JP2002535959A - Materials and methods containing condition-holding domains - Google Patents

Abstract

(57) SUMMARY Materials and methods related to the condition retention domain (CRD) are disclosed. Also disclosed are fusion proteins containing CRDs and cells expressing such fusion proteins. Further, the present invention provides a novel method for producing a target protein in vivo using a fusion protein containing a condition retaining domain, and a method for identifying a novel CRD.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION

For example, many important uses, including materials and methods for gene therapy, the production of biological materials, and biological research, have led to the development of cells that produce therapeutically, commercially or experimentally valuable proteins. It depends on whether you can guide. Allostery-based switches triggered by tetracycline, RU486 or ecdysone, and rapamycin, coomamycin, FK506 dimer, synthetic
A variety of regulatable expression systems are involved, including those involving switches based on dimerization triggered by a dimerizing agent such as FKBP binding agents and / or CsA, or the like. Ever developed. For example, Clackson, "Control of mammalian gene expression by small molecules" Current Opinion in Chemical Biology, 1: 210-2
See 18, 1997. In these expression systems, protein production is regulated at the transcriptional level. An inherent limitation of all such systems is the inability to achieve fine temporal control over secretion of the target protein. For example, the maximum therapeutic level of secretion of the target protein is delayed for hours or even days until the transcribed mRNA has reached a level high enough to produce significant amounts of secreted protein. Also, mR
Until the NA is completely degraded, secretion cannot return to low baseline levels, even after removal of the inducing agent, and this degradation can again take hours or days. For many applications, this level of control is inadequate. In those cases,
It would be desirable to induce protein production on a much faster time scale than can be achieved using transcription-based methods.

[0002]

SUMMARY OF THE INVENTION

The present invention employs a unique approach to regulated production of a target protein that is not based on regulated transcription, but rather on regulated release or secretion of the target protein. The compositions and methods of the present invention are useful for biological research and gene therapy applications.

[0003] An important feature of the present invention is that condition-holding domains (abbreviated as "CRD"), fusion proteins containing them, ligands that bind to CRD and allow release or secretion of said fusion proteins, such Includes recombinant nucleic acids encoding fusion proteins, vectors containing such recombinant nucleic acids, cells and other materials transduced with these vectors, and important methods utilizing these. Important fusion proteins of the invention contain at least two mutually heterologous domains, one of which is the CRD.

[0004] More specifically, the fusion proteins of the invention contain at least one condition-retention domain (CRD) and at least one additional domain heterologous thereto, usually together with a secretory signal sequence. Is designed to be After being translated in the endoplasmic reticulum (ER), proteins containing a secretory signal sequence pass through other secretory compartments, such as the cis, intermediate and trans parts of the Golgi, during secretion. But,
Proteins containing one or more CRDs will, in principle, remain in the secretory machinery unless there is a ligand that binds to the protein. Representative examples of CRD include retinol binding protein and F36M, as described in detail below.
Human FKBP12 variants such as hFKBP12 are included. Concatemer formation of multiple CRDs may allow the user to adjust the degree of aggregation or retention.

[0005] Typically, the fusion protein also includes a secretory signal sequence for targeting the fusion protein to a secretory compartment, such as the ER or any part of the Golgi apparatus. Many secretory signal sequences are known. For example, human growth hormone is a source of a secretory signal sequence suitable for use in the present invention.

In addition, the fusion protein further includes an enzyme cleavage site so that the portion of the fusion protein containing the CRD can be cleaved from the portion of the fusion protein containing a peptide sequence heterologous to the CRD. Is preferred in many embodiments. Preferably, the enzyme cleavage site comprises a peptide sequence that is recognized by a trans Golgi-specific endoprotease, such as furin. For example, the cleavage site for furin is given by the peptide sequence SARNRQKR.

The portion of the fusion protein that is heterologous to the CRD may include any protein or protein domain of interest to the practitioner. For example, the heterologous moiety may include a target protein such as insulin, parathyroid hormone or β-endorphin.

[0008] To further illustrate this, one exemplary fusion protein of the invention contains a signal sequence, a condition-retaining domain, a furin cleavage site, and a polypeptide sequence comprising the selected target protein sequence. One example of such a fusion protein is
In order from N-terminal to C-terminal, a signal sequence derived from human growth hormone, three F36M
Includes hFKBP12 domain, human stromelysin-3 furin cleavage site, and selected target protein sequence. The fusion protein may also include several target proteins, each separated by an enzyme cleavage site. For example, such a fusion protein comprises a signal sequence from human growth hormone, one or more CRDs such as F36M hFKBP12, a furin cleavage site, a target protein, another furin cleavage site, and another target protein. This type of construct allows for the simultaneous release of two or more target proteins.

[0009] Further, the fusion proteins of the invention may optionally include a lysosomal targeting signal or other polypeptide sequence targeting it for degradation. Placing such a peptide sequence along with the CRD on one side of the cleavage site and a selected target polypeptide on the other side of the cleavage site allows the CRD of the fusion protein
It can help ensure cell removal of the containing portion.

[0010] Accordingly, one of the objects of the present invention is a fusion protein as described herein. Another object of the invention is a recombinant nucleic acid encoding such a fusion protein. The recombinant nucleic acids may be operably linked to expression control node sequences that allow their expression in a host cell into which they have been transduced or otherwise contain them. Any promoter can be used to drive expression of these fusion proteins, including strong promoters such as the CMV enhancer, other viral promoters such as the RSV promoter, or tissue-specific promoters such as MCK. Good.

[0011] Another object is a vector comprising a recombinant nucleic acid of the invention, generally operably linked to an expression control node sequence. Such vectors include "viral" vectors that contain part or all of the viral genome in addition to the recombinant nucleic acid encoding the fusion protein of the present invention. Viral vectors can be designed and used to produce recombinant viruses having the recombinant nucleic acids of the invention. A wide variety of such virus systems are known in the art, including, for example, adenovirus, AAV, retrovirus, hybrid adeno-AAV, lentivirus, and others, and are applicable to the practice of the present invention.

[0012] The recombinant nucleic acids of the present invention may be transduced into the host cell by any available means, for example, to allow the host cell to achieve regulated secretion of the target protein. The cell is preferably a eukaryotic cell, generally an animal cell, and in many embodiments a mammalian cell, which may be human or non-human. Transduction of the cells may be performed in situ within the host organism, or while maintained in vitro. The cells can be primary cells or derived from a cell line.

[0013] Thus, the present invention provides a cell, comprising introducing a recombinant nucleic acid of the present invention into a cell to produce a genetically engineered cell capable of expressing the encoded fusion protein described above. To provide for the regulated secretion of the target gene. This recombinant nucleic acid may be introduced into a cell maintained in vitro or a cell present in a living body in the form of a virus or other forms. The resulting genetically engineered cells containing one or more of these recombinant nucleic acids and their progeny can be used in human gene therapy, similar livestock applications, generation of cell or animal models (including transgenic applications), It can be used in a variety of important applications discussed elsewhere in the specification, including assay applications, and in vitro production of the desired protein (eg, for recovery and use). Such cells are useful, for example, in methods that include modulating the secretion of a target gene by adding a ligand, preferably a cell-permeable ligand, to the cell (or administering the ligand to a living organism containing the cell). It is. Particularly important animal models include rodents (especially mice and rats) and non-human primate models. For human gene therapy applications, the cells are generally human cells, and the peptide sequence of each of the various domains present in the fusion protein is preferably a peptide sequence of human origin or derived from it as much as possible Will.

The present invention also provides a method for identifying a novel CRD. The CRD may be identified by a two-hybrid method. The method provides a genetically engineered host cell comprising (a) and (b): (a) a reporter gene linked to a regulatable expression control element, and (b) a first recombination gene. A recombinant nucleic acid comprising a polylinker linked to first and second types of recombinant nucleic acid sequences, wherein the nucleic acid sequence encodes a DNA binding domain and the second recombinant nucleic acid sequence encodes a transcription activation domain ,
Here, the association between the DNA binding domain and the transcription activation domain activates the expression of the reporter gene. As described herein, the construct includes a single polylinker linked to two independent translation cassettes. This allows each to have the same CRD
It allows the expression of two fusion proteins linked to the candidate, one being a DNA binding domain and the other a transcriptional activation domain. Also provided are genetically engineered host cells comprising (a)-(c): (a) a reporter gene linked to a regulatable expression control element, (b) linked to a candidate condition-carrying domain. (C) a second recombinant nucleic acid encoding a fusion protein comprising a DNA binding domain linked to said candidate condition-carrying domain, wherein: The association of the two fusion proteins activates the expression of the reporter gene.

[0015] The invention further provides a method of identifying a ligand capable of binding to a condition-maintaining domain. Page 46 (English) See Part 3 of the “Methods for Identifying CRDs” below.
One such method uses cells that have been genetically engineered to express a reporter gene when the CRD-containing assembly is disassembled (disassembled) by a suitable ligand. The method comprises the steps of: (a) contacting the engineered cell with a candidate ligand under appropriate conditions to allow gene expression; and (b) the presence and / or amount of reporter gene expression. And (c) correlate the presence and / or amount of reporter gene expression upon cell contact with one or more candidate ligands.

The invention also provides a method of directly screening for CRDs that allows for ligand-dependent secretion of target proteins or ligand-dependent localization of membrane proteins. For these screening assays, a fusion protein encoding a member of a library of candidate CRDs linked to a signal sequence and an enzyme cleavage site is expressed. These domains are further linked to either the secretory target protein or the extracellular membrane domain of a membrane protein. The fusion protein is expressed under conditions that allow secretion of the target protein or localization of the membrane protein.
After treating cells containing the fusion protein with a ligand that binds CRD, the ligand-dependent presence of secreted or membrane proteins is assessed. The target protein secretion and / or membrane protein localization is then correlated with one or more individual members of the CRD library.

[0017]

[Detailed description]

For convenience in definition , the intended meaning of some terms and phrases used herein are described below.

