH”), α-myeloid cell-stimulating hormone (“α-MSH”), β-MSH and β-reportropin. In addition, the POMC precursor molecule has the potential to produce other endorphins, including α-endorphins and gamma-endorphins. Treatment of the POMC precursor occurs differently in various tissues depending on the location of the cleaving enzyme, such as prohormone convertase in the tissue.

In the pituitary gland, POMCs are cleaved to produce ACTH and β-endorphins, which are no longer processed. In contrast, within the hippocampus, ACTH is converted to β-MSH. Although the cell types described above synthesize the same initial gene product, the final profile of hormone secretion can vary widely.

The present invention contemplates the use of a DNA sequence encoding any suitable endorphin that retains analgesic activity. Also contemplated are analogs and fragments of these endorphins that possess analgesic activity. Thus, endorphins to be produced by the cells of the invention may be characterized by amino acid insertions, deletions, substitutions and alterations at one or more positions within the naturally occurring amino acid sequence of the endorphins of interest. We preferred conservative alterations or substitutions (ie, alterations or substitutions that have minimal effect on the secondary or tertiary structure of endorphins and the analgesic properties of the endorphins). Such conservative substitutions are described in Dayhoff, Atlas of Protein Sequence and Structure, 5, (1978) and Argos, Embo J. 3, pp. 779-85 (1989).

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

The present invention contemplates the use of a DNA sequence encoding the entire POMC precursor molecule. This embodiment uses cleavage enzymes (ie prohormone convertase 2) of the host cell to produce some or all series of endorphins that can retain analgesic properties.

The present invention also contemplates the use of DNA fragments of the POMC gene encoding endorphins of particular interest.

The amino acid sequences of these DNAs and POMCs are well known. See Cochet et al., Nature, 297, pp. 335-9 (1982); Takahashi et al., Nucl. Acids Res., 11, pp. 6847-58 (1983).

We preferred DNA sequences encoding POMCs lacking the ACTH coding region. Preferred endorphins encoded by this construct are β-endorphins.

Some enkephalins are synthesized as part of proenkephalin A, which contains six repeats consisting of the methionine enkephalin sequence, which is the larger protein in the adrenal gland, and one leucine-enkephalin structure. Methionine-enkephalin-arginine-glycine-leucine possesses significant anti-traumatic activity. See, eg, Sagen et al., Brain Res., 502, pp. 1-10 (1989).

Other enkephalins, ie dynopine and neoendorphin, are derived from proenkephalin B, the distinct molecule. Furthermore, additional "hidden" peptides are encoded within the structure of these precursor proteins and can be released in "progenitor hormone form". See, eg, Harrison's "Principles Of Internal Medicine", 12th edition, pp. 1168-69 (1991).

The present invention contemplates the use of a DNA sequence encoding any suitable enkephalin that retains analgesic activity. Also contemplated are analogs and active fragments possessing analgesic properties. Thus, such analogs or fragments may retain amino acid insertions, deletions, substitutions at one or more positions in the naturally occurring amino acid sequence. Such variants may be produced as described above.

The present invention encompasses the use of DNA sequences encoding the desired enkephalins in their "mature" form. The present invention also contemplates the use of DNA sequences encoding whole proenkephalin A precursors or whole proenkephalin B precursors. In addition, we contemplate the use of DNA encoding a fusion or fragment of these sequences to yield one or more enkephalin-like molecules that possess analgesic properties upon expression.

We prefer the use of DNA sequences encoding whole proenkephalin A precursor molecules. The amino acid sequences of the DNA and proenkephalin A are well known [Folkesson supra]. This embodiment uses cleavage enzymes of a host cell, such as prohormone convertase, to produce some or all series of enkephalins that can retain analgesic properties. Preferred enkephalins encoded by this construct are methionine-enkephalins.

There are three naturally occurring catecholamines that act as neurotransmitters in the central nervous system: norepinephrine ("NE"), epinephrine ("N") and dopamine. NE is associated with posterior ganglion sympathetic nerve endings. NE exerts its effect immediately topically before and after its immediate release.

Catecholamines are synthesized from the amino acid tyrosine, which are subsequently hydroxylated to form dihydroxyphenylalanine (dopa), which is then decarboxylated to form dopamine, and then the beta position of the side chain is followed by dopamine beta hydroxylase. Hydroxylation to form NE. Harrison, supra, pp. 380]. NE is N-methylated to E by phenylethanolamine-N-methyltransferase ("PNMT").

The hydroxylation of tyrosine by tyrosine hydroxylase (“TH”) is a rate determining step in NE synthesis. Modulation of dopa and NE synthesis in the adrenal bone marrow can be accomplished by varying the amount and activity of TH.

In addition, modulation of the synthesis of E from NE can occur by changing the amount and activity of phenylethanolamine-N-methyltransferase ("PNMT"). PNMT can be induced by glucocorticoids from the adrenal cortex (see above).

Catecholamines are maintained at high concentrations, mostly E, in adrenal bone marrow chromaffin tissue. In addition, opioid peptides are stored in the adrenal gland.

NE and E have similar activity at the α ₂ receptor, and thus both may contribute to analgesic. Bylund, FASEB J., 6, pp. 832-39 (1992). Enkephalin peptides primarily comprising methionine-engepalin preferentially activate delta (δ) opioid receptors. Reisine and Bell, Trends Neurosci., 16, pp. 506-10 (1993). Activation of the α ₂ adrenergic and δ opioid receptors in the spinal cord results in anti-traumatic use, respectively, and is potentially synergistic. Yaksh and Malmberg, Progress in Pain Research and Management, Vol. 1, Seattle, IASP Publishers (Fields and Lisbeskind), pp. 141-71 (1994). The activation of alpha opioid versus delphi opioid receptors in experimental animals has few adverse side effects, including constipation and intoxication [Lee et al., J. Pharmacol. Exp. Ther., 267, pp. 883-87 (1993). Associative delivery of different opioid functional and adrenofunctional agents may reduce the degree of resistance seen for a single agent and may lead to persistent pain relief. Yaksh and Reddy, Anesthesiol., 54, pp . 451-67 (1981).

The present invention contemplates the use of analogues or fragments thereof to obtain a DNA sequence encoding a catecholamine biosynthetic enzyme or a catecholamine having analgesic properties. Preferred catecholamines in the present invention are NE and E.

In one embodiment, the host cell is transformed with a gene necessary to carry out the production of NE or E as needed. The selection of the heterologous gene sequence required depends on the supplementation of the catecholamine synthase 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, a gene encoding PNMT is also required. Genes encoding PNMT are known. Baetre et al., Proc. Nat'l Acad. Sci., 83, pp. 5455-58 (1986)].

Genes encoding TH are known (see, eg, US Pat. No. 5,300,436, incorporated herein by reference). Altered TH variants are also known (US Pat. No. 5,300,436). In addition, truncated forms of TH containing the required C-terminal catalytic domains are also known. See, eg, Daubner et al., Protein Science, 2, pp. 1452-60 (1993).

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

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

In addition to the preferred DNA sequences herein, a number of degenerative DNA sequences encoding the analgesics of interest will also be considered.

In addition, secondary compounds that possess potential analgesic action may be produced by the cells of the invention. Such compounds include galanine and somatostatin. Neuropeptide Y, neurotensin and cholecystokinin may also be produced by the transformed cells of the invention. The cells of the present invention can be genetically engineered using standard techniques to normally produce some or all of these compounds or to produce some or all of these compounds.

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

For example, the complete amino acid sequence of the compound of interest can be used to construct a detranslated gene. DNA oligomers containing nucleotide sequences encoding desired analgesic compounds can be synthesized. For example, some small oligonucleotides encoding portions of each desired polypeptide can be synthesized and then linked. Individual oligonucleotides typically have a 5 'or 3' extension for assembly.

DNA encoding each desired analgesic compound may or may not include a DNA sequence encoding a signal sequence. If such a signal sequence is present, the signal sequence should be that recognized by the cell selected for expression of the analgesic compound. The signal sequence may be a prokaryotic signal sequence or a eukaryotic cellular signal sequence or a combination thereof. In addition, the signal sequence may be a signal sequence of a natural compound. In general, the signal sequence is preferably encoded, most preferably using the native signal sequence.

Once assembled, the DNA sequence encoding the desired compound is inserted into one or more expression vectors and operably linked to an expression control sequence suitable for expression in the transformed cell of interest.

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

The choice of expression control sequences and expression vectors will depend on the choice of cell. Various expression host / vector combinations can be used. Examples of expression vectors useful for eukaryotic hosts include vectors comprising expression control sequences of SV40, bovine papilloma virus, adenovirus and cytomegalovirus.

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

Various expression control sequences can be used in these vectors. Such useful expression control sequences include expression control sequences associated with structural genes of the expression vectors described above. Examples of useful expression control sequences include early and late promoters of SV40 or adenovirus, promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, promoters of acid phosphatase, such as Pho5, promoters of yeast α-mating system And eukaryotic cells or other sequences in which they are known to regulate gene expression of viruses and various combinations thereof.

It is to be understood that not all of the above vectors and expression control sequences act equally to express the DNA sequences described herein. All cells do not work equally using the same expression system. However, those skilled in the art will be able to select from these vectors, expression control sequences and cells without performing inappropriate experiments. For example, in the selection of a vector, a host cell should be considered, because the selected vector must replicate within the host cell. Consideration should also be given to the expression of any other protein encoded by the vector, such as the number of copies of the vector, the ability to control the copy number, and an antibiotic marker.

In the selection of expression control sequences, several factors must be considered. Examples of these include the relative strength of the sequence, its controllability and compatibility with substantial DNA sequences encoding the analgesic compounds of interest, with particular consideration of possible secondary structures. Host cells should be selected in consideration of their compatibility with the selected vector, the toxicity of the product encoded by the DNA sequence, their secretion properties, the ability to fold the polypeptide correctly, and their culture requirements. If host cells are encapsulated, cell viability when encapsulated and transplanted should also be considered.

Within these variables, one of skill in the art can select various vector / expression control sequence / host combinations that express the DNA sequence of interest in culture.