“Selectively hybridizable” refers to the presence of other DNA molecules under hybridization conditions that can be empirically selected and readily determined by those skilled in the art.
It means that the two DNA molecules are capable of undergoing the formation of a detectable mutual hybrid. Such treatments can be performed at temperatures ranging from room temperature to 65-75 ° C.
Includes high stringency conditions, such as strong washing with a buffer containing 0.2-6 × SSC and / or 0.1% -1% SDS. For example, FM Aus
ubel et al., Ed., Short Protocols in Molecular Biology, Units 6.3 and 6.4.
(John Wiley and Sons, New York, Third Edition, 1995).

“Cell”, “host cell” or “recombinant host cell” refers not only to the particular cell being discussed, but also to its progeny. Such progeny may not actually be identical to the parent cell, as mutations or environmental influences may result in certain alterations in subsequent generations, but the scope of the terms used herein is not Is still included.

“Cell line” means a population of cells capable of continuous or long-term growth (proliferation) and division in vitro. Often the cell line is a clonal population from one mother cell. Furthermore, it is known in the art that spontaneous or induced changes in karyotype can occur during storage or transfer of such clonal populations. Therefore,
Although cells from one cell line may not be exactly the same as the progenitor cell or culture, the cell lines referred to also include such variants.

“Composite”, “fusion” and “recombinant” are not otherwise found in direct (covalent) association in nature, ie they are naturally the same contiguous Not found in the polypeptide or gene, at least in the same order or orientation as present in the complex, fusion or recombinant product;
Or at least two dissimilar to each other in the sense that they are not found at the same interval
A material such as a nucleic acid, nucleic acid sequence or polypeptide comprising two components.
Typically, such materials include components derived from at least two different proteins or genes, or from at least two non-contiguous portions of the same protein or gene. In general, a "complex" refers to the joining together of different protein or nucleic acid parts to form a single functional unit,
In general, "fusion" refers to two or more functional units linked together. "Recombinant" is generally used in reference to nucleic acids or nucleic acid sequences.

A “coding sequence”, ie, a sequence that “encodes” a particular polypeptide or RNA, when placed under the control of appropriate expression control sequences, is in vitro or in vitro.
A nucleic acid sequence that is transcribed (in the case of DNA) and translated into a polypeptide (in the case of mRNA) in vivo. The boundaries of the coding sequence are generally determined by a start codon at the 5 '(amino) terminus and a translation stop codon at the 3' (carboxy) terminus. Coding sequences include, but are not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. A transcription termination sequence will usually be located 3 ′ to the coding sequence.

“Construct”, eg, “nucleic acid construct” or “DNA construct” refers to a nucleic acid or nucleic acid sequence. "Derived from (or derived from)" means a peptide or nucleotide sequence selected from within a given sequence. A peptide or nucleotide sequence derived from (derived from) a sequence of a given name may further contain a small number of alterations (modifications) relative to the parent sequence, which in most cases are present in the parent sequence. Refers to a deletion, substitution or insertion of no more than about 15%, preferably no more than about 10%, and often no more than about 5% of the amino acid residues or bases. For DNA,
If two DNA molecules and one DNA molecule can selectively hybridize to each other,
One DNA molecule may be derived from another DNA molecule. Also for a polypeptide or polypeptide sequence, if the DNA encoding the two polypeptides or sequences of this and the reference polypeptide or polypeptide sequence can selectively hybridize to each other, the first polypeptide or sequence will It may also be derived from a peptide or sequence. Typically, the derived peptide sequence will differ from the parent sequence by substitution of up to 5 amino acids, often up to 3 amino acids, and very often 0 or 1 amino acid. The derived nucleic acid sequence will differ from the parent sequence by substitution of up to 15 bases, often up to 9 bases, and very often 0 to 3 bases. In some cases, amino acids or bases are added or deleted, rather than substituted.

“Domain” refers to a portion of a protein or polypeptide. In the art, the term "domain" can refer to a portion of a protein that has a distinct secondary structure. However, as is clear from the context used herein, the term "domain" as used herein does not necessarily imply a certain secondary structure. Rather, a peptide sequence is referred to herein as a "domain" simply to mean a polypeptide sequence that is derived from, or has the intended or observed activity from, a particular source. Domains may be derived from naturally occurring proteins or may include non-naturally occurring sequences.

“Expression control elements” or simply “control elements” refer to DNA sequences, such as initiation signals, enhancers, promoters and silencers, that direct or control the transcription of the DNA sequence to which they are operably linked. The control elements of a gene may be located in introns, exons, coding regions and 3 'flanking sequences. Some regulatory elements are "tissue-specific", that is, they preferentially affect the expression of a selected DNA sequence in a particular cell (e.g., cells of a particular tissue), while others are more or less Active in most cell types. If expression in one particular cell type is significantly higher than expression in another cell type, gene expression occurs preferentially in that particular cell type. The control elements include so-called "leaky" promoters,
It primarily regulates the expression of the selected DNA in one tissue, but causes expression in other tissues as well. Furthermore, the control element can act constitutively or inductively. For example, inducible promoters are clearly more active in responding to a stimulus than without the stimulus. The stimulus is
Hormones, cytokines, heavy metals, phorbol esters, cyclic AMP (cAM
P), retinoic acid or derivatives thereof, and the like. Nucleotide sequences that include one or more expression control elements are sometimes referred to as "expression control sequences."

“Gene” means a nucleic acid molecule or sequence that includes an open reading frame and includes at least one exon and (optionally) one or more intron sequences.

The term “(gene) -engineered cell” means a cell that has been modified by the introduction of a recombinant or heterologous nucleic acid (eg, one or more DNA constructs or their RNA counterparts). Progeny of these cells that retain some or all of the genetic alterations are also included.

“Heterologous” when referring to nucleic acid or peptide sequences means a sequence that does not normally associate with and / or does not normally associate with a particular cell. Thus, a "heterologous" region of one nucleic acid construct is a segment of a nucleic acid within or associated with another nucleic acid molecule that is not found to be associated with another molecule in nature. For example, a heterologous region of a construct may include a coding sequence in which the two sequences on each side are not found to be naturally associated with the coding sequence. Another example of a heterologous coding sequence is a construct in which the coding sequence itself is not found in nature (eg, a synthetic sequence having different codons than the native gene). Also, in the case of a cell transduced with a nucleic acid construct not normally present in the cell, the cell and the construct will be considered heterologous to each other for the purposes of the present invention.

The term “interact” means an interaction between molecules that can be detected directly or indirectly, for example, one that can be detected by a yeast two-hybrid assay or an immunoprecipitation method. The term "interact" also includes "binding" interactions between molecules.
Interactions can occur in nature in, for example, protein-protein, protein-nucleic acid, protein-small molecule or small molecule-nucleic acid.

“Nucleic acid” refers to a polynucleotide such as deoxyribonucleic acid (DNA) and, where appropriate, ribonucleic acid (RNA). The term also refers to RNA or DNA derivatives, variants and analogs made from nucleotide analogs and, as applicable to the described embodiment, single-stranded (sense or antisense) and double-stranded. It should be understood that the term also includes a strand polynucleotide.

A “polylinker”, sometimes referred to as a “multiple cloning site”, is a region in a vector that contains multiple sites for restriction enzyme digestion, thereby allowing the vector to be used for cloning foreign genes. This is the area to be suitable.

“Protein”, “polypeptide” and “peptide” are used interchangeably. "Recombinant virus" is a virus particle in which the packed nucleic acid contains a heterologous portion.

The “secretory machinery” of a cell (also called the secretory apparatus) refers to the compartment of a cell where secreted and membrane proteins are targeted and processed. These compartments include the endoplasmic reticulum (ER) and the cis, middle and trans Golgi. In this document, the term ER is often used generically to mean “secretory compartment”.

“Target protein” is a protein of interest, the secretion of which is regulated by the methods of the present invention. The target protein can be, for example, a hormone, endorphin, and the like.

“Transfection” refers to the introduction of a naked nucleic acid molecule into recipient cells. "Infection" refers to the process of introducing a nucleic acid into a cell with a virus containing the nucleic acid. "Proliferative infection" refers to the process by which a virus enters, replicates, and then is released from a cell (also called a "lytic" infection). "Transduction" includes introducing a nucleic acid into a cell by any method.

“Transgene” refers to a nucleic acid sequence introduced into a cell. Daughter cells from cells into which the transgene has been introduced are also said to contain the transgene (if not deleted). The polypeptide or RNA encoded by the transgene may be partially or totally heterologous, ie, foreign, to the animal or cell into which it is introduced. Alternatively, the transgene may be homologous to the endogenous gene of the transgenic animal or cell into which it is introduced, but designed to be inserted into the animal genome to alter the genome of the cell into which it is inserted. Or inserted (eg, at a different location than that of the native gene). The transgene can also be present in the episome. The transgene can include one or more expression control elements necessary or desirable for optimal expression of the selected coding sequence, and any other nucleic acids (eg, introns).

The term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is an episome, a nucleic acid capable of extrachromosomal replication. Vectors capable of autonomous replication and / or expression of linked nucleic acids are often used. A vector operably linked to an expression control sequence and capable of directing the expression of a contained gene may be referred to as an “expression vector”. An expression vector is typically in the form of a "plasmid", which generally refers to a circular double-stranded DNA loop that is not attached to a chromosome in the form of a vector. As used herein, "plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector. However, the invention also includes other forms of vectors that perform equivalent functions and are known or will become known in the art. Viral vectors are nucleic acid molecules that contain viral sequences that can be packed into viral particles.

Condition-retaining domain: The condition-retaining domain is retained in the ER or other secretory compartment in the absence of the ligand and is released from the secretory machinery when the ligand is bound, ie, in the presence of the ligand. Any domain (releasing). The use of CRD is thought to take advantage of the phenomenon of ER "quality control" so that incorrectly folded or assembled proteins are retained in the ER rather than translocating to the Golgi. After all,
Most misfolded proteins were degraded, but some were found to accumulate in substantial steady-state quantities (eg, VSV-G protein: AM de Silva et a
l. (1990) J. Cell Biol. 111, 857-866. Kopito, RR (1997) Cell 88, 427-4
See also 30). Several types of domains described below can function as condition-holding domains.

1) CRD can be a natural example of a protein that is retained in the secretory machinery in the absence of certain small molecules : An example of this type of condition-retaining domain is retinol binding protein (RBP) Or it is derived from it. Retinol binding protein is retinol (vitamin A)
Is a serum protein of approximately 20 kD, which is a specific carrier of (Melhus, H. et al. (199
2) J. Biol. Chem. 267, 12036-12041). It is retained in the ER as a complex with another protein, transerythrin. Retinol is RBP
Upon binding, the complex is released from its molecular chaperone and is free to enter the Golgi apparatus. Thus, retinol binding protein, in the absence of ligand,
And acts as a secreted silane in its presence. Although retinol binding protein is predominantly expressed in hepatocytes, several groups have shown that retinol-mediated secretion of RBP is independent of cell type and does not require hepatocyte-specific cofactors. Therefore, this is generally useful as a CRD (eg, Melhus, H. et.
al. J. Biol. Chem. 267: 12036-12041, 1992).

Another example of a protein that is retained in the ER in the absence of a small molecule ligand is IgM. Retention of the soluble μ-chain in the ER is dependent on a single unpaired cysteine residue. Secretion of IgM usually requires binding of the light chain to the mu heavy chain, whereas secretion of the IgM intermediate can be induced by the addition of 2-mercaptoethanol or other reducing agents (Alberini et al., (Nature 347: 485-487, 1990). Thus, the soluble μ chain is
It can function as a CRD that is secreted in the presence of a thiol-reactive small molecule.

[0041] 2) CRD was selected to have the property of being selectively retained in the absence of a given small molecule may be a genetically modified mutant of the native protein: de Protein It is known that stabilizing mutations can lead to ER retention. While not wishing to be bound by any one theory, including that theory, we have found that certain mutations in human FKBP Phe36 are poorly expressed, possibly due to instability (eg, F36A) I learned that It is believed that such proteins are retained to some extent in the secretory apparatus. The ER is exited using a high affinity ligand that binds to the protein.

3) CRD can be a small molecule reversibly self-assembling protein : It is known that large protein aggregates are retained in the ER. In such cases, ER retention occurs due to the formation of aggregates rather than protein misfolding. A natural example of assembly-dependent ER retention is found in the Z mutation of α ₁ -antitrypsin. In this secreted M form of this plasma protease, a glutamic acid residue is located at position 342 in the reactive central loop of the molecule. In the mutant Z form, the glutamic acid has been replaced by lysine, which allows the reactive loop to insert itself into the A-sheet of the adjacent α ₁ -antitrypsin molecule,
A linear assembly is formed that is ineligible for transport. This aggregate accumulates in the ER, but can be released (released) by addition of a peptide that inserts into the A-sheet and prevents polymerization (Hammond and Helenius, Current Opinion in Cell Biology 7: 523-52).
9, 1995; Lomas et al., Nature 357: 605-607, 1992/06/18).

The mutated form of α-galactosidase A found in Fabry lymphoblasts provides another example of small molecule-dependent release of aggregates from the ER. Whereas the wild-type form of this enzyme passes through the secretory pathway efficiently, the mutant proteins assemble in the endoplasmic reticulum and contribute at least partially to the enzyme deficiency in patients with Fabry disease. Recently, Fan et al. Described 1-deoxy, a competitive inhibitor of α-galactosidase A.
-Reported that the addition of galactonojirimycin (DJG) enhances α-galactosidase A activity of Fabry lymphoblasts by acting as a “chemical chaperone” and thus promoting transport and processing of the mutant enzyme (Fan
et al., Nature Medicine 5: 112-115, 1999).

[0044] In one preferred embodiment, the CRD is from human FKBP12. In particular, the FKBP mutant
F36M, when fused to a signal sequence and a heterologous target sequence in mammalian cells,
Functions as a condition holding domain. In the absence of ligand, the fusion protein containing FKBP F36M and the signal sequence will self-assemble and accumulate in the endoplasmic reticulum. Upon addition of the ligand, the fusion protein disassembles and travels through the ER, resulting in secretion of the fusion protein or its cleavage product. Another FKBP mutant that functions as a CRD is FKBP W59V.

A wide variety of ligands, including both natural and synthetic ligands for CRD, can be used in the present invention to dissociate fusion protein molecules. The criteria for selecting a ligand are (A) the physiological tolerance of the ligand (i.e., the ligand does not have undue toxicity to the cells or animals used), (B) a moderate therapeutic dose range, (C) the gene Suitability for oral administration for veterinary use, including therapeutic use
(I.e., adequate stability in the gastrointestinal system and absorption into the vasculature), (D) ability to penetrate cell and other membranes, and, if necessary, (E) moderate binding affinity for CRD (desired About the use of). Preferably, the compound is physiologically relatively inert except for its affinity for CRD. To the extent that the ligand must not bind to the native protein or other material in the targeted cell, the response will usually be better. Preferably, the ligand is other than a peptide or nucleic acid, and preferably has a molecular weight of about 5000 daltons or less, more preferably about 1200 daltons or less.

In various embodiments where the ligand binding domain for the candidate ligand is endogenous to the cell to be manipulated, the peptide sequence of the ligand binding domain is changed to allow the endogenous and modified ligand binding domain to It is often preferable to use a discriminating ligand. Such a ligand should preferentially bind to the modified ligand binding domain as compared to the native peptide sequence, eg, the sequence from which the modified domain is derived. This method can avoid the inappropriate intrinsic activity of the ligand. Important guidance and illustrations for this purpose are provided in the various references cited herein.

[0047] Substantial structural modifications of the ligand to the ligand binding domain are made as long as the modified compound functions as the ligand binding domain of interest, ie, the compound has sufficient binding affinity and specificity for the functions disclosed herein. It is allowed as long as it has sex. Some of the compounds are macrocycles, such as macrolides, but in certain embodiments linear and branched compounds may be preferred. Suitable binding affinity significantly lower than ^10-4, preferably ^10-6 or less, and more preferably is reflected as about ^10-7 following Kd values, but 10 ^-9 or 10 ^-10 or less of the binding affinity Is also possible and in some cases would be most desirable.

Examples of ligand binding domain / ligand pairs include retinol binding protein or variant thereof and retinol or derivative thereof, cyclophilin or variant thereof and cyclosporin or analog thereof, FKBP or variant thereof And FK506, FK520, rapamycin, analogs thereof or a pair of synthetic FKBP ligands. In the case of a ligand binding domain comprising or derived from immunophilin or cyclophilin, the complex of the ligand binding domain and the ligand preferably does not specifically bind to calcineurin or FRAP. A wide variety of FK506 derivatives and synthetic FKBP ligands with no observable immunosuppressive activity are known. Similarly, various rapamycin analogs that bind to FKBP but are not immunosuppressive are known. See, for example, WO 98/02441 for non-immunosuppressive rapalogs. These and other ligands can be used as well, depending on the choice of CRD. A number of assays are known in the art for identifying ligands that bind to a CRD identified by screening as described below.

[0049] The ligand binding domain / ligand pair can be identified as FKBP domains (eg, F36M FKBP) and FKP
This is exemplified by BP ligands. Generally, the ligand was mutated from wild-type FKBP.
(I.e., having a peptide sequence that is not naturally present in the cell to be genetically engineered)
Preferably, it binds preferentially to FKBP. Ligands to FKBP proteins, including F36M FKBP, are native FKs such as rapamycin, FK506 or FK520.
It may contain or be derived from a BP ligand, or may be a synthetic FKBP ligand
(E.g., PCT / US95 / 10559; Holt, et al., J. Amer. Chem. Soc., 1993 , 115, 9925.
-9938; Holt, et al., Biomed. Chem. Lett., 1993 , 4, 315-320; Luengo, et a
l., Biomed. Chem. Lett., 1993 , 4, 321-324; Yamashita, et al., Biomed. Ch.
em. Lett., 1993 , 4, 325-328; PCT / US94 / 01617; as disclosed in PCT / US94 / 08008). EP 0 455 427 A1; EP 0
465 426 A1; US 5,023,263; WO 92/00278; WO 94/18317; WO 97/31898; WO 96/4
1865; and Van Duyne et al (1991) Science 252, 839.

Typical types of ligands for the FKBP-derived ligand binding domain include the following genus I:

[0051]

Embedded image

In the formula, n = 1 or 2; X ＝ O, S, NH or CH ₂ ; B ¹ and B ² independently represent H, or usually 1 to about 12 (optional substituents) Aliphatic, heteroaliphatic, aryl or heteroaryl (the meaning of these terms is defined below); Y = O, S, NH, -NH (C = O) -, -NH (C = O) -O-, -NH (SO ₂ )-or NR ³ ;
Or represents a direct (ie, covalent) bond from R ² to carbon 9; R ¹ , R ² , and R ³ typically have from 1 to about 36 carbons (without counting the carbons of the optional substituents) An aliphatic, heteroaliphatic, aryl or heteroaryl group; two or more of B ¹ , B ² and R ² may be covalently bonded to form a C ₃ -C ₇ cyclic or heterocyclic moiety And the term "aliphatic" as used herein includes both saturated and unsaturated, straight, branched, cyclic or polycyclic aliphatic hydrocarbons, which may include one or more substituents May be optionally substituted.

The term “substituent” refers to an optionally substituted aliphatic, aryl,
Heteroaryl and heterocyclic moieties, and R ⁸ , -OR ⁸ , -SR ⁸ , -CN, -CHO,
= O, -COOH, -COR ⁸ , -OS (O) ₂ R ⁸ , -SO ₂ -NHR ⁸ , -NHSO ₂ R ⁸ , sulfate, sulfonate, (or its ester, carbamate, urea, oxime or carbonate), -NH ₂ (or substituted amine, amide, urea, carbamate or guanidino derivative), encompasses halo, trihaloalkyl, -SO ₂ -CF _3, and a functional group such as -OSO ₂ F, wherein R ⁸ is, H , Aliphatic, aryl, heteroaryl or heteroaliphatic. Aliphatic, heteroaliphatic, aryl and heterocyclic substituents can themselves be substituted or unsubstituted (eg, mono, di and trialkoxyphenyl; methylenedioxyphenyl or ethylenedioxyphenyl; halophenyl; or - phenyl _{-C (Me) 2 -CH 2 -O} -CO- [C 3 -C 6] alkyl or alkylamino). Additional examples of the substituent are exemplified by specific examples shown in Examples described later. (Unless otherwise specified, alkyl, other aliphatic, alkoxy, and acyl groups preferably contain 1-8, and often 1-6, consecutive carbon atoms.) The term "aliphatic" Is thus meant to encompass alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties.