In one embodiment, the cells (eg, RIN cells) are transformed sequentially using four separate expression vectors containing the POMC gene, proenkephalin A gene, TH gene and DBH gene. In such transformed host cells, amplification of the copy number of the heterologous gene is more difficult to obtain. Therefore, fewer expression vectors are preferred. Most preferably, a single expression vector containing all four heterologous genes is used.

In certain embodiments, RIN cells are transiently transfected with three expression vectors. The first vector contains the POMC gene operably linked to the CMV promoter. Preference is given to using truncated forms of the POMC gene that carry the deleted ACTH coding region. The second vector contains the proenkephalin A gene operably linked to the CMV promoter. ProA constructs contain the Kozak sequence immediately upstream of the starting codon. The third vector contains the TH gene (preferably truncated and has a Kozak consensus sequence immediately upstream of the start codon) and the DBH gene. In this embodiment, the TH gene is operably linked to a CMV promoter. The DBH gene is operably linked to an internal ribosomal entry site promoter sequence. RIN cells were then sequentially transfected with their respective expression vectors according to known methods.

In another embodiment, a single expression vector comprising the proenkephalin A gene, POMC gene, TH gene and DBH gene is constructed. Preferably, the ACTH region of the POMC gene is deleted. The TH gene is preferably cleaved.

Expression of multiple genes from a single transcript is preferably by expression from multiple transcription units. One method of obtaining multiple gene expressions from a single eukaryotic transcript uses the sequence of picornavirus mRNA known as the internal ribosomal entry site ("IRES"). These positions serve to facilitate protein translation from sequences located downstream from the initial AUG of the mRNA.

Macejak and Sarnow are immunoglobulin heavy chain binding proteins (BiP, also known as CRP 78, glucose-regulated proteins with a molecular weight of 78,000) mRNAs directly provide internal ribosomal binding to mRNAs in a 5'-cap dependent manner in mammals This means that translational initiation by internal ribosomal binding mechanisms is used by the cellular mRNAs. Nature 353, pp. 90-94 (1991).

WO 94/24870 describes the use of two or more IRESs for initiating translation from a single transcript, as well as the use of a copy of the same IRES in a single construct.

The present invention also contemplates the use of "suicide" genes in transformed cells. The cells carry TK (thymidine kinase) as a safety measure, and it is desirable to allow the host cells to die in vivo by treatment with gancyclovir.

The use of the "suicide" gene is known in the art. See, eg, Anderson, published PCT application WO93 / 10218; Hamre, published PCT application WO 93/02556. The recipient's own immune system provides the first level of protection from adverse reactions to the transplanted cells. When encapsulated, the polymer capsule itself may be an immuno-isolate. The presence of the TK gene (or other suicide gene) in the expression construct provides an additional level of safety for the receptors of the transplanted cells.

Preferred vectors for use in the present invention are vectors which allow the DNA to encode analgesic compounds to amplify the copy number. Such amplifiable vectors are well known in the art. Examples of these include DHFR amplification (see, eg, Kaufman, US Pat. No. 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 91982) or glutamine synthase (“GS”) amplification (see, eg, US Pat. No. 5,122,464 and European Application Publication 338,841). Such amplification can be used to increase the production of the desired analgesic compound.

Other techniques for increasing the production of the desired analgesic compound are also contemplated. For example, consider subcloning of existing polyclonal cell lines. Cells are cloned by limiting dilution to single cells in each well. Cell clones are cultures and these clones are tested to select the highest producing clone of analgesic material.

Another technique for increasing the production of the desired analgesic compound involves cloning of altered forms of biosynthetic enzymes (ie cleaved TH 1-155) with higher activity than wild type. Some cleaved forms of TH have 4-6 fold increased activity than the wild type of TH [see, eg, Daubner et al., "Expression and characterization of catalytic and regulatory domains of rat tyrosine hydroxylase", Protein Science, 2 , pp. 1452-60 (1993).

In addition, the use of selected tyrosine free media to increase tetrahydrobiopterin cofactor levels can potentially increase tyrosine hydroxylase activity. See, eg, Horellou et al., "Retroviral transfer of a human. tyrosine hydroxylase cDNA in various cell lines; regulated release of domain in mouse anterior pituitary AtT-20 cells ", Proc. Natl. Acad. Sci. USA, 86, pp. 7233-37 (1989).

The amount of β-endorphin is preferably 1 to 10,000 pg / 10 ⁶ cells / hour. The amount of methionine-enkephalin produced is preferably 1 to 10,000 pg / 10 ⁶ cells / hour. The amount of catecholamine produced is preferably 1 to 1,000 pmole / 10 ⁶ cells / hour.

Cells of the invention can be transplanted into mammals, including humans, to treat pain. When implanted without encapsulation, any suitable transplantation method can be used, including those described in Sagen et al., US Pat. No. 4,753,635; Also incorporated herein by reference.

It may be desirable to encapsulate the genetically modified cells of the invention prior to transplantation. These encapsulated cells form a living artificial organ (“BAO”). BAO can be designed for transplantation in the receptor or can be made to work externally. BAO useful in the present invention typically have a jacket surrounding one or more semipermeable outer surface membranes or cell-containing cores. The jacket allows for diffusion through BAO of nutrients, biologically active molecules and other selected products. The BAO is biocompatible.

In some cases, the membrane may contribute to immunocontaining the cell by blocking cellular or molecular effectors of immunological rejection. By using the immune isolation membrane, transplantation of heterologous or heterologous cells into a subject can be performed without performing immunosuppression. When biologically active molecules are released from isolated cells, they enter the body of the receptor through the surrounding semipermeable membrane. When metabolic function is provided by the isolated cells, the metabolized substance enters BAO from the body of the receptor through the cell-acting membrane.

Various types of membranes can be used to construct the BAO. In general, membranes used in BAO are either microporous membranes or ultrafiltration grade membranes. Various membrane materials have been proposed for use in BAO, examples being PAN / PVC, polyurethane, polysulfone, polyvinylidene and polystyrene. Typical membrane geometries include flat sheets, which can be assembled in a "sandwich" form with a layer of living cells located between two essentially flat membranes holding a seal formed around the perimeter of the device. Alternatively, hollow fiber devices can also be used, in which live cells are located inside the tubular membrane. Hollow fiber BAO can be formed stepwise by loading live cells into the lumen of the hollow fiber and sealing the ends of the fiber. Hollow fiber BAO can also be formed by coextrusion, where live cells are coextruded with a polymer solution that forms a membrane around the cells.

BAO is described, for example, in US Pat. Nos. 4,892,538, 5,106,627, 5,156,844, 5,158,881 and 5,182,111, and in PCT applications PCT / US94 / 07015, WO 92/19195, WO 93/03901 and 91/00119. It is hereby incorporated by reference.

BAO may contain other ingredients that promote long term survival of encapsulated cells. For example, WO 92/19195 describes an implantable immunoisotope biocompatible vehicle carrying a hydrogel matrix to enhance cell survival.

The encapsulation film of BAO can be made of the same material as the core, or of different materials. In either case, a membrane or peripheral membrane surrounding the site of the BAO, which is permeable and biocompatible, will be formed. In addition, the membrane may be composed of an immunoisolator if necessary. The core contains isolated cells, which may be suspended in liquid medium or immobilized in a hydrogel matrix.

The choice of material used to construct the BAO is determined by a number of factors, which are described in detail in WO 92/19195 (Dionne). Briefly, various capsules of polymers and polymers can be used to make such capsule jackets. The polymer film forming the BAO and the growth surface therein may be selected from the group consisting of polyacrylates (including acrylic copolymers), polyvinylidene, polyvinyl chloride copolymers, polyurethanes, polystyrenes, polyamides, cellulose acetates, cellulose nitrates, Polysulfone, polyphosphazene, polyacrylonitrile, poly (acrylonitrile / covinyl chloride) as well as derivatives, copolymers and mixtures thereof.

The BAO can be formed by any suitable method known to those skilled in the art. One method is the co-extrusion of coagulants, which may include polymer casting solutions and biological tissue fragments, suspensions of intracellular organelles or cells and / or other therapeutic agents. See WO 92/19195 (Dionne) and US Pat. No. 5,158,881, 5,283,187 and 5,284,761; Incorporated herein by reference].

The jacket may have a single skin layer or a double skin layer. Single layer hollow fibers can be prepared by quenching only one of the surfaces of the polymer solution when coextruded. Bilayer hollow fibers can be prepared by quenching both surfaces of the polymer solution when coextruded.

Various capsule structures are possible, such as cylindrical, disc or spherical.

The jacket of the BAO will determine the pore size that determines the nominal molecular weight cutoff (nMWCO) of the selective permeable membrane. Molecular weights greater than nMWCO do not physically pass through the membrane. Nominal molecular weight cuts are defined as 90% rejection under convective conditions. In situations where it is desirable for BAO to be immune sequestered, the membrane pore size is chosen to enable specific factors produced by the cell to diffuse out of the vehicle, but exclude host host immune response factors from entering the BAO. Typically, nMWCO is 50 to 200 kD, preferably 90 to 150 kD. In addition, the most suitable membrane composition will minimize the reactivity of the effector molecule with the outer membrane component of BAO, which is known to be present at the selected transplant site.

The core of the BAO is configured to provide a suitable local environment for the particular cells isolated therein. The core may comprise sufficient liquid medium to maintain cell growth. Liquid cores are particularly suitable for maintaining transformed cell lines such as PC12 cells. Alternatively, the core may comprise a gel matrix. The gel matrix may consist of a hydrogel (alginate, "Vitrogen®", etc.) or extracellular matrix components (WO 92/19195 (Dionne)).

Compositions that form hydrogels fall into three general classes. The first class holds net negative charges (eg alginates). The second class possesses net positive charges (eg collagen and laminin). Examples of commercially available extracellular matrix components include Matrigel® and Vitrogen®. The third class is net neutral (eg highly crosslinked polyethylen oxide or polyvinyl alcohol).