The term “alkyl” as used herein includes both straight and branched chain alkyl groups. Similar provisions apply to other general terms such as "alkenyl", "alkynyl" and the like. Additionally, terms such as "alkyl,""alkenyl,""alkynyl," and the like, as used herein include both substituted and unsubstituted groups.

The term “alkyl” refers to groups having usually 1 to 8, preferably 1 to 6, carbon atoms. For example, "alkyl" refers to methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, t
It can mean ert-pentyl, hexyl, isohexyl and the like. Suitable substituted alkyls include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 3-fluoropropyl, hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, and the like. .

The term “alkenyl” refers to groups having usually 2 to 8, preferably 2 to 6, carbon atoms. For example, `` alkenyl '' can mean 2-propenyl, 2-butenyl, 3-butenyl, 2-methyl-2-propenyl, 2-hexenyl, 5-hexenyl, 2,3-dimethyl-2-butenyl and the like. it can. The term "alkynyl" also refers to groups having usually 2 to 8, preferably 2 to 6 carbon atoms, including, but not limited to, 2-propynyl, 2-butynyl, 3-butynyl, 2- Pentynyl, 3-methyl-4-pentynyl, 2
-Hexynyl, 5-hexynyl and the like.

As used herein, the term “cycloalkyl” refers to a group having 3 to 7, preferably 3 carbon atoms.
~ 6 groups. Suitable cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like.

The term “heteroaliphatic” as used herein refers to an aliphatic moiety that contains, for example, one or more oxygen, sulfur, or nitrogen atoms instead of carbon atoms. The term "heterocycle" as used herein, preferably has a total of 3 to 7 ring atoms,
A cycloaliphatic group having one or more heteroatoms, including, but not limited to, oxetane, tetrahydrofuranyl, tetrahydropyranyl, aziridine, azetidine, pyrrolidine, piperidine, morpholine, piperazine, and the like.

As used herein, the terms “aryl” and “heteroaryl” refer to a stable monocyclic or polycyclic, heterocyclic, heterocyclic, polycyclic ring having from 3 to 14 carbon atoms, which may be substituted or unsubstituted. , And the unsaturated portion of the polyheterocycle. Non-limiting examples of useful aryl ring groups include phenyl, halophenyl, alkoxyphenyl,
Dialkoxyphenyl, trialkoxyphenyl, alkylenedioxyphenyl, naphthyl, phenanthryl, anthryl, phenanthro and the like. Examples of typical heteroaryl rings include 5-membered monocyclic groups such as thienyl, pyrrolyl, imidazolyl, pyrazolyl, furyl, isothiazolyl, furazanyl, isoxazolyl, and thiazolyl; A ring monocyclic group; and benzo [b] thienyl, naphtho [2,3-b] thienyl, thianthrenyl, isobenzofuranyl, chromenil, xanthenyl, phenoxathienyl, indolizinyl, isoindolyl, indolyl, indazolyl,
Purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, benzothiazole, benzimidazole, tetrahydroquinoline, cinnolinyl, pteridinyl, carbazolyl, β-carbolinyl, phenanthrinyl, acridinyl, perimidinyl, phenanthronyl, phenanthronyl, phenazinylyl , Phenothiazinyl, phenoxazinyl, etc. (eg, Katrit
zky, see Handbook of Heterocyclic Chemistry). Aryl or heteroaryl moiety, hydroxy, C ₁ -C ₈ alkoxy, C ₁ -C ₈ branched or straight-chain alkyl, acyloxy, carbamoyl, amino, N-
It may be substituted with 1 to 5 members selected from the group consisting of acylamino, nitro, halo, trihalomethyl, cyano, and carboxyl.

A “halo” substituent according to the present invention may be a fluoro, chloro, bromo, or iodo substituent. As noted above, R ¹ can be aliphatic, heteroaliphatic, aryl or heteroaryl, and has usually 1 to about 36 carbon atoms, excluding any substituents.

In some embodiments, R ¹ is optionally linked, ie, covalently, to R ² , B ^1, or B ² to form a macrocyclic structure. In some embodiments, -XR ¹ is a moiety represented by the formula:

[0062]

Embedded image

Here, R ⁴ is H 2, aliphatic, heteroaliphatic, aryl or heteroaryl. Aliphatic moieties can be branched, unbranched, cyclic, saturated or unsaturated, substituted or unsubstituted, and include, for example, methyl, ethyl, isopropyl, t-butyl, cyclopentyl, cyclohexyl, and the like. The heteroaliphatic moiety can be branched, unbranched or cyclic and includes heterocyclic groups such as morpholino, pyrrolidinyl and the like. Examples of ortho-, meta- or para-substituents for the phenyl group at this position include one or more of the following: halo (eg, chloro or fluoro); hydroxyl, amino,
_{-SO 2 NH 2, -SO 2 NH} ( aliphatic), -SO ₂ N (aliphatic) _2, -O- aliphatic -COOH, -O- aliphatic
-NH ₂ (which may contain one or two N- aliphatic or N- acyl substituent), C ₁ -C ₆ alkyl, acyl, acyloxy, C ₁ -C ₆ alkoxy (e.g., methoxy , Ethoxy, methylenedioxy, ethylenedioxy) and the like. Heteroaryl groups are as previously described, and include indolyl, pyridyl, pyrrolyl, and the like. Specific examples of the R ⁴ moiety include the following groups:

[0064]

Embedded image

Z = 1 to 6 Q = -NH ₂ , -NHalkyl, -N-dialkyl, -COOH, Matah -OH R ⁵ is a branched, unbranched or cycloaliphatic moiety having 1 to 8 carbon atoms. Which may be optionally substituted, for example, -CH-, -CHCH _2- , -CH ₂ CH-, -CHCH ₂ CH _2- , -CH ₂ CHCH _2- , -CH (CH ₃ )- CH ₂ —CH—, —CH (CH ₂ CH ₃ ) —CH ₂ —CH—, —CH ₂ CH ₂ CH—, —C (CH ₃ ) CH ₂ — and the like.

R ⁶ is an aliphatic, heteroaliphatic, heterocyclic, aryl or heteroaryl moiety, which may be substituted or unsubstituted. Exemplary substituents for R ^6, branched, unbranched or cyclic C ₁ -C ₈ aliphatic or heteroaliphatic group may be mentioned, including substituted or unsubstituted alkene, heterocycle, phenyl, etc. Is included.

R ⁷ is H or, in some embodiments, — (CH ₂ ) _z —CH = CH ₂ , — (CH ₂ ) _z —COOH, — (CH ₂ ) _z —
CHO,-(CH ₂ ) _z -OH,-(CH ₂ ) _z -NH ₂ ,-(CH ₂ ) _z -NH-alkyl,-(CH ₂ ) _z -SH, or an amino group (which may be substituted or unsubstituted) And a substituent such as a tertiary amine). In embodiments where R ⁶ is aryl, R ⁷ can be in the o-, m- or p-position. z
Is an integer of 0 to 4.

As mentioned above, B ¹ , B ² and R ² may be aliphatic, heteroaliphatic, aryl or heteroaryl. Representative groups include branched, unbranched or cyclic, preferably saturated or unsaturated aliphatic moieties having 1 to about 12 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, cyclo propyl, -CH ₂ - cyclopropyl, allyl, n- butyl, sec- butyl, isobutyl, tert- butyl, cyclobutyl, -CH ₂ - cyclobutyl, n- pentyl, sec- pentyl, isopentyl, t
ert- pentyl, cyclopentyl, -CH ₂ - cyclopentyl, n- hexyl, sec- hexyl, cyclohexyl, -CH ₂ - cyclohexyl), the aliphatic moiety is optionally -OH, -C = O, -COOH , -CHO, allyl, NH ₂ (or substituted amine, amide, urea or carbamate), ether (or thioether, in each case aliphatic or aromatic), aryl, or heteroaryl moieties, It may also optionally contain a heteroatom in place of one or more CH ₂ or CH units; or substituted or unsubstituted aryl (eg, mono, di, and trialkoxyphenyl; methylenedioxyphenyl or ethylenedioxyphenyl). Oxyphenyl; halophenyl; or -phenyl-C (Me) ₂ -CH ₂ -O-CO
- a [C ₃ ~C _6] alkyl or alkylamino) or heteroaromatic moiety. Y
In such embodiments where R ² is —O-phenyl and B ¹ is H ² , B ² is preferably not cyclopentyl. In another embodiment, Y is NH, - (C = O) -CH (B 1) NHR 2 moiety, inter alia, natural or synthetic α- amino acids and their N- alkyl D- or L- form, N -Acyl, N-aryl and N-aroyl derivatives. Specific examples of the XR ¹ , G, B ¹ , B ² and YR ² groups for the various structures described above are further described, for example, in WO 96/06
097, WO 97/31899, WO 97/31898, including those set forth in the compounds described in the Tables of Monomers and Dimers and Other Disclosures.

One preferred type of compound is a compound of the genus I wherein n is 2. Another preferred class of compounds are compounds of genus I, wherein B ¹ is H; B ² is branched, unbranched or cyclic, preferably having 1 to 8 carbon atoms, more preferably 1 to 6 carbon atoms. saturated or unsaturated aliphatic moiety (e.g., methyl, ethyl, n- propyl, isopropyl, cyclopropyl, -CH ₂ - cyclopropyl, allyl, n- butyl, sec- butyl,
Isobutyl, tert- butyl, cyclobutyl, -CH ₂ - cyclobutyl, n- pentyl, se
c- pentyl, isopentyl, tert- pentyl, cyclopentyl, -CH ₂ - cyclopentyl, n- hexyl, sec- hexyl, cyclohexyl, -CH ₂ - a cyclohexyl, etc.), the aliphatic moiety is optionally, for example , -OH, -C = O, -COOH, -CHO,
May be substituted with allyl, NH ₂ (or substituted amine, amide, urea or carbamate) or ether (or thioether, in each case aliphatic or aromatic), and one or more CH ₂ or CH units And YR ² is an aryl or heteroaryl moiety and may be optionally substituted [YR ² is, for example, an o-, m- or p-alkoxyphenyl; 3,5-, 2,3-, 2,4-, 2,5-, 3,4- or 3,5-dialkoxyphenyl, or a moiety such as, for example, 3,4,5-trialkoxyphenyl Wherein the alkoxy group is independently selected from methoxy and ethoxy, one or more of which may have a hydroxy or amino moiety.