Any suitable method of sealing a BAO includes methods using polymeric adhesives and / or crimping, knotting and heat sealing. These sealing techniques are known in the art. In addition, any suitable "dry" sealing technique can also be used. In this method, substantially nonporous fittings are provided through the passage into which the cell-containing solution is introduced. After filling, the BAO is sealed. Such methods are described in co-pending US application 08 / 082,407, which is incorporated herein by reference.

One or more in vitro assays are preferably used to establish the function of the BAO prior to implantation in vivo. Assays or diagnostic tests that are well known in the art can be used for this purpose (see, eg, Methods In Enzymology, Abelson, Academy Press (1993)). For example, ELISA (enzyme-linked immunosorbent assay), chromatography or enzyme assay or bioassay specific to the secreted product can be used. If necessary, a suitable sample (eg, serum) can be collected from the receptor and analyzed to monitor the secretion of the implant over time. If the receptor is a primate, microdialysis may also be used.

The number of BAOs and the BAO size that should be sufficient to produce a therapeutic effect on the transplant is determined by the amount of biologically active required for the particular application. When secreted cells release therapeutic drugs, considerations and criteria according to standard administration known in the art are used to determine the amount of secretory material required. Factors to consider are described in WO 92/19195 (Dionne).

Implantation of BAO is performed under sterile conditions. In general, the BAO enables the delivery of secreted products or functions to the host appropriately, the delivery of nutrients to the encapsulated cells or tissues, and the access to the BAO for recovery and / or replacement. Is implanted at the site of the host. Preferred hosts are primates, with human beings most preferred.

Several different implant locations are contemplated. These transplant sites include the central nervous system including aqueous and vitreous fluids in the brain, spinal cord, and eyes. Preferred locations in the brain include striatum, cerebral cortex, hypothalamus nucleus and basal ganglia in the Minert. Other preferred locations are cerebrospinal fluid, most preferred subarachnoid space and lateral ventricles. The present invention also contemplates implantation into renal subcapsular and intraperitoneal and subcutaneous positions or any other therapeutically useful position.

The invention will be described in more detail by the following examples. These examples are illustrative only and are not intended to limit the scope of the invention in any sense.

Summary of the Invention

The present invention provides cell lines which are genetically engineered to produce one or more analgesic compounds selected from the group consisting of endorphins, enkephalins and catecholamines. The cell line can be used to treat pain.

There is an advantage of using cell lines over the use of early cells. High cost and low efficiency tests should be performed for individual isolation of initial cells to ensure cell safety and performance standards. Cell banks reduce the need for testing. There is no need to isolate early cells. The production of the desired analgesic is more stable because the performance of the initial cell may vary depending on the age, sex, health or hormonal status of the animal that donates it. In addition, higher quantities of the desired product can be obtained, as well as specifically modified peptides can be engineered into cell lines. This allows for simultaneous delivery of multiple analgesics. The expression of one or more such analgesics can be controlled (by using a controllable promoter to drive expression). In addition, for safety reasons, a "suicide" gene may be incorporated into the cell line. In addition, for the purpose of encapsulation, proliferative cells possess the advantage of separating the cells or dead cells from which they die.

Construction of Polycistronic Expression Vectors

Composition of IgSP-POMC Fusion

SmaI-SalI fragments containing human POMC exon 3 were subcloned into a pBS cloning vector (stratagen). Takahashi, supra; Cochet, supra. The resulting plasmid was called pBS-hPOMC-027 (see FIG. 1).

PCR fragments were generated using two oligonucleotide primers called oCNTF-003 (SEQ ID NO: 1) and oIgSP-018 (SEQ ID NO: 2) and a pNUT plasmid containing the human CNTF gene. Baetge et al., Proc. Natl. Acad. Sci. USA, 83, pp. 5454-58 (1986)]. Two primers pCNTF-003 and oIgSP-018 contained the synthetic BamHI and SamI restriction sites at the 5 'end, respectively.

The 196 base pair (bp) PCR fragment was digested with the restriction endonuclease BamHI and SamI-Isoskizomer XmaI and electrophoresed through 1% Seaplaque agarose. The 193 bp HindIII / XmaI DNA fragment was cut and purified using the FMC Spinbind DNA Purification Kit (FMC Bioproducts, Rockland, Maine, USA).

In addition, pBS-hPOMC-027 was digested with BamHI and XmaI, and purified through 1% Seaplaque agarose using FMC spinbind DNA purification kit (FMC Bioproducts, Rockland, Maine, USA). The ligation mixture was transformed into E. coli DH5α (Zibco BRL, Gettysburg, Maryland, USA).

Positive subclones were initially identified by gel digestion (Promega Protocol and Application Guide (1991)). Small lysate DNA was then prepared using the FMC spinbind DNA purification kit (FMC Bioproducts, Rockland, Maine, USA) and digested with restriction enzymes BamHI and SmaI. The positive subclone was called pBS-IgSP-hPOMC-028 (see FIG. 1). The nucleotide sequence of the fusion junction in pBS-IgSP-hPOMC-028 was determined by dideoxynucleotide sequencing using the Sequinase kit (USBC, Cleveland, USA). The sequence of the IgSP-hPOMC fusion is shown in SEQ ID NO: 3.

Construction of IgSP-POMC Expression Vector

IgSP-hPOMC DNA fragments in pBS-IgSP-hPOMC-028 include pcDNA3 (Invitrogen Corp., San Diego, California, USA) and pCEP4 (Invitrogen Co., Ltd., San Diego, CA, USA). ) And subclones into sense and antisense orientations.

NotI-SalI IgSP-hPOMC fragments derived from pBS-IgSP-hPOMC-028 were linked with NotI-XhoI digested pCEP4 to create a sense orientation clone called pCEP4-hPOMC-030. BamHI-SalI IgSP-hPOMC fragments derived from pBS-IgSP-hPOMC-028 were linked with BamHI-XhoI digested pCEP4 to produce an antisense orientation clone called pCEP4-hPOMC-031 (see FIG. 2). Insertion orientation in pCEP4-hPOMC-030 and -031 was confirmed by dideoxynucleotide sequencing using the Sequinase kit (USBC, Cleveland, USA) as well as BamHI, NotI, SalI and NotI / SalI restriction digestion.

BamHI-SalI IgSP-hPOMC fragments derived from pBS-IgSP-hPOMC-028 were linked to BamHI-XhoI digested pcDNA3 to generate a sense orientation clone referred to as pcDNA3-hPOMC-034 (see FIG. 2). NotI-HindIII IgSP-hPOMC fragments derived from pBS-IgSP-hPOMC-028 were linked with NotI-HindIII digested pcDNA3 to create an antisense orientation clone called pcDNA3-hPOMC-035. Restriction digestion with SmaI, BamHI, EcoRI and BamHI / EcoRI was used to confirm insertion orientation in pcDNA3-hPOMC-034, while restriction digestion with HindIII, NotI and SalI was confirmed for insertion orientation in pcDNA3-hPOMC-035.

Configuration 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 XmaI restriction enzyme and treated with pfu DNA polymerase (Promega, Madison, Wisconsin, USA). Subsequently, pBS-IgSP-hPOMC-028 treated with XmaI-pfu DNA polymerase was digested with STuI restriction enzyme and 1% using FMC spinbind DNA purification kit (FMC Bioproducts, Rockland, Maine, USA). Purification from Seaplaque agarose. Positive subclones were identified by BamHI / HindIII restriction digestion and named pBS-IgSP-hPOMCΔACTH-029 (FIG. 1). The nucleotide sequence of the ACTH deletion region in pBS-IgSP-hPOMC-ΔACTH-029 was confirmed by dideoxynucleotide sequencing. The sequence of the IgSP-hPOMC-ΔACTH fusion is shown in SEQ ID NO: 4.

Construction of ACTH-deleted IgSP-POMC Expression Vectors

IgSP-hPOMC-ΔACTH DNA fragments in pBS-IgSP-hPOMC-ΔACTH-029 were subcloned into sense and antisense orientations into pcDNA3 (Invitrogen, San Diego, CA, USA) and pCEP4 (Invitrogen). NotI-Sal IgSP-hPOMC-ΔACTH fragments derived from pBS-IgSP-hPOMC-ΔACTH-029 were linked with NotI-XhoI digested pCEP4 to generate a sense orientation clone referred to as pCEP4-hPOMC-ΔACTH-032. 3). BamHI-SalI IgSP-hPOMC-ΔACTH fragments derived from pBS-IgSP-hPOMC-ΔACTH-029 were linked to BamHI-XhoI digested pCEP4 to create an antisense orientation clone referred to as pCEP4-hPOMC-ΔACTH-033 (see: 3). Insertion orientation in pCEP4-hPOMC-ΔACTH-032 and -033 was confirmed by dideoxynucleotide sequencing using the Sequinase kit (USBC, Cleveland, USA) as well as BamHI and EcoRI restriction digestion.

BamHI-SalI IgSP-hPOMC-ΔACTH fragments derived from pBS-IgSP-hPOMC-ΔACTH-029 were linked to BamHI-XhoI digested pcDNA3 to generate a sense orientation clone referred to as pcDNA3-hPOMC-ΔACTH-036 (see: 3). NotI-HindIII IgSP-hPOMC-ΔACTH fragments derived from pBS-IgSP-hPOMC-ΔACTH-029 were linked with NotI-HindIII digested pcDNA3 to generate an antisense orientation clone referred to as pcDNA3-hPOMC-ΔACTH-037 (see: 3).

Restriction digestion with PvuII and EcoRI was used to confirm insertion orientation in pcDNA3-hPOMC-ΔACTH-036, while restriction digestion with SalI and EcoRI was confirmed for insertion orientation in pcDNA3-hPOMC-ΔACTH-037.

Cloning of Full Length and Cleaved TH cDNA

Total RNA from PC12 cells was prepared using guanidium thiocyanate-based TRI preparations (Molecula Research Center Inc., Cincinnati, Ohio). 500 ng of PC12 total RNA was 10 mM Tris.HCl (pH8.3), 50 mM KCl, each dNTP 4 mM, 5 mM MgCl ₂ , 1.25 μM oligo (dT) 15-mer, 1.25 μM random hexamer, 31 units of RNase Reverse transcription was carried out at 42 ° C. for 30 minutes in 20 μl of a reaction solution containing a Guard RNase inhibitor (Pharmacia, Sweden) and 200 units of SuperScript II reverse transcriptase (Zibco BRL, Gettysburg, Maryland, USA). 2 μl of the reverse-transcribed cDNA was added to 10 mM Tris.HCl (pH 8.3), 50 mM KCl, each dNTP 800 nM, 2 mM MgCl ₂ , 400 nM Primer # 1 and # 2 and Termus Aguaticus (Taq) DNA polymer. It was added to 25 μl of the PCR reaction mixture containing Lase (Müllermann, Germany).

To generate full length TH cDNA, oligonucleotide primers orTH-052 (SEQ ID NO: 5) and orTH-053 (SEQ ID NO: 6) were used. For cleaved TH, primers orTH-54 (SEQ ID NO: 7) and orTH-053 (SEQ ID NO: 6) were used instead. These oligonucleotides were constructed based on published TH sequence information. See Grima et al., Nature, 326, pp. 707-11 (1987); U.S. Patent 5,300,436 and Daubner, supra.

Primers orTH-052 (SEQ ID NO: 5) and orTH-054 (SEQ ID NO: 7) had synthetic HindIII restriction sites at the 5 'end, and orTH-053 had BamHI at the 5' end. The PCR reaction mixture was denatured at 94 ° C. for 30 seconds (first cycle of 2 minutes); Annealing at 50 ° C. for 1 minute; And 30 amplification cycles consisting of elongation at 72 ° C. for 3.5 minutes (final cycle 5 minutes). The 1537 bp full length mouse TH PCR fragment and 1087 bp truncated mouse TH PCR fragment were digested with restriction endonucleases BamHI and HindIII and dissolved on a 1% Seaplaque agarose gel. The 1531 bp and 1081 bp HindIII / BamHI fragments were cut and purified using the FMC Spinbind DNA Purification Kit (FMC Bioproducts, Rockland, Maine, USA).

In addition, pcDNA3 expression vectors were digested with BamHI and HindIII and purified from 1% Seaplaque agarose using FMC spinbind DNA purification kit (FMC Bioproducts, Rockland, Maine, USA). The ligation mixture was transformed into E. coli DH5α (Zibco BRL, Gettysburg, MD, USA).

Positive subclones were screened using gel digestion (Promega Protocol and Application Guide (1991)). Identification of the correct clone was further demonstrated by BamHI / HindIII double digestion.

Positive sub-clones for full length rat TH and truncated mouse TH in pcDNA3 were referred to as pcDNA3-rTH-044 (FIG. 4) and pcDNA3-rTHΔ-045 (FIG. 4), respectively. Both full-length murine TH PCR clones and truncated murine TH PCR clones were determined by dideoxynucleotide sequencing using the Sequenase kit (USBC, Cleveland, USA). The sequence of the rTHΔ construct is shown in SEQ ID NO: 16.

To optimize the translational efficiency of truncated rat TH, oligonucleotide orTH-078 (SEQ ID NO: 8) was designed so that the consensus Kozak sequence was located immediately upstream of the start codon ATG. pcDNA3-rTHΔ-45 was prepared in 50 μl of a PCR reaction mixture containing the same reagent composition as described above except that the oligonucleotide primers were replaced with orTH-078 (SEQ ID NO: 8) and orTH-053 (SEQ ID NO: 6). Used as a template. The 1097 bp PCR product was cloned into pcDNA3 in the same manner as described above. The resulting subclone was called pcDNA3-rTHΔKS-75 (see FIG. 4). The sequence of the rTHΔKS construct is shown in SEQ ID NO: 17.

Composition of the rTH-IRES-bDBH Fusion Gene

Recombinant PCR techniques were used to generate the rTH-IRES-bDBH fusion gene. Oligonucleotides oIRES-057 (SEQ ID NO: 9) and obDH-065 (SEQ ID NO: 10) were specific for the IRES and bBDH gene sequences, respectively, and contained 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) were complementary. In addition, the oligonucleotide primer oIRES-bBDH-064 (SEQ ID NO: 11) had its 5 '16 nucleotides identical to the IRES sequence and 3' 18 nucleotides identical to the bDBH sequence; In the case of oIRES-bDBH-066 (SEQ ID NO: 12), the opposite was true.

Two first PCR reactions were performed using the oligonucleotide pairs oIRES-057 / oIRES-bDBH-066 and oIRES-bDBH-064 / obDBH-065 with an insert containing the IRES sequence shown in template PCTI-001 (SEQ ID NO: 30). ) And pBS-bDBH-006 (containing bovine DBH gene cloned from bovine adrenal chromaffin cells (see Lamoroux et al., EMBO J., 6, pp. 3931-37 (1987))), respectively, on plasmids It was performed by. 100 ng of template DNA was added to 10 mM Tris.HCl (pH 8.3), 50 mM KCl, 800 nM dNTP, 2 mM MgCl ₂ , 400 nM of primer # 1 and # 2 and 2.5 units of Termus Aguaticus (Taq) 50 μl of a PCR reaction mixture containing DNA polymerase (Möllinger Mannheim, Germany) was added.

The PCR reaction mixture was denatured at 94 ° C. for 30 seconds (first cycle of 2 minutes); Annealing at 50 ° C. for 1 minute; And 30 amplification cycles consisting of elongation at 72 ° C. for 30 seconds (final cycle 5 minutes). The PCR product was dissolved on 1% Trivigel 500 (Tribigen). Two agarose plugs each containing one of the first PCR products were transferred to a tube containing 50 μl of the same PCR reaction mixture as above, except using oligonucleotides pIRES-057 and obDH-065.

The second PCR reaction showed that the PCR reaction mixture was denatured at 94 ° C. for 30 seconds (first cycle 2 minutes); Annealing at 60 ° C. for 30 seconds (2 minutes at 37 ° C. of the second to fourth cycles); And 30 amplification cycles consisting of elongation at 72 ° C. for 30 seconds (final cycle 2 minutes). The 2407 bp IRES-bDBH fusion PCR product and the cloning vector pcDNA3-rTHΔ-45 were digested with BamHI and NotI restriction enzymes, and were digested with FMC spinbind DNA purification kit (FMC Bioproducts, Rockland, Maine, USA). Subsequent purification from% Seaplaque agarose gel.

Linkage of IRES-bDBH / BamHI / NotI and pcDNA3-rTHΔ-045 / BamHI / NotI produces an rTHΔ-IRES-bDBH expression vector called pcDNA3-rTHΔ-IRES-bDBH-066, while IRES-bDBH / BamHI / The linkage of NotI and pcDNA3-rTHΔKS-075 / BamHI / NotI produced an rTHΔ-IRES-bDBH expression vector called pcDNA3-rTHΔKS-IRES-bDBH-076, where ATG, the starting codon in rTHΔ, was present before the consensus Kozak sequence. It was. The sequence of the rTHΔ-IRES-bDBH construct is shown in SEQ ID NO: 18. The sequence of the rTHΔ-IRES-bDBH construct is shown in SEQ ID NO: 19. The ligation mixture was transformed into DH5α (Zibco BRL, Gettysburg, MD, USA). The positive clones were identified using gel digestion (Promega, Madison, WI) and restriction digestion using HindIII, BamHI, HindIII / BamHI, SmaI and NotI.

4114 bp NruI-XhoI fragment containing CMV promoter-rTHΔKS-IRES-bDBH was cleaved from pcDNA3-rTHΔKS-IRES-bDBH-076 and pZeoSV cloning vector digested with ScaI and XhoI (San Diego, CA, USA) Subcloning into multiple cloning positions in Invitrogen coporation). The resulting expression vector was termed pZeo-Pcmv-rTHΔKS-IRES-bDBH-088 (FIG. 6).

Composition of the IgSP-hPOMC ACTH-rTHD-IRES-bDBH Fusion Gene

A 4100 bp NruI-NotI fragment comprising a CMV promoter, rTHD-IRES-bDBH fusion gene and BGH polyadenylation sequence was cleaved from pcDNA3-rTHΔ-IRES-bDBH-066 and pBS (Lazola, CA, USA) Stratagen) cloning in the cloning vector.

The resulting plasmid, pBS-Pcmv-rTHΔ-IRES-bDBH-067 (FIG. 7), was used as an intermediate construct into which the recombinant PCR IgSP-hPOMCΔACTH-IRES fragment was inserted.

Oligonucleotide oIgSP-068 (SEQ ID NO: 13) containing the synthetic EcoRV restriction site was specific for the IgSP sequence.

Oligonucleotide primer orTHΔ-073 (SEQ ID NO: 14) was specific for the rTHΔ sequence and contained an endogenous SmaI restriction site.

Oligonucleotide primers ohPOMC-IRES-069 (SEQ ID NO: 15) and ohPOMC-IRES-070 (SEQ ID NO: 20) were complementary to each other. In addition, the oligonucleotide primer ohPOMC-IRES-069 had its 5 '18 nucleotides identical to the hPOMC sequence and its 3' 12 nucleotides identical to the IRES sequence; In the case of ohPOMC-IRES-070, the opposite was true.

Oligonucleotide primers oIRES-rTHΔ-071 (SEQ ID NO: 21) and oIRES-rTHΔ-072 (SEQ ID NO: 22) were complementary to each other. In addition, the oligonucleotide primer oIRES-rTHΔ-071 had its 5 ′ 15 nucleotides identical to the rTHΔ sequence and its 3 ′ 18 nucleotides identical to the IRES sequence; The opposite was true for oRIRES-rTHΔ-072.

Three sets of first PCR reactions were performed.

PCR reaction A: template pBS-IgSP-hPOMCDACTH-029, oligonucleotide oTgSP-068 / ohPOMC-IRES-069;

PCR reaction B: template pCTI-001, oligonucleotide ohPOMC-IRES-070 / oIRES-rTHΔ-071; And

PCR reaction C: template pcDNA3-rTHΔ-045, oligonucleotide orIRES-rTHΔ-072 / orTHΔ-073.