Another preferred class of compounds is compounds of the genus I, wherein B ¹ , B ² and YR ² are the same or different lower aliphatic moieties. Another preferred class of compounds are those compounds of genus I that include a -NB ¹ R ² moiety, wherein B ¹ is H and B ² is lower aliphatic.

Another preferred class of compounds are those of the genus I, wherein G is an alicyclic or heterocyclic group with optional substituents. Another preferred type of compound is a compound of the genus I wherein X is oxygen and R ¹ is
Some containing ^{R 4 R 5 R 6 R 7} . Wherein R ⁴ is an optionally substituted aliphatic, alicyclic, aryl, heteroaryl, or heterocyclic group, R ⁵ is a branched or unbranched lower aliphatic group, and R ⁶ is An optionally substituted aliphatic, cycloaliphatic, heteroaliphatic, heterocyclic, aryl or heteroaryl.

Another preferred class of compounds are compounds of genus I, wherein R ¹ comprises R ⁴ R ⁵ R ⁶ R ⁷ as described in the immediately preceding section and YR ² is substituted or unsubstituted And aryl or heteroaryl. The aryl or heteroaryl of YR ² is phenyl; o-, m- or p-substituted phenyl (substituents are halo such as chloro, lower alkyl,
Or alkoxy such as methoxy or ethoxy); disubstituted phenyl
(Examples, such as 2,4-, 3,4- or 3,5-dimethoxy or diethoxyphenyl,
Or a dialkoxyphenyl such as methylenedioxyphenyl or 3-methoxy-5-ethoxyphenyl); or a trisubstituted phenyl (eg, a trialkoxyphenyl such as 3,4,5-trimethoxy or triethoxyphenyl, 3,5 -Dimethoxy-4-chlorophenyl etc.).

[0073] Such compounds may also include substituted proline and pipecolic acid derivatives, numerous examples of which are described in the literature. Using the same synthetic techniques described in the patent and scientific literature cited herein, using substituted prolines and pipecolates, as exemplified in the patent literature cited herein,
C-2 to C-6 positions (see most FK506 number positions in the references cited below)
Can be prepared.

For representative examples of substituted prolines and pipecolic acids, see: Chung, et al., J. Org. Chem., 1990 , 55, 270; Shuman, et al., J. Org. Che.
m., 1990 , 55, 738; Hanson, et al., Tetrahedron Lett., 1989 , 30, 5751; Ba
iley, et al., Tetrahedron Lett., 1989 , 30, 6781.

For various guidance on chemical conversion, synthesis, formulation and release of various compounds, including additional information on FKBP ligands and / or ligands of other ligand binding domains, see, eg, WO 94 / 18317 and Belshaw et al,
1996, PNAS 93: 4604-4607 (for methods and materials based on ligands for immunophilins such as FKBP, cyclophilin and / or FRB domains); WO 96
/ 06097 and WO 97/31898 (Further ligands for FKBP and its variants); WO 93
/ 33052, WO 96/41865 and Rivera et al `` Human-adapted system for pharmacological control of gene expression '' Nature Medicine 2 (9): 1028-1032 (1997) (rapamycin analog); WO 94/18317 ( Cyclophilin / cyclosporin); Licitra et al, 1996,
Proc. Natl. Acad. Sci. USA 93: 12817-12821 (DHFR / methotrexate); and Farrar et al, 1996, Nature 383: 178-181 (DNA gyrase / coomamycin). Many alterations and modifications to the ligands and ligand binding domains and methods of their design, selection and / or characterization that may be employed in the present invention are disclosed in the cited references.

Cleavage Enzyme In designing the fusion protein of the present invention, it is often preferable to place an enzyme cleavage site between the CRD and the target protein. If the fusion protein exits the ER after addition of the ligand, the enzyme cleavage site allows the target protein to be released from the CRD and secreted. Ideally, the cleavage site should be specific for enzymes present in the cell compartment between the ER and the plasma membrane (eg, the Golgi apparatus).

A typical cleavage enzyme is furin, also known as PACE. Furin is a member of the KEX2 / subtilisin family of proprotein convertases, enzymes that convert proproteins and prohormones into their active forms (Nakayama Kazuhisa, Biochem. J. (1997) 327: 625-635). It is a protein present in the trans-Golgi apparatus, but, like many Golgi proteins such as TGN38, circulates constitutively between the cell surface and the TGN (trans-Golgi network). Furin has a ubiquitous (ubiquitous) tissue distribution and its substrates are numerous and diverse. However, almost all share a common cleavage sequence, RX (K / R) R. Proteins that are substrates for furin include: human proneurotrophin-3 (MSMRVRR), human proinsulin-like growth factor I (KPAKSAR),
Human pro-parathyroid hormone (KSVKKR), human stromelysin-3 (ARNRQKR)
. Furin can also cleave membrane-bound substrates such as human insulin pro-receptor (RPSRKRR) and human hepatocyte growth factor pro-receptor (TEKRKKR). Cleavage sites from any furin substrate can be used in the fusion proteins of the invention.
In some cases, the cleavage site will be a non-natural peptide sequence that includes a common furin cleavage sequence. One particular advantage of utilizing furin as a cleavage enzyme is that its recognition sequence is located exclusively N-terminal to its cleavage site. This allows the portion of the protein that encodes the target protein to be released from cells that are not modified by the presence of additional amino acids.

The furin family includes other members that may also be useful in the practice of the present invention. Many of these proteins have a unique tissue distribution. For example, PC1 / PC3 and PC2 are found only in neuroendocrine tissues such as pancreatic islets, pituitary gland and brain, while PC4 is mainly expressed in testis germ cells. PACE4 and PC5 / PC6
And LPC / PC7 / PC8 / SPC7 are ubiquitously expressed (Nakayama, 1997). Cleavage sites for these enzymes may also be used in the practice of the invention as long as the fusion protein is expressed in a suitable cell type.

In addition, any mammalian protease with a specific cleavage sequence, such as subtilisin, could be used to cleave a fusion protein of the invention once it was targeted to a desired location in the cell. For example, subtilisin could be targeted to TGN by fusing it to a localization sequence from a resident Golgi protein such as TGN38. Alternatively, motifs known to target furin to TGN, including SDSEEDE in the YKGL and Ser-containing clusters, may be sufficient to target cytoplasmic proteases to the Golgi. The cells may be genetically engineered to express enzymes tailored to cleave sequences present only in the CRD-containing fusion protein. For example, Ballinger et al. Report a mutant form of subtilisin in which the subtilisin enzyme has been genetically engineered to acquire furin specificity (Ballinger MD, et al. Biochemistry.
2; 35 (42): 13579-85), Ballinger MD, et al. Biochemistry. 1995/10/17; 34 (4
1): 13312-9).

Secretory Signal Sequence When translating a secreted protein on the ribosome, a signal sequence called 16-30
The amino acid sequence of the residues directs the ribosome to the ER membrane. This sequence then initiates a signal that transports the nascent (nascent) chain across the ER membrane and into the ER. Generally, such sequences are located at the N-terminus of the protein and include one or more positively charged amino acids followed by an extension of 6-12 hydrophobic residues. Numerous signal sequences are known, and any signal sequence that normally directs the transport of secreted or transmembrane proteins to the ER can be used in the fusion proteins of the invention. Typical signal sequences are from preproalbumin, prelysozyme, human growth hormone, proinsulin, acetylcholine receptor or an IgG light chain. For use in the present invention, the signal sequence is such that the protein's N
Coded at the end. This signal sequence then directs the ribosome to the ER, where the translated protein containing the CRD assembles until the ligand is added to the cell.

Target Proteins The fusion proteins of the present invention may contain any target protein that may be desired to be secreted or transported quickly and efficiently. Preferably, the target protein will be a therapeutic protein. The target protein can confer a desired phenotype. This is a membrane-bound or membrane-spreading protein (membrane-
spanning protein), a secreted protein, or a cytoplasmic protein. Proteins expressed alone or in combination can be involved in homing, cytotoxicity, proliferation, differentiation, immune response, inflammatory response, coagulation, thrombolysis, hormonal regulation, angiogenesis, and the like. The polypeptide may be a naturally occurring or non-naturally occurring peptide sequence.

Various secretory products include hormones such as insulin, human growth hormone, glucagon, pituitary release factor, ACTH, melanotropin, relaxin, leptin; EG
F, IGF-1, TGF-α, TGF-β, PDGF, G-CSF, M-CSF, GM-CSF, a member of the FGF family, growth factors such as erythropoietin, thrombopoietin, megakaryocyte growth factor, nerve growth factor; Proteins that stimulate or inhibit angiogenesis, such as angiostatin, endostatin and VEGF and variants thereof; interleukins such as IL-1 to IL-15; TNF-α and -β; interferon-α, -β And -γ
Enzymes such as tissue plasminogen activator, members of the complement cascade, perforin, superoxide dismutase and other factors; antithrombin-III, factor V, factor VII, VIIIc, vWF, IX, α Coagulation-related factors such as ₁ -antitrypsin, protein C and protein S; endorphin, dynorphin, osteogenic factor, CFTR and the like.

The protein may be a native surface membrane protein or a protein so made by the introduction of a suitable signal peptide and transmembrane sequence. A variety of such proteins include homing receptors [eg, L-selectin (Mel-14)], hematopoietic cell markers (eg, CD3, CD4, CD8, B cell receptors, TCR subunit α, β, γ Or δ, CD10, CD19, CD28, CD33, CD38, CD41, etc.) Interleukin receptor IL-2R,
Receptors such as IL-4R; Receptors for other ligands including various hormones, growth factors, etc .; Receptor antagonists of these receptors and their soluble forms; Ions (eg,
Channel proteins for inflow or outflow of H ⁺ , Ca ²⁺ , K ⁺ , Na ⁺ , Cl ⁻ and the like; CFTR, tyrosine activation motif, Zap-70 and the like.