The first three sets of PCR reactions were 100 ng of template DNA, 10 mM Tris.HCl (pH8.3), 50 mM KCl, 800 nM of each dNTP, 2 mM MgCl ₂ , 400 nM of primers # 1 and # 2 and 2.5 The reaction was carried out in 50 μl of a PCR reaction mixture containing a unit of Termus Aquaticus DNA polymerase (Möllinger Mannheim, Germany).

The PCR reaction mixture was denatured at 94 ° C. for 30 seconds (first cycle of 2 minutes); Annealing at 60 ° C. for 30 seconds; And 30 amplification cycles consisting of elongation at 72 ° C. for 30 seconds (final cycle 5 minutes).

The PCR product was dissolved on 1% Trivigel 500 (Tribigen). Two agarose plugs each containing one of the first PCR products were transferred to a tube containing 50 μl of the same PCR reaction mixture as described above, except using oligonucleotides ohPOMC-IRES-070 and orTHΔ-073. .

The second PCR reaction showed that the PCR reaction mixture was denatured at 94 ° C. for 30 seconds (first cycle 2 minutes); Annealing at 60 ° C. for 30 seconds (2 minutes at 37 ° C. of the second to fourth cycles); And 30 amplification cycles consisting of elongation at 72 ° C. for 30 seconds (final cycle 2 minutes).

The PCR product was treated as described above. Agarose plugs containing the PCR product from the second PCR reaction and PCR reaction A were combined and a third PCR using oIgSP-068 / rTHΔ-073 was performed. 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 XmaI restriction enzymes and FMC spinbind DNA purification kit (USA, Maine, Rockland) Purified from 1% Seaplaque agarose gel using FMC Bioproducts, Inc.

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

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

4491 bp NotI fragment containing IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES-bDBH gene was cleaved from pBS-IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES-bDBH-068 and pcDNA3 (Invigo, San Diego, CA, USA) Subclones into NotI positions in multiple cloning positions). Restriction digestion using NotI and SmaI inserted the IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES-bDBH gene in the sense orientation to generate pcDNA3-IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES-bDBH-069 (see FIG. 9). .

Construction of IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES-bDBH-IRES-Zeosin Expression Vectors

Recombinant PCR techniques were used to generate the IRES-Xeosin fusion gene. Oligonucleotides oIRES-074 (SEQ ID NO: 24) and ozeocin-077 (SEQ ID NO: 25) were specific for the IRES and zeocin gene sequences, respectively, and contained NotI and XhoI at the 5 'end. Oligonucleotides oIRES-Xeosin-075 (SEQ ID NO: 26) and oIRES-Xeosin-076 (SEQ ID NO: 27) were complementary to each other. In addition, the oligonucleotide oIRES-zeocin-075 had its 5 '15 nucleotides identical to the zeosin sequence and its 3' 18 nucleotides identical to the IRES sequence; In the case of oIRES-Zeosin-076, the opposite was true.

Two first PCR reactions were performed on oligonucleotide pairs oIRES-074 / oIRES-Zeocin-075 and oIRES-Zeosin- on templates pCTPI-001 and pZeoSV (Invitrogen Coporation, San Diego, CA, respectively), respectively. It was performed using 076 / ozeocin-075.

100 ng of template DNA was added to 10 mM Tris.HCl (pH 8.3), 50 mM KCl, 800 nM dNTP, 2 mM MgCl ₂ , 400 nM of primer # 1 and # 2 and 2.5 units of Termus Aguaticus (Taq) 50 μl of a PCR reaction mixture containing DNA polymerase (Möllinger Mannheim, Germany) was added.

The PCR reaction mixture was denatured at 94 ° C. for 30 seconds (first cycle of 2 minutes); Annealing at 50 ° C. for 1 minute; And 30 amplification cycles consisting of elongation at 72 ° C. for 30 seconds (final cycle 5 minutes).

The PCR product was dissolved on 1% Trivigel 500 (Tribigen). Two agarose plugs, each containing one of the first PCR products, were transferred to a tube containing 50 μl of the same PCR reaction mixture as above, except using oligonucleotides oIRES-074 and ozeosine-077. .

The second PCR reaction showed that the PCR reaction mixture was denatured at 94 ° C. for 30 seconds (first cycle 2 minutes); Annealing at 60 ° C. for 30 seconds (2 minutes at 37 ° C. of the second to fourth cycles); And 30 amplification cycles consisting of elongation at 72 ° C. for 30 seconds (final cycle 2 minutes).

974 bp IRES-Zeosin fusion PCR product and cloning vector pcDNA3 were digested with NotI and XhoI restriction enzymes and 1% Seaplaque using FMC spinbind DNA purification kit (FMC BioProducts, Rockland, Maine, USA). Purification from agarose gel.

Linkage of IRES-Zeosin / NotI / XhoI and pcDNA3 / NotI / XhoI produced an intermediate cloning vector called pcDNA3-IRES-Xeosin-072 (see FIG. 10).

Positive clones were identified by gel digestion (Promega, Madison, WI) and restriction digestion using HindIII, SmaI, XhoI, NotI and NotI / XhoI.

To generate the final IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES-bDBH-zeocin expression vector, the 4491 bp NotI fragment containing the IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES-bDBH gene was selected as pBS-IgSP-hPOMCΔACTH-IRES- It was cleaved from rTHΔ-IRES-bDBH-068 (FIG. 8; SEQ ID NO: 23) and subcloned into pcDNA3-IRES-Xeosin-072 (FIG. 10) to the NotI position in multiple cloning positions.

Restriction digestion using NotI and SmaI results in the insertion of the IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES-bDBH gene in the sense direction to form pcDNA3-IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES-bDBH-IRES-zeosin-073. Confirmed. The sequence of this construct is shown in SEQ ID NO: 28 (see FIG. 11).

Composition of ProA + KS Fusion

The construct contained the coding region of the human proenkephalin A gene with the consensus Kozak sequence present immediately upstream of the start codon ATG. The sequence of this construct is shown in SEQ ID NO: 29.

Construction of hProA + KS Expression Vectors

HindIII / BamHI fragments containing the hProA + KS fusion were substantially linked into BamHI and HindIII digested pcDNA3 expression vectors as described above. After screening as described above, positive clones were referred to as pcDNA3-hProA + KS-091 (see FIG. 12). The construction of the pBS-CMV ProA vector is described in Mothi, J. and Lindberg, I., Endocrinology, 131, pp. 2287-96 (1992).

Cell transformation

RIN and AtT-20 cells were transformed as follows.

RINa and AtT-20 based cell lines were grown in DMEM (Zibco) containing 10% fetal bovine serum and pen-strep-fungozone (Zibco) based media. The cells were plated (750,000 cells / dish) in a P100 Petri dish in 10 ml of basal medium. After 18-24 hours, the cells were transfected with the calcium phosphate method using a kit made from Stratagen (San Diego, Calif., USA). 10 μg of the plasmid DNA vector DNA was diluted in 450 μl of deionized sterile water. 50 μl of 10 × buffer (solution # 1) was then added to the plasmid DNA. 500 μl of solution # 2 was added immediately to the DNA containing solution and mixed gently. The mixture was incubated at room temperature for 20 minutes, and then 1.0 ml solution was added to the cells in a petri dish. The cells were incubated overnight and then washed twice after 18 hours with Hank's balanced salt solution containing no calcium and magnesium. The cells were then cultured in basal medium + selection drug. The cells were selected from 600 μg / ml Geneticin (Zibco) or 400 μg / ml Hygromycin (Möllinger Mannheim) or 500 μg / ml Zeosin (Invitrogen, San Diego, CA, USA). The cells were sequentially transfected and selected to select the final cell line.

The RINa cells were transfected with plasmid pCEP4-hPOMC-030 containing the POMC gene. The plasmid was a hygromycin resistance vector. The cells were also transformed with plasmid pcDNA3-hProA + KS-091. The plasmid was a geneticin resistance vector. Finally, the cells were transfected with plasmid pZeo-PCMV-rTHΔKS-IRES-bDBH-088 conferring zeocin resistance.

AtT-20 cells were transfected with the plasmids pBS-CMV-ProA and pCEP4-POMC-ΔACTH-32 conferring genesis and hygromycin resistance, respectively. Finally, the cells were infected with the plasmid pZeo-Pcmv-rTHΔKS-IRES-bDBH-088.

We tested a number of media for cell growth. Surprisingly, we have found that in certain serum-free media, the cell lines produced enhanced neurotransmitters compared to serum-containing media. We preferred CHO-Ultra (BioWhittaker) for the growth of AtT-20 cells and Ultra-Culture (BioWhittaker) for the growth of RINa cells.

The production of various analgesics from one transformed RINa cell line (RINa / ProA / P030 / P088) is shown in Table 2 below. All values refer to unstimulated cells. The production of β-endorphin and methionine-enkephalin was pg / 10 ⁶ cells / hour. β-endorphin and methionine-enkephalin were measured by immunoassay using the Inkstar kit (Stillwater, Minnesota, USA). Catecholamine production was pmole / 10 ⁶ cells / hour. The values in parentheses are from the cells pre-incubated for 18 hours with 100 μM tetrahydrobiopterin. Catecholamines are described in Lavoie et al., "Two PC12 pheochromocytoma lines seald in hollow fiber-based capsules tonically release l-dopa in vitro", Cell transplantation, 2, pp. 163-73 (1993)]. GABA production from these RINa cells was 28 ng / 10 ⁶ cells / hour.

There was no encoded enkephalin fragment that was not fully processed from the pro-enkephalin precursor molecule. These encoded enkephalins retained opioid binding activity. We digested these encoded enkephalins to determine opioid activity. The trypsin digestion protocol is as follows: 2 μg / ml of trypsin (Washington # 34E470) solution was added to the medium on ice. The sample was stirred and then incubated for 20 minutes in a 37 ° C. water bath. After digesting for 20 minutes, the sample was returned to ice and 100 ng / ml of Carboxypeptidase B (Sigma # C-7011) was added. The samples were stirred and mixed and returned to the water bath at 37 ° C. for 15 minutes. The sample was placed once more on ice and 10 μg / ml of trypsin inhibitor was added. At this stage, samples were extracted for methionine-enkephalin or frozen immediately for later extraction. This resulted in complete enzymatic cleavage, freeing all methionine-enkephalins from the longer encoded fragments. Methionine-enkephalin radioimmunoassay of the digested samples yielded the entire methionine-enkephalin from the supernatant. The transformed RINa cells were shown to have a 5 times larger encoded enkephalin compared to fully treated methionine-enkephalin.

Fiber Capsule Formation and Characteristics

Hollow fibers were spun from a 12.5-13.5% poly (acrylonitrile vinylchloride) solution using the wet spinning technique. Cabasso, Hollow Fiber Membranes, Vol. 12, Kirk-Othmer Encyclopedia of Chemical Technlogy, Wiley, New york, 3rd edition, pp. 492-517 (1980), US Pat. No. 5,158,881; Incorporated herein by reference].

The resulting membrane fibers may be bilayer or monolayer PAN / PVC fibers. To make implantable capsules, the fibers were cut into fragments of the first 5 cm length and the spaced ends of each fragment were sealed with acrylic glue. The encapsulation hub assembly provides a membrane of length as described above, seals one end of the fiber with a drop of LCM 24 (photocurable acrylic glue commercially available from ICI), cures the glue to blue light, and with a second LCM drop. Prepared by repeating the above steps. The opposite end is already attached to the hub assembly with a brittle neck. The assembly has a silicon septum into which the cell solution can be introduced. The fibers can be attached to the hub assembly by applying LCM22 to the outer diameter of the hub assembly, pulling the fibers over and curing with blue light. The hub / fiber assembly was placed in a sterile bag and sterilized by ETO.

After sterilization and degassing using ethylene oxide, the fibers were first removed by filtration of 70% ethanol, then HEPES buffered saline solution by ultrafiltration through the walls of the fibers under vacuum.

Preparation and Encapsulation of Transformed Cells

As described below, transformed cells were prepared and encapsulated:

Matrix solutions were prepared using commercially available alginates, collagen or other suitable matrix materials. The cell solution was diluted with the ratio of 2 parts of the matrix solution to 1 part of the cell solution containing the transformed cells. We preferred Vitrogen (Celtics of Santa Clara) as the matrix for AtT-20 cells.

We preferred organogen (Organogenesis, Canton, Massachusetts, USA) as the matrix for RINa cells. RINa-based cells were prepared for encapsulation by the following method. The cells of the proliferative phase were grown in basal medium consisting of DMEM + 10% fetal bovine serum. These cells could be removed from tissue culture flasks by washing twice in Hank's balanced salt solution containing no calcium and magnesium. The cells were then incubated for 1 minute in 0.25% trypsin + EDTA. This was removed and cells were washed from the flask with Hank's balanced salt solution containing no calcium and magnesium. The cells were placed in 10 ml of basal medium and centrifuged at 100 × g for 2 minutes. The cells were resuspended in 10 ml of a preferred serum-free medium (BioWittaker, Walkersville, MD, USA). Surprisingly, the RINa cells secreted more analgesic material when cultured in serum-free medium than in serum-containing medium.

The cells were centrifuged twice at 100 × g prior to concentrating the cells 1: 1 with the desired organogen matrix. Organogen is 1% bovine dry collagen obtained as a sterile solution. Eight parts of the solution and one part of 10X DPBS were mixed. 0.5 N sodium hydroxide was added (about 250 μl) until a physiological pH was obtained.

The final concentration of the cell + matrix solution used for encapsulation was 20,000 to 50,000 cells / μl. The cells were counted on a hemocytometer by standard methods.

The cell / matrix suspension was placed in 1 ml syringe. A Hamilton 1800 series 50 μl syringe adjusted to 15 μl of bubbles was inserted into a 1 mL syringe containing the cell solution and 30 μl was withdrawn. The cell solution was injected into the lumen of a modacrylic hollow fiber membrane having a molecular weight cutoff of about 50,000 to 100,000 Da through a silicone seal of the hub / fiber assembly. Ultrafiltration should be observed along the entire length of the fiber. After one minute, the hub was removed and the cell solution exposed a fresh surface of the sub-hub that was not wet. One drop of LCM24 was applied and the adhesive was cured with blue light. The device was first placed in HEPES buffered NaCl solution and then placed in CaCl ₂ solution for 5 minutes to crosslink alginate. Each implant was about 5 cm in length and 1 mm in diameter and contained about 2.5 × 10 ⁶ cells.

After filling and sealing the device, a silicone tether (specialty silicon fabrication, Paso Robles, Calif .; inner diameter = 0.69, outer diameter = 1.25) was placed on the adjacent end of the fiber. A radiopaque titanium plug was inserted into the lumen of the silicon tether to serve as a marker for radiographic measurements. The device was then placed in a 100 mm tissue dosing dish containing 1.5 ml of PC-1 medium, stored in 37 ° C. and 5% carbon dioxide incubator for in vitro analysis, and kept stored until transplantation.

Encapsulated cells were then implanted into the human subarachnoid space as follows:

Surgical Method

After establishing an IV approach and administering prophylactic antibiotics (cepazoline sodium, 1 g IV), the patient is generally placed in the supine or knee-thorax-relaxing position while bending the lumbar spine to the ventral side on the operating table. I was. The operating room was manufactured aseptically, covered with the lumbar region exposed from the S-1 to L-1 levels, and allowed to internalize the lumbar spine by C-cancer fluoroscopy. Partial infiltration with 1% lidocaine was used to anesthetize the skin and other deep connective tissue including the yellow ligaments leading to the epicardium and the lower part.

A 3-5 cm incision was made from 1-2 cm to the left and right sides of the central line, and an incision was made to the lumbar fascia using electrocautery for hemostasis. 18 gauges of tohihi using traditional bone markers including fluoroscopy induction as well as iliac tomb and lumbar spine treatments ) A needle was introduced into the subarachnoid space between L-3 and L-4. The needle was guided to enter the hollow at an induced angle at the top, which is 30-35 ° or less relative to the spinal cord in the sagittal plane or transverse plane. Appropriate location of the end of the needle was confirmed by collecting several ml of cerebrospinal fluid (CSF) for pre-transplanting catecholamines, enkephalins, glucose, measuring protein levels and counting cells.

The Touhi needle hub is reexamined to confirm that the opening at the end is oriented upwards (the opening direction is marked with gold on the occlusion opening on the needle hub) and until the guide line extends 4 to 5 cm into the astrocyte space. (Determined by measuring in advance) The needle was passed down through the lumen of the lumen. It should be noted that when passing the lead line there is no resistance to the entry of the lead line from the needle, and the patient does not complain of significant neurological signs or impending neuromuscular or spinal cord injury indicating the direction of the lead line is wrong.

After the guide line appeared to be properly positioned in the subarachnoid space, the tonee needle was pulled out separately and separated from the guide line. It was confirmed by fluoroscopy that the location of the guide line in the centerline of the spinal conduit before the expected position of the cauda equina and that no entanglement or unexpected bending occurred. After removal of the tow needle, the guideline must be dense and move through the fibrous yellow ligament, allowing it to move freely in and out of the subarachnoid space with very little resistance due to the rough surface of the guideline.

A 7 French dilator was then placed on the guide line, using the guide line to guide softly or strongly through the fascia, peri spinal muscle and yellow ligaments, and then directed the track of the guide line toward the subarachnoid space. 7 The expansion of the French dilator was stopped and the dilator passed through the yellow ligament and was removed from the induction line as soon as a loss of resistance was detected. This is to avoid advancing and manipulating relatively rigid dilators in the subarachnoid space to any significant extent.

After the guide line tracks were "extended" by the 7 French expanders, the 6 French expanders and cannula packs were assembled and placed over the guide lines. The 6 French dilator and cannula were carefully advanced into the subarachnoid space until the opening end of the cannula was located 7 cm in the subarachnoid space. As with the 7 French dilator, the assembled 6 French dilator and cannula were guided by guide lines in the lumen of the dilator. The location within the subarachnoid space was determined by preliminary measurement of the device and confirmed by fluoroscopy as a whole. Careful attention should be given to the manipulation of the dilator and cannula in the subarachnoid space to avoid erroneous orientation and possible neurological damage.

When the proper position of the cannula was ensured, the guide line and the 6 French dilators were removed sequentially from within the lumen of the cannula. Depending on the position of the patient on the operating table, the CSF flows through the cannula from the point of view and can be very light and prevents excessive flow of CSF by clogging the cannula's lid or very quickly positioning the cannula implant. It was.

Encapsulated (promoted cells) are located in the transport medium and are provided in a fully assembled sterile double membrane container containing a tubular silicone tether. Prior to implantation through the cannula into the subarachnoid space, the capsule was transferred into an insertion kit tray where it was properly placed and maintained in the transport medium while performing a complete investigation of damage or major disorders, while cutting the ends of the silicone tether. Its length was adjusted to a pusher and the hema clip (trademark) connecting its outer end was removed.

The tether portion of the capsule was laminated onto a stainless steel pusher by inserting the small diameter guideline portion of the pusher when the membrane portion of the device was carefully introduced into the cannula. The capsule was advanced until the end of the membrane reached a point of 2-10 mm in the cranial end of the cannula in the subarachnoid space. This position was obtained by preliminary measurement of the cannula and cannula-terter-pusher assembly, which ensured that the membrane portion of the capsule was protected by the cannula for the entire time of advancing into position.

After placing the capsule in the cannula, the pusher was used to position (without advancing or retracting) the subarachnoid space and the cannula was completely removed from the capsule and pusher. By using this method, the final position of the capsule is positioned entirely within the CSF where the 5 cm long membrane portion of the device contains the horsetail in the subarachnoid space mask. It is secured to its tail end by a about 1-2 cm long silicone tether moving within the astrological space before the tether exits through the cerebral meninges and yellow ligaments. The tether continues outward through this periphery of the spine from this level, leaving about 10 to 12 cm of glass tether material useful to exit the lumbar fascia and secure the device.