A target protein is a protein involved in a metabolic pathway or a regulatory protein,
It may be an intracellular protein such as a steroid receptor or a transcription factor. By way of further example, in a T cell, it may be desirable to introduce a gene encoding one or both chains of the T cell receptor. For B cells, the immunoglobulin heavy and light chains could be provided for secretion. For skin cells such as keratinocytes, especially keratin stem cells, protection against infection is achieved by secreting α, β or γ interferons, anti-chemotactic factors, proteases specific to bacterial cell wall proteins, and various antiviral proteins. Could be provided.

In various situations, one may wish to direct a cell to a particular site. Such sites include anatomical sites, such as lymph nodes, mucosal tissue, skin, synovium, lungs or other internal organs, or functional sites, such as blood clots, damaged sites, surgical sites , Inflammation, infection and the like. Regulated expression of a membrane protein that recognizes or binds to a particular site of interest, for example, provides a way to direct genetically engineered cells to that site. In this way, a localized concentration of secreted product can be achieved, or cell recovery, elimination, infection protection, antitumor activity, etc. can be performed. Proteins of interest include homing receptors such as L-selectin, GMP-
140, CLAM-1, etc., or adressins, such as ELAM-1, PNAd, LNAd, etc., and cell surface proteins that respond to the localization gradient of clot binding proteins or chemotactic factors.

[0086] In one embodiment of the invention, binding of the ligand to the CRD modulates transcription of the target gene. In this aspect, the target gene may encode any protein, including those described above.

Disposable Targeting Sequences In many embodiments of the invention, it may be desirable to discard the CRD after its cleavage from the target protein. Disposal of the CRD will prevent its secretion from cells and its accumulation in the bloodstream. One approach to achieving this goal is to target the CRD within a lysosomal compartment, where the CRD will be degraded. Normal cell traffic
During trafficking, lysosomal proteins are sorted from the trans-Golgi network, where these proteins are directed to the endosomal pathway and then to the lysosome. Marking a resident soluble lysosomal enzyme such as cathepsin D to target the lysosomal pathway by attaching a phosphate group to carbon 6 of one or more mannose residues on a particular N-linked oligosaccharide This oligosaccharide is then recognized by the mannose-6-phosphate receptor in lysozyme. The phosphotransferase recognition sequence of cathepsin D consists of two discontinuous sequences, amino acids 188-230 containing a critical lysine residue at position 203 and amino acids 265-292 (Barans
ki et al., Cell 1990, 63: 281-291). Baranski et al. Have demonstrated that splicing these sequences into appropriate positions on the secreted protein pepsinogen results in phosphorylation of sugars on the chimeric molecule (Baranski et al., Supra). Another group has shown that fusion of the entire sequence of cathepsin B to MyoD results in targeting of the resulting complex to lysosomes (Li et al., J. Cell Biol., 135: 1043-1).
057, 1996/11). Also, a chimeric protein consisting of soluble CD4, procathepsin D and the C-terminal tail of three lysosomal membrane proteins was able to direct the HIV glycoprotein gp160 to lysosomes for degradation (Lin et al., FASEB J., 7:10
70-1080, 1993/8). Lysosomal membrane proteins such as lamp-1 and LAP are directed to lysosomes via a tyrosine-based targeting motif at their C-terminal tail (Williams et al., J. Cell Biol., 111: 955-966, 1990; Klionski et
al., J. Biol. Chem., 265: 5349-5352, 1990). Fusion of these tail resident plasma membrane proteins to the extracellular and transmembrane domains is sufficient to target them to lysosomes.

[0088] Any of the lysosomal targeting signals described above can be used to target a CRD of the invention for disposal. For soluble proteins, the preferred method is to fuse a resident lysosomal protein containing a mannose-6-phosphate signal to the CRD. Examples of such proteins are the cysteine proteases of the cathepsin family: cathepsins B, D, H, L, S, C and K. Other lysosomal enzymes that can be used include carboxypeptidase, prolyl carboxypeptidase and deamidase (cathepsin A). In the case of a membrane-bound CRD, a preferred targeting sequence would be that present on a lysosomal membrane protein, such as a tyrosine-based internalization motif. These motifs are short linear extensions of several amino acids in the cytoplasmic region of the protein to be targeted.
Tyrosine-based motifs are centered on critical tyrosine residues within the sequence NPXY or YXXO, where X is any amino acid and O is an amino acid with a bulky hydrophobic group. In many proteins, glycine in front of the tyrosine of the YXXO-type signal enhances the targeting of these proteins to the lysosome.
Sequences used in some applications of the present invention include proteins such as Lamp-1, LAP (lysosomal acid phosphatase), CD63, Lamp-2 or CD3-γ, all of which are normally targeted to lysosomes. It may be derived from. For more information on tyrosine-based selection motifs, see, for example, Marks et al., Trends in Cell Bio.
logy, 7: 124-128, 1997.

Design and Assembly of DNA Constructs The constructs are designed according to the principles, examples and materials, methods disclosed in the patent and scientific literature cited herein, with modifications and further embodiments as described herein. do it. The components of the construct can be prepared in a conventional manner as follows. The coding sequence and regulatory regions may be isolated, ligated as necessary, cloned as appropriate in a cloning host, and analyzed by restriction enzyme digestion or sequencing, or other suitable means. In particular, individual fragments containing all or part of the functional unit may be isolated using PCR, in which case one or more mutations may be isolated by "primer repair", ligation, in vitro mutagenesis, etc. May be introduced as appropriate. In the case of a DNA construct encoding a fusion protein, the DNA sequence encoding the individual domains and subdomains encodes a fusion protein that can be translated into a single polypeptide with all component domains in a cell or cell lysate. Combine to make up a single open reading frame. The DNA construct encoding the fusion protein may then be placed in a vector for transducing host cells to allow expression of the protein. For biochemical analysis of the encoded chimera, it may be desirable to generate a plasmid that directs expression of the protein in bacteria or in a reticulocyte-lysate system. For use in producing proteins in mammalian cells, sequences encoding the protein are introduced into expression vectors that direct expression in these cells. Expression vectors suitable for such uses are well known in the art. Many types of such vectors are commercially available.

Promoters The fusion proteins described herein may be used in combination with any promoter that directs their expression in mammalian cells. Promoter is human
A strong promoter such as the CMV promoter or a weak promoter such as a promoter of a human endogenous gene may be used. Other promoters that can be used include the Rous sarcoma virus (RSV) promoter, the retroviral LTR from mouse Moloney leukemia virus (MMLV), the muscle creatine kinase (MCK) enhancer, the SV40 promoter and the CMV enhancer from major immediate early genes. But not limited to them. The following table shows the Genbank accession numbers for the above promoters.

Promoter Genbank accession number CMV AF 067197 RSV M 83237 MMLV LTR M 77239 SV40 U 47120 MIE gene CMV enhancer K 03104 MCK enhancer X 67536 In many cases, the choice of promoter depends on the configuration of the fusion protein used for that purpose. right. Thus, if practitioners want to express CRD-containing fusion proteins at high levels, they will use a strong promoter, such as CMV. Alternatively, one would choose a tissue-specific promoter, such as the MCK enhancer (for expression in muscle) for tissue-specific expression.

Transfection of Constructs into Cells The invention is particularly useful in applications involving the (gene) manipulation of animal cells and the use of the engineered animal cells. The animal cell may be an insect, insect or mammalian cell, among others. For example, a variety of mammalian cells can be used, including cells of horses, cows, sheep, dogs, cats, mice and non-human primates, but human and mouse cells are of particular interest. Different types of cells across different species, including hematopoiesis, nerves, glia, mesenchyme, skin, mucous membranes, stroma, muscle (including smooth muscle cells)
, Spleen, retinal endothelium, epithelium, endothelium, liver, kidney, gastrointestinal, lung, fibroblasts and other cell types can be used. Of particular interest are muscle cells (including skeletal, cardiac and other muscle cells), cells of the central and peripheral nervous system, and hematopoietic cells
(Can include erythrocytes, lymphocytes or any nucleated cell that may be involved in the myeloid lineage)
) And myoblasts and fibroblasts. Also of interest are stem cells and progenitor cells such as hematopoietic, neural, stroma, muscle, liver, lung, gastrointestinal and mesenchymal stem cells.

The cells can be autologous, syngeneic, allogeneic, and in some cases heterologous with respect to the intended host organism. Cells are major histocompatibility complex
("MHC") may be modified by altering properties. It inactivates β2-microglobulin to prevent the formation of functional class I MHC molecules and the inactivation of class II molecules, provides for the expression of one or more MHC molecules, and is involved in cytotoxic activity. The cytotoxicity can be increased or inactivated by increasing or inhibiting the expression of the gene to be expressed.

In some cases, one may be interested in particular clonal or oligoclonal cells in which the cells have particular specificities, such as T cells and B cells with particular antigen specificity or homing target site specificity. unknown.

The construct encoding the fusion protein and containing the target gene of the present invention may comprise 1 or 2
The above nucleic acid molecules or constructs can be introduced into cells, and in many cases,
One or more markers that allow the selection of host cells containing the construct are also introduced. Constructs can be prepared by conventional methods, whereby the coding sequence and regulatory regions are isolated, ligated, if appropriate, cloned in a suitable cloning host, digested with restriction enzymes or sequenced or otherwise suitable. What is necessary is just to analyze by means. In particular, individual fragments containing all or part of a functional domain may be isolated by PCR, and one or more mutations may be isolated therefrom by "primer repair", ligation, in vitro mutagenesis, etc. It may be introduced as appropriate.

The completed construct, which has been proven to have the appropriate sequence, can then be introduced into a host cell by any convenient means. The construct can be introduced into a vector capable of episomal replication (eg, a BPV or EBR vector) or a vector designed to integrate into the chromosome of a host cell. The construct is constructed using a non-replicating defective viral genome, e.g., adenovirus, adeno-associated virus (AAV).
Or herpes simplex virus (HSV) and others (including retroviral vectors)
Packaging for infecting or transducing cells
You can also. Alternatively, the construct may be introduced by protoplast fusion, electroporation, biolistics, calcium phosphate transfection, lipofection, microinjection of DNA, and the like. The host cell may, in some cases, be cultured and grown and expanded prior to introduction of the construct,
Next, an appropriate process for introducing the construct and incorporating the construct is performed. Then
Cells may be screened for growth and / or markers present in the construct. Various markers that can be used successfully include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc., and Tac,
There are various cell surface markers such as CD8, CD3, Thy1 and NGF receptor.