CSF outflow was minimized by injecting fibrin glue (TICEL®) into the track occupied by the tether in the periphery of the spine and completely sealing the upper fascia opening of the track with purse-string sutures. . The glass end of the tether was then fixed with a nonabsorbable suture and completely covered with two hermetic layers consisting of epidermis and subcutaneous tissue.

The patient was then transferred to a room for neurosurgical recovery and rested in a straight bed for 24 hours after surgery. In addition, antibiotic prophylaxis was performed for 24 hours after transplantation.

order

Below is an overview of the sequences mentioned in the sequence listing:

SEQ ID NO: 1 DNA sequence of oligo oCNTF-003

SEQ ID NO: 2: DNA sequence of oligo oIgSP-018

SEQ ID NO: 3: DNA sequence of the IgSP-hPOMC fusion

SEQ ID NO: 4: DNA sequence of the 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 oIRES-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 rTHΔ1-155

SEQ ID NO: 17 DNA sequence of rTHΔ + KS

SEQ ID NO: 18 DNA sequence of rTHΔ-IRES-bDBH

SEQ ID NO: 19 DNA sequence of rTHΔKS-IRES-bDBH

SEQ ID NO: 20 DNA sequence of oligo ohPOMC-IRES-070

SEQ ID NO: 21 DNA sequence of oligo oIRES-rTHΔ-071

SEQ ID NO: 22 DNA sequence of orIRES-rTHΔ-072

SEQ ID NO: 23 DNA sequence of an IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES-bDBH-068 fusion

SEQ ID NO: 24 DNA sequence of oIRES-074

SEQ ID NO: 25 DNA sequence of oligo ozeosine-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 of IgSP-hPOMCΔACTH-IRES-rTHΔ-IRES-bDBH-IRES-Zeocin-073

SEQ ID NO: 29 DNA sequence of ProA + KS

SEQ ID NO: 30 DNA sequence of an IRES fragment

Deposit

As described in the examples, transformed RINa / ProA / POMC / TH-IRES-DBH cells (RINa / ProA / P030 / P088) were deposited to produce catecholamines, enkephalins and endorphins. The deposit was deposited on June 7, 1995, by the United States Bacterial Culture Collection (ATCC), Rockville, Maryland, USA under the BUDIFEST Treaty, and was assigned CRL 11921 by accession number.

The description so far is intended to illustrate and explain the invention. The present invention is not limited to the scope for the purpose of such illustration only. The scope of the invention will be determined by the appended claims.

Sequence Listing

(1) General Information:

(i) Applicant: Cytoteraphytics, Inc. (in all designated countries except US)

Show 웡 (in the United States)

Joel Seidoff (in the United States)

(ii) Name of the Invention: Pain Cell Line

(iii) number of sequences: 30

(iv) Letter Address:

(A) To: James F. Harley, Junior / Ivory El. Elfies

Fish and Nib

(b) Street: Avenue of the Americas 1251

(C) City: New York

(D) State: New York

(E) Country: United States

(F) Zip code: 10020-1104

(v) computer readable form:

(A) Media type: floppy disk

(B) Computer: IBM PC compatible model

(C) operating system: PC-DOS / MS-DOS

(D) Software: Patented Release # 1.0, Version # 1.30

(vi) current application data

(A) Application number:

(B) Applicant:

(C) Classification:

(vii) Former application data:

(A) Application number: US 08 / 481,917

(B) Application date: June 7, 1995

(viii) Agent Information:

(A) Statement: Ivor El. Elfies

(B) registration number: 39,529

(C) Reference Number: CIT-29 CIP PCT

(ix) Communications Information:

(A) Phone: (212) 596 9600

(B) Fax: (212) 596 9090

(2) information for SEQ ID NO:

(i) sequence properties:

(A) Length: 33 base pairs

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: cDNA

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) Clone: oCNIF-003

(xi) SEQ ID NO 1:

(2) Information about SEQ ID NO:

(i) sequence properties:

(A) Length: 23 base pairs

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: cDNA

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) Clone: oIgSP-018

(xi) SEQ ID NO: 2:

(2) Information about SEQ ID NO:

(i) sequence properties:

(A) Length: 849 base pairs

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genome)

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) Clone: IgSP-hPOMC

(ix) sequence characteristics:

(A) Characteristic symbol: 5'UTR

(B) Presence location: 1..43

(ix) sequence characteristics:

(A) Characteristic symbol: exon

(B) Presence location: 44..89

(ix) sequence characteristics:

(A) Characteristic symbols: intron

(B) Presence location: 90..168

(ix) sequence characteristics:

(A) Characteristic symbol: 3'UTR

(B) Presence location: 807..849

(ix) sequence characteristics:

(A) Symbol indicating features: misc_feature

(B) Presence location: 43..186

(C) Other information: / product = "IgSp area"

(ix) sequence characteristics:

(A) Symbol indicating features: misc_feature

(B) Presence location: 187..806

(C) Other information: / product = "hPOMC area"

(xi) SEQ ID NO: 3:

(2) Information about SEQ ID NO:

(i) sequence properties:

(A) Length: 525 base pairs

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genome)

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) IgSP-hPOMCDACTH

(ix) sequence characteristics:

(A) Characteristic symbol: 5'UTR

(B) Presence location: 1..43

(ix) sequence characteristics:

(A) Characteristic symbol: exon

(B) Presence location: 44..89

(ix) sequence characteristics:

(A) Characteristic symbols: intron

(B) Presence location: 90..168

(ix) sequence characteristics:

(A) Characteristic symbol: exon

(B) Presence location: 169..482

(ix) sequence characteristics:

(A) Characteristic symbol: 3'UTR

(B) Presence location: 483..525

(ix) sequence characteristics:

(A) Symbol indicating features: misc_feature

(B) Presence location: 44..188

(C) Other information: / product = "IgSp area"

(ix) sequence characteristics:

(A) Symbol indicating features: misc_feature

(B) Presence location: 189..482

(C) Other information: / product = "hPOMC area"

(xi) SEQ ID NO 4:

(2) Information about SEQ ID NO:

(i) sequence properties:

(A) Length: 30 base

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: cDNA

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) clone: orTH-052

(xi) SEQ ID NO: 5:

(2) Information about SEQ ID NO:

(i) sequence properties:

(A) Length: 30 base

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: cDNA

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) clone: orTH-053

(xi) SEQ ID NO 6:

(2) Information about SEQ ID NO:

(i) sequence properties:

(A) Length: 30 base

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: cDNA

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) clone: orTH-054

(xi) SEQ ID NO 7:

(2) Information about SEQ ID NO:

(i) sequence properties:

(A) Length: 33 base pairs

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: cDNA

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) clone: orTH-078

(xi) SEQ ID NO: 8:

(2) Information about SEQ ID NO:

(i) sequence properties:

(A) Length: 30 base pairs

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: cDNA

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) clone: oTRES-057

(xi) SEQ ID NO: 9:

(2) information for SEQ ID NO: 10:

(i) sequence properties:

(A) Length: 30 base pairs

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: cDNA

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) clone: obDBH-065

(xi) SEQ ID NO: 10:

(2) information for SEQ ID NO:

(i) sequence properties:

(A) Length: 30 base pairs

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: cDNA

(vii) direct origin:

(B) Clone: oTRES-bDBH-064

(xi) SEQ ID NO: 11:

(2) information for SEQ ID NO: 12:

(i) sequence properties:

(A) Length: 30 base pairs

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: cDNA

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) clone: oIRES-bDBH-066

(xi) SEQ ID NO: 12:

(2) Information about SEQ ID NO:

(i) sequence properties:

(A) Length: 30 base

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: cDNA

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) Clone: oIgSP-068

(xi) SEQ ID NO: 13:

(2) information for SEQ ID NO: 14:

(i) sequence properties:

(A) Length: 25 base pairs

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: cDNA

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) clone: orTHD-073

(xi) SEQ ID NO: 14:

(2) Information about SEQ ID NO: 15:

(i) sequence properties:

(A) Length: 30 base

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: cDNA

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) clone: ohPOMC-IRES-069

(xi) SEQ ID NO: 15:

(2) information for SEQ ID NO: 16:

(i) sequence properties:

(A) Length: 1030 base pairs

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genome)

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) clone: rTHD

(ix) sequence characteristics:

(A) Characteristic symbol: 5'UTR

(B) Presence location: 1..6

(ix) sequence characteristics:

(A) Characteristic symbol: exon

(B) Presence location: 7..1017

(ix) sequence characteristics:

(A) Characteristic symbol: 3'UTR

(B) Presence location: 1018..1030

(xi) SEQ ID NO 16:

(2) information for SEQ ID NO: 17:

(i) sequence properties:

(A) Length: 1037 base pairs

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genome)

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) clone: rTHDKS

(ix) sequence characteristics:

(A) Characteristic symbol: 5'UTR

(B) Presence location: 1..13

(ix) sequence characteristics:

(A) Characteristic symbol: exon

(B) Presence location: 14..1024

(ix) sequence characteristics:

(A) Characteristic symbol: 3'UTR

(B) Presence location: 1025..1037

(xi) SEQ ID NO: 17:

(2) information for SEQ ID NO: 18:

(i) sequence properties:

(A) Length: 3425 base pairs

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genome)

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) clone: rTH-IRES-bDBH

(ix) sequence characteristics:

(A) Characteristic symbol: 5'UTR

(B) Presence location: 1..6

(ix) sequence characteristics:

(A) Characteristic symbol: exon

(B) Presence location: 7..1017

(ix) sequence characteristics:

(A) Characteristic symbols: intron

(B) Presence location: 1018..1617

(ix) sequence characteristics:

(A) Characteristic symbol: exon

(B) Location: 1618..3411

(ix) sequence characteristics:

(A) Characteristic symbol: 3'UTR

(B) Presence location: 3412..3425

(ix) sequence characteristics:

(A) Symbol indicating features: misc_feature

(B) Presence location: 1025..1617

(C) Other information: / product = "IRES sequence"

(xi) SEQ ID NO: 18:

(2) Information about SEQ ID NO: 19:

(i) sequence properties:

(A) Length: 3432 base pairs

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genome)

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) clone: rTHDKS-IRES-bDBH

(ix) sequence characteristics:

(A) Characteristic symbol: 5'UTR

(B) Presence location: 1..13

(ix) sequence characteristics:

(A) Characteristic symbol: exon

(B) Presence location: 14..1024

(ix) sequence characteristics:

(A) Characteristic symbols: intron

(B) Presence location: 1025..1624

(ix) sequence characteristics:

(A) Characteristic symbol: exon

(B) Presence location: 1625..3418

(ix) sequence characteristics:

(A) Characteristic symbol: 3'UTR

(B) Presence location: 3419..3432

(ix) sequence characteristics:

(A) Symbol indicating features: misc_feature

(B) Presence location: 1032..1624

(C) Other information: / product = "IRES sequence"

(xi) SEQ ID NO: 19:

(2) information for SEQ ID NO: 20:

(i) sequence properties:

(A) Length: 30 base

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: cDNA

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) clone: ohPOMC-IRES-070

(xi) SEQ ID NO: 20:

(2) Information about SEQ ID NO: 21:

(i) sequence properties:

(A) Length: 30 base pairs

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: cDNA

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) clone: oIRES-rTHD-071

(xi) SEQ ID NO: 21:

(2) Information about SEQ ID NO: 22:

(i) sequence properties:

(A) Length: 30 base pairs

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: cDNA

(iii) putative sequence: no

(iv) Anti Sense: No

(vii) direct origin:

(B) clone: oIRES-rTHD-072

(xi) SEQ ID NO: 22:

(2) Information about SEQ ID NO: 23:

(i) sequence properties:

(A) Length: 4499 base pairs

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genome)

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) clone: pomc-th-doh fusion

(ix) sequence characteristics:

(A) Characteristic symbol: 5'UTR

(B) Presence location: 1..43

(ix) sequence characteristics:

(A) Characteristic symbol: exon

(B) Presence location: 44..89

(ix) sequence characteristics:

(A) Characteristic symbols: intron

(B) Presence location: 90..168

(ix) sequence characteristics:

(A) Characteristic symbols: intron

(B) Presence location: 169..482

(ix) sequence characteristics:

(A) Characteristic symbols: intron

(B) Presence location: 483..1080

(ix) sequence characteristics:

(A) Characteristic symbol: exon

(B) Presence location: 1081..2091

(ix) sequence characteristics:

(A) Characteristic symbols: intron

(B) Presence location: 2092..2691

(ix) sequence characteristics:

(A) Characteristic symbol: exon

(B) Presence location: 2692..4485

(ix) sequence characteristics:

(A) Characteristic symbol: 3'UTR

(B) Location: 4486..4499

(xi) SEQ ID NO: 23:

(2) Information about SEQ ID NO: 24:

(i) sequence properties:

(A) Length: 30 base pairs

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: cDNA

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) clone: oIRES-074

(xi) SEQ ID NO: 24:

(2) Information about SEQ ID NO: 25:

(i) sequence properties:

(A) Length: 30 base pairs

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: cDNA

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) Zeocin-077

(xi) SEQ ID NO: 25:

(2) information for SEQ ID NO: 26:

(i) sequence properties:

(A) Length: 30 base pairs

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: cDNA

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) Clone: OIRES-Zeosin-075

(xi) SEQ ID NO: 26:

(2) information for SEQ ID NO: 27:

(i) sequence properties:

(A) Length: 30 base pairs

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: cDNA

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) Clone: oIRES-Zeosin-076

(xi) SEQ ID NO: 27:

(2) information for SEQ ID NO: 28:

(i) sequence properties:

(A) Length: 5540 base pairs

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genome)

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) Clone: POMCDACTH-IRES-THD-IRES-DBH-IRES-Zeocin

(ix) sequence characteristics:

(A) Characteristic symbol: 5'UTR

(B) Presence location: 1..118

(ix) sequence characteristics:

(A) Characteristic symbol: exon

(B) Presence location: 119..164

(ix) sequence characteristics:

(A) Characteristic symbols: intron

(B) Presence location: 165..243

(ix) sequence characteristics:

(A) Characteristic symbol: exon

(B) Presence location: 244..557

(ix) sequence characteristics:

(A) Characteristic symbols: intron

(B) Presence location: 558..1155

(ix) sequence characteristics:

(A) Characteristic symbol: exon

(B) Presence location: 1156..2166

(ix) sequence characteristics:

(A) Characteristic symbols: intron

(B) Presence location: 2167..2766

(ix) sequence characteristics:

(A) Characteristic symbol: exon

(B) Presence location: 2767..4560

(ix) sequence characteristics:

(A) Characteristic symbols: intron

(B) Presence location: 4561..5159

(ix) sequence characteristics:

(A) Characteristic symbol: exon

(B) Presence location: 5160..5534

(ix) sequence characteristics:

(A) Characteristic symbol: 3'UTR

(B) Presence location: 5535..5540

(xi) SEQ ID NO: 28:

(2) information for SEQ ID NO: 29:

(i) sequence properties:

(A) Length: 829 base pairs

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genome)

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) Clone: ProAKS

(ix) sequence characteristics:

(A) Characteristic symbol: 5'UTR

(B) Presence location: 1..16

(ix) sequence characteristics:

(A) Characteristic symbol: exon

(B) Presence location: 17..820

(ix) sequence characteristics:

(A) Characteristic symbol: 3'UTR

(B) Presence location: 821..829

(xi) SEQ ID NO: 29:

(2) Information about SEQ ID NO: 30:

(i) sequence properties:

(A) Length: 598 base pairs

(B) Type: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(ii) Type of sequence: DNA (genome)

(iii) putative sequence: no

(iv) Antisense: No

(vii) direct origin:

(B) clone: IRES sequence

(ix) sequence characteristics:

(ix) sequence characteristics:

(A) Characteristic symbols: intron

(B) Presence location: 1..598

(xi) SEQ ID NO: 30:

Indications for deposited microorganisms

(PCR Rule 13bis)

Indications for deposited microorganisms

(PCR Rule 13bis)

Indications for deposited microorganisms

(PCR Rule 13bis)

Claims (29)

Cells stably transformed to produce one or more analgesic compounds selected from the group consisting of endorphins, enkephalins and catecholamines. The cell of claim 1, wherein said endorphin is β-endorphin. The cell of claim 1, wherein said enkephalin is methionine-enkephalin. The cell of claim 1, wherein the catecholamine is norepinephrine or epinephrine. The cell of claim 1, wherein said cell is a RIN cell. The cell of claim 1, wherein the cell is an AtT-20 cell. The cell according to any one of claims 1 to 6, wherein said cell further produces a compound selected from the group consisting of galanine, somatostatin, neuropeptide Y, neurotensin or cholecystokinin. A cell transformed with DNA encoding POMC, DNA encoding TH, DNA encoding DBH, and DNA encoding ProA, each DNA molecule operably linked to an expression control sequence. The cell of claim 8, wherein said cells were transformed with pCEP4-POMC-030, pcDNA3-hProA + KS-091, and pZeo-pCMV-rTHΔKS-IRES-bDBH-088. The cell of claim 8, wherein said cells were transformed with pCEP4-h, POMC-ΔACTH-032, pBS-CMV-ProA, and pZeo-pCMV-rTHΔKS-IRES-bDBH-088. The cell of claim 8, wherein said cells were transformed with pcDNA3-hPOMCΔACTH-IRES-rTHD-bDBH-IRES-Zeocin-073 and pcDNA3-ProA + KS-091. A first vector containing DNA encoding POMC operably linked to an expression control sequence; A second vector containing DNA encoding pro-enkephalin A operably linked to an expression control sequence; One or more enkephalins, one or more transformed with a third vector containing DNA encoding TH operably linked to an expression control sequence and DNA encoding a dopamine beta hydroxylase operably linked to an expression control sequence Transformed cells producing endorphins and one or more catecholamines. A method of treating pain comprising transplanting a cell according to any one of claims 1 to 12 to a transplant site of a patient. The method of claim 13, wherein said cells are encapsulated within a semipermeable membrane to form a biological artificial organ. The method of claim 14, wherein the living artificial organ is an immunoisotope. 16. The method of any one of claims 13-15, wherein said transplant location is the central nervous system. 16. The method of any one of claims 13-15, wherein said implantation location is a subarachnoid space. DNA encoding POMC operably linked to a first expression control sequence, DNA encoding pro-enkephalin A operably linked to a second expression control sequence, DNA encoding TH operably linked to a third expression control sequence And transforming the cells with DNA encoding dopamine beta hydroxylase operably linked to a fourth expression control sequence, thereby producing cells that secrete one or more enkephalins, one or more endorphins, and one or more catecholamines. How to. 19. The method of claim 18, wherein said first, second, third and fourth expression control sequences are identical. A method of using the cell of any one of claims 1 to 12 for the manufacture of a medicament for the treatment of pain. The method of claim 20, wherein the cell is transplanted. 23. The method of claim 21 or 22, wherein said cells are encapsulated within a semipermeable membrane to form a biological artificial organ. 23. The method of claim 22, wherein said living artificial organ is an immunoisotope. 23. The method of any one of claims 21-22, wherein the transplant location is the central nervous system. 24. The method of any one of claims 21-23, wherein the implantation location is a subarachnoid space. (a) a biocompatible permeable jacket surrounding the core; And (b) A biological artificial organ comprising a core comprising one or more biological cells transformed to produce one or more analgesic compounds selected from the group consisting of endorphins, enkephalins and catecholamines. 27. The living body artificial organ according to claim 26, for use in treating pain. Encapsulating a core comprising one or more biological cells transformed to produce one or more analgesic compounds selected from each group consisting of endorphins, enkephalins and catecholamines into a biocompatible permeable jacket Method of manufacturing organs. A method of using a living artificial organ comprising the cells of any one of claims 1 to 12 for the manufacture of a medicament for the treatment of pain.