In some cases, it may be possible to have a target site for homologous recombination, in which case it is desirable to integrate the construct at a particular locus. For example, an endogenous gene (same locus or another locus) may be deleted and / or replaced with a recombinant targeting construct of the invention. In the case of homologous recombination, generally either Ω or O-vector may be used. For example, Thomas & Capecchi, Cell (1987) 51, 503-5
12; Mansour, et al., Nature (1988) 336, 348-352; Joyner, et al., Nature
(1989) 338, 153-156.

The construct can be introduced as a single DNA molecule encoding all of the genes, or as different DNA molecules having one or more genes. Each construct can be introduced simultaneously or sequentially with the same or different markers.

A bacterial or yeast origin of replication, a selectable and / or amplifiable marker,
Useful elements that can be used to prepare stocks of construct DNA and perform transfections, such as promoter / enhancer elements for expression in eukaryotic or prokaryotic cells, and expression control (regulatory) elements in mammals. Included vectors are well known in the art and many are commercially available.

Transfection of Constructs into Animals Any means for introducing genetically engineered cells or heterologous DNA into animals, preferably mammals, which may be human or non-human, can be used to target various DNA constructs. Can be applied to the implementation of the present invention to supply it to the user. For the purposes of this discussion, the various DNA constructs described herein are referred to collectively as transgenes.

Cells transduced ex vivo or in vitro with the ex vivo genetically engineered DNA construct are cultured under selective conditions, and cells selected as having the desired construct are then expanded, and For example, analysis may be performed using the polymerase chain reaction method to determine the presence of the construct in the host cell, and / or using an assay method for the production of the desired gene product. After transducing the heterologous gene construct, the modified host cells can be identified, selected, expanded, characterized, etc., as desired, and then, as planned, e.g., grown in culture or introduced into the host organism. It may be used for introduction.

Depending on the nature of the cell, the cell allows the host organism, eg, a mammal, to migrate by a wide variety of methods, generally to the desired tissue or compartment, or to the desired location of the cell. It may be introduced by injection (injection) or transplantation into the tissue or compartment. Examples of injection or implantation sites include the vasculature, bone marrow, muscle, liver, skull or spinal cord, peritoneum and skin. For example, hematopoietic cells can be administered by infusion into the vasculature, usually having a cell number of about 10 ⁴ or more, generally about 10 ¹⁰ or less. The number of cells used will depend on the circumstances, the purpose of the transfer, the life span of the cells, the protocol used (eg, number of doses), the growth potential of the cells, the stability of the therapeutic agent, the physiological need of the therapeutic agent, and the like. In general, for example for myoblasts or fibroblasts, cell number of about 10 ⁴ or more,
It is less than about 10 ⁹ and can be administered as a dispersion and is generally injected at or near the site of interest. The cells are usually contained in a physiologically acceptable medium.

Cells engineered according to the present invention may be (micro) encapsulated, eg, using conventional biocompatible materials and methods, prior to implantation into a host organism or patient for production of a therapeutic protein. You can go to See, for example, Hguyen et al, Tissue transplantation systems and methods for sustaining high cell density surviving in a host, US Pat. No. 5,314,471.
No. (Baxter International, Inc.); Uludag and Sefton, 1993, J Biomed. Mate
r. Res. 27 (10): 1213-24 (Hep G2 cells / hydroxyethyl methacrylate-methyl methacrylate membrane); Chang et al, Hum Gene Ther 4 (4): 433-40 (hGH expressing mouse Ltk cells / immunoprotection) Perm-selective alginate microcapsules; Reddy et
al, 1993, J Infect Dis 168 (4): 1082-3 (alginate); Tai and Sun, 1993,
FASEB J 7 (11): 1061-9 (hGH-expressing mouse fibroblast / alginate-poly-L-
Lysine-alginate membrane); Ao et al, 1995, Transplantation Proc. 27 (6): 334
9, 3350 (alginate); Rajotte et al, 1995, Transplantation Proc. 27 (6):
3389 (alginate); Lakey et al, 1995, Transplantation Proc. 27 (6): 3266
(Alginate); Korbutt et al, 1995, Transplantation Proc. 27 (6): 3212 (
Alginate); Dorian et al, U.S. Patent No. 5,429,821 (alginate); Emeri
ch et al, 1993, Exp Neurol 122 (1): 37-47 (polymer encapsulated PC12 cells); Sag
en et al, 1993, J Neurosci 13 (6): 2415-23 (bovine chromaffin cells encapsulated in a semipermeable polymer membrane and implanted in the rat spinal cord / subarachnoid space); Aebischer
et al, 1994, Exp Neurol 126 (2): 151-8 (see also polymer-encapsulated rat PC12 cells implanted in monkeys; see also Aebischer, WO 92/19595); Savelkoul et al, 1994.
, J Immunol Methods 170 (2): 185-96 (encapsulated hybridoma producing antibodies; encapsulated transfection cell line expressing various cytokines); Winn
et al, 1994, PNAS USA 91 (6): 2324-8 (engineered BHK cells expressing human nerve growth factor encapsulated in an immunoisolating polymer device and implanted in rats);
Emerich et al, 1994, Prog Neuropsychopharmacol Biol Psychiatry 18 (5): 93
5-46 (polymer-encapsulated PC12 cells implanted in rats); Kordower et al, 199
4, PNAS USA 91 (23): 10898-902 (monkey transplanted polymer-encapsulated engineered BHK cells expressing hNGF); and Butler et al WO 95/04521 (encapsulation equipment). The cells can then be introduced in encapsulated form into an animal host, preferably a mammal, more preferably a human patient in need thereof. Preferably, the encapsulating material is semi-permeable, allowing release of the target protein produced by the encapsulated cells to the host. In many embodiments, semi-permeable encapsulation causes the encapsulated cells to be separated immunologically from the host organism into which they have been introduced. In such embodiments, the cells to be encapsulated express one or more fusion proteins that include a component domain from a protein of the host species and / or from a viral protein or a protein from a species other than the host species. May be. The cells may be from one or more individuals other than the recipient and may be from a species other than the species of the recipient organism or patient.

As an alternative to ex vivo modification of cells by in vivo genetic engineering, it may be desirable in many situations to modify cells in vivo. A variety of techniques have been developed for in vivo genetic manipulation of target tissues and cells, including viral and non-viral systems.

In one method, the delivery (delivery) of the DNA construct to the cell is accomplished by transfection, ie, to “naked DNA”, lipid-complexed or liposome formulated DNA, or other formulated DNA cells. By delivery of Prior to formulation of the DNA (e.g., with lipids) or, in other cases, prior to transfer in the final expression vector, a plasmid containing the transgene carrying the desired DNA construct is first experimentally expressed. Optimization (eg, inclusion of introns in the 5 'untranslated region and removal of unnecessary sequences, Felgner et al., Ann NY Aca Sci.
126-139, 1995). Thereafter, for example, the DNA may be formulated using various lipids and liposome materials using known methods and materials, and then sent to the recipient mammal. For example, Canonico et al, Am J Respir Cell Mol Biol 10: 24-29, 1994 (in vivo introduction of rabbits of aerosolized recombinant human α ₁ -antitrypsin gene complexed into cationic liposomes into the lung); Tsan et al, Am J Physiol 268 (Lung Cell
Mol Physiol 12): L1052-L1056, 1995 (transfer of genes into rat lung by tracheal gas injection of plasmid DNA alone or complexed with cationic liposomes); Alt
on et al., Nat Genet. 5: 135-142, 1993 (Nebulization of cDNA-liposome complex
(Nebulization) Gene transfer into mouse airways by delivery). In each case, the delivery of the vector or the naked or formulated DNA involves the transfer of the virus particles to Ringer's solution,
After transfer to phosphate buffered saline or other similar carrier, it can be performed by bronchoscopic instillation or by nebulization.

Viral systems include those based on viruses such as adenovirus, adeno-associated virus, hybrid adeno-AAV, lentivirus and retrovirus, which allow transduction by infection, and in some cases, virus or Allows integration of the transgene into the host genome. For example, Dubuensky et al. (1984) Proc. Na
tl.Acad.Sci. USA 81, 7529-7533; Kaneda et al., (1989) Science 243, 375.
-378; Hiebert et al. (1989) Proc. Natl. Acad. Sci. USA 86, 3594-3598; Ha
tzoglu et al, (1990) J. Biol. Chem. 265, 17285-17293 and Ferry, et al.
(1991) Proc. Natl. Acad. Sci. USA 88, 8377-8381. Virus injection
(Eg, intravascular, intramuscular), by inhalation or other parenteral forms. Non-viral delivery methods, such as via complexes with liposomes or by injection, catheter or biolistic, are also used. For example, see WO 96/41 for further guidance on formulation and delivery of recombinant nucleic acids to cells and organisms.
865, PCT / US97 / 22454 and USSN 60/084819.

If desired, use one of the transcription factor constructs to activate the virus so that the virus is produced and transduced into neighboring cells by using an attenuated or modified retrovirus having a target transcription initiation region. Can be

The use of recombinant viruses to deliver nucleic acid constructs is of particular interest. The transgene may be introduced into any of a variety of viruses useful in gene therapy.

In a clinical setting, a gene delivery system (ie, a recombinant nucleic acid in a vector, virus, lipid formulation or other form) can be introduced into a patient, for example, by any of a number of known methods. For example, a gene delivery system formulation can be introduced systemically, for example, by intravenous injection or infusion, inhalation, and the like. In some systems, the means of delivery results in specific or selective transduction of the construct into the desired target cells. This may be by local or topical administration (see U.S. Patent No. 5,328,470), by stereotactic injection (e.g., Chen et al., (1994) PNAS USA 91: 3054-3057), or by determinants of the delivery vehicle. Can be achieved. For example, some viral systems have tissue or cell type specificity for infection. In some systems, cell-type or tissue-type expression is achieved through the use of cell-type or tissue-specific expression control elements that control (regulate) gene expression.

In a preferred embodiment of the invention, the expression construct is of interest to a viral delivery system comprising a recombinant retrovirus, adenovirus, adeno-associated virus (AAV), hybrid adenovirus / AAV, herpes virus or lentivirus. It is induced by the introduction of a genetic construct (although other uses are made using recombinant bacteria or eukaryotic plasmids). Although various viral vectors can be used in the practice of the present invention, AAV
And adenovirus-based methods are of particular interest for the introduction of foreign genes in vivo, particularly into humans and other mammals. The following additional guidance on the selection and use of viral sites is provided to practitioners, both ex vivo and in vivo, especially for applications involving all organisms, including both the development and use of human gene therapy and animal model systems. May be helpful.

Viral Vectors : Adenovirus Vectors The viral gene delivery systems useful in the present invention utilize adenovirus-derived vectors. With knowledge of the genetic makeup of adenovirus, a 36 kb linear double-stranded DNA virus, it is possible to replace large pieces of adenovirus DNA with foreign sequences up to 8 kb. In contrast to retroviruses, infection of host cells with adenovirus DNA does not result in chromosomal integration because the adenovirus DNA may replicate episomally and may be genotoxic. There is no. In addition, adenovirus is structurally stable, and no genomic rearrangement has been detected even after large-scale amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenovirus has
It appears to be associated only with mild illnesses such as acute respiratory illness in humans.

Adenoviruses are particularly suitable for use as gene transfer vectors because of their medium size genome, ease of handling, high titer, wide target cell range, and high infectivity. Both ends of the viral genome contain 100-200 base pair (bp) inverted terminal repeats (ITRs), which are cis elements required for viral DNA replication and packaging. The early (E) and late (L) regions of this genome contain different transcription domains that divide by the onset of viral DNA replication. E1 area
(E1A and E1B) encode proteins responsible for the transcriptional regulation of the viral genome and a few cellular genes. Expression of the E2 region (E2A and E2B) results in protein synthesis for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell shut off (Renan (1990) Rad
iotherap. Oncol. 19: 197). The product of the late gene, which contains most of the viral capsid proteins, is only expressed after significant processing of a single primary transcript generated by the major late promoter (MLP). MLPs (located at 16.8 mu) are particularly effective in the late phase of infection, and all mRNAs resulting from this promoter have a 5 'tripartite leader (TL) sequence that makes them preferred mRNAs for translation.

The adenovirus genome encodes a gene product of interest, but can be engineered to be inactivated in its normal lytic virus life cycle in terms of its ability to replicate (
For example, Berkner er al., (1988) BioTechniques 6: 616; Rosenfeld et al., (1
991) Science 252: 431-434; and Rosenfeld et al., (1992) Cell 68: 143-155.
See). Adenovirus Ad type 5 d1324 strain or other adenovirus strains (e.g.,
Suitable adenovirus vectors from Ad2, Ad3, Ad7, etc.) are well known to those skilled in the art. Recombinant adenovirus is unable to infect non-dividing cells and has a broad spectrum of cell types, such as bronchial epithelium (Rosenfeld et al., (1992), cited above), endothelial cells (Lemarchand et al., ( 1992) PNAS USA 89: 6482-6486), hepatocytes (Herz and
Gerard, (1993) PNAS USA 90: 2812-2816) and muscle cells (Quantin et al., (1
992) PNAS USA 89: 2581-2584) can be advantageous in certain situations because it can be used to infect Adenovirus vectors have also been used in vaccine development (Grunhaus and Horwitz (1992) Seminar in Virology 3: 237; Graham and
Prevec (1992) Biotechnology 20: 363). Experiments involving the administration of recombinant adenovirus to different tissues include tracheal instillation (Rosenfeld et al. (1991); Rosenfeld e.
t al. (1992) Cell 68: 143), intramuscular injection (Ragot et al. (1993) Nature 361: 647)
), Peripheral vein injection (Herz and Gerard (1993) Proc. Natl. Acad. Sci. USA 9
0: 2812) and stereotaxic inoculation into the brain (Le Gal La Salle et al. (1993) Science
254: 988).

In addition, the virus particles are relatively stable, easy to purify and concentrate, and can be modified to affect the infectivity spectrum as described above. In addition, adenoviruses are easy to grow and handle, and exhibit a wide host range in vitro and in vivo. Viruses of this genus can be obtained at high titers, eg, 10 ⁹ -10 ¹¹ plaque forming units (PFU) / ml, and are highly infectious. The life cycle of adenovirus does not require integration into the host genome. Foreign genes delivered by adenovirus vectors are episomal and therefore have low genotoxicity to host cells. No side effects have been reported in wild-type adenovirus vaccine studies (Couch et al., 1963; Top et al., 1971), demonstrating safety and therapeutic potential as an in vivo gene transfer vector. I have. Furthermore, the ability to retain the adenovirus genome for foreign DNA is greater (up to 8 kilobases) than other gene delivery vectors (Berkner et al., Supra; Haj-Ahmand and Graham (1986) J. Virol. 57: 267)
. Most of the replication deficient adenoviral vectors currently used and therefore preferred in the present invention lack all or part of the viral E1 and E3 genes, but retain up to about 80% of the adenovirus genetic material. (For example, Jones et a
l., (1979) Cell 16: 683; Berkner et al., supra; Graham et al., Methods in
Molecular Biology, EJ Murray, Ed. (Humana, Clifton, NJ, 1991) vol. 7, p.
p. 109-127). Expression of the inserted gene can be under the control of, for example, the E1A promoter, the major late promoter (MLP) and associated leader sequences, the viral E3 promoter or an exogenous promoter sequence.

Apart from the requirement that the adenovirus vector be replication-deficient or at least conditionally defective, the nature of the adenovirus vector is not believed to be essential for the successful practice of the present invention. . The adenovirus may be of any of the 42 different known serotypes or AF subgroups. Subgroup C type 5 adenovirus is a preferred starting material for obtaining conditional replication defective adenovirus vectors for use in the methods of the present invention. This is because adenovirus type 5 is a human adenovirus for which much biochemical and genetic information is known and has been used historically for most constructs that employ adenovirus as a vector. is there.
As mentioned above, typical vectors of the invention are replication deficient and adenovirus E1
Will have no territory. Therefore, it may be most convenient to introduce the nucleic acid of interest at a location where the E1 coding sequence has been removed. However, the position of insertion of the nucleic acid of interest in a region within the adenovirus sequence is not critical to the invention. For example, the nucleic acid of interest may also be a deletion in the E3 replacement vector reported by Karlson et al. (1986).
Instead of the E3 region, or a helper cell line or helper virus may be inserted into the E4 region to compensate for the E4 deletion.

A preferred helper cell line is 293 (ATCC Accession No. CRL 1573). This helper cell line is also referred to as a "packaging cell line" and is used by Frank Graham (Grah
am et al. (1987) J. Gen. Virol. 36: 59-72 and Graham (1977) J. General V.
irology 68: 937-940) and provides E1A and E1B to trans.
However, the helper cell line may also be derived from human cells, such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from cells of other mammalian species that allow human adenovirus. Such cells include, for example, Vero cells or other monkey embryonic mesenchymal or epithelial cells.

A variety of adenovirus vectors have been demonstrated to be useful for introducing genes into mammals, including humans. Replication-deficient adenovirus vectors have been used to express marker proteins and CFTR in lung epithelium. Adenovirus vectors have been the subject of much research in recent years because of their ability to infect dividing cells efficiently, their affinity for the lungs, and the relative ease of producing high-titer stocks. However, various vectors have been used to deliver genes to the lungs of human patients (Zabner et al., Cell 75: 207-216, 1993; Crystal, et al.,
Nat Genet. 8: 42-51, 1994; Boucher, et al., Hum Gene Ther 5: 615-639, 199
Four). First-generation E1a-deficient adenoviral vectors have been modified using second generations containing temperature-sensitive E2a viral proteins, resulting in lower expression of viral proteins, thereby targeting virus-infected cells to the immune system. (Goldman et al., Human Gene Therapy 6: 839-851, 1995). More recently, viral vectors in which the entire open reading frame of the virus has been deleted have been reported (Fisher er al., Virology 217: 11-22, 1996). Furthermore, viral IL-10 expression has been shown to inhibit the immune response to adenovirus antigens (Qin et al., Human Gene Therapy 8: 1365-1374, 1997).

Adenoviruses may also be cell type-specific, ie, infect only a limited type of cells and / or express the transgene only in a limited type of cells. For example, U.S. Pat.No. 5,698,443 (Henderson and Schuur, 19
The virus contains the gene under the transcriptional control of a transcription initiation region that is specifically controlled by the target host cell, as described in US Pat. Thus, replication-competent adenoviruses are restricted to certain types of cells, for example, by inserting cell-specific response elements and regulating the synthesis of proteins required for replication (e.g., E1A or E1B). be able to.

A number of adenovirus-type DNA sequences are available from GenBank. For example,
Human adenovirus type 5 is GenBank Accession No. M 73260. The adenovirus DNA sequence may be obtained from any of the 42 types of human adenovirus currently identified. Various adenovirus strains are available from the American Type Culture Collection (Rockville, Md.) Or upon request from a number of commercial and academic institutions. The transgenes described here can be inserted into any adenovirus vector in the same manner as used to insert CFTR or other genes into the vector (restriction enzyme cleavage, linker ligation or terminal recruitment and ligation).
) May be introduced by any delivery protocol.

The adenovirus-producing cell line may contain one or two of the adenovirus gene E1, E2a and E4 DNA sequences for packaging of an adenovirus vector in which one or more genes are mutated or deleted. The above can be included. This is, for example, the PCT / US95 / 15947 of Kadan et al. (WO 96/18418); PCT / US95 of Kovesdi et al.
/ 07341 (WO 95/346671); PCT / FR94 / 00624 (WO 94/28152); Perrocaude of Imler et al.
PCT / FR94 / 00851 (WO 95/02697) such as t; PCT / US95 / 14793 (WO 96/14061) such as Wang
It is described in.

Another viral vector system useful for delivery of AAV vector DNA is adeno-associated virus (AAV). Adeno-associated virus is a naturally occurring virus that requires another virus, such as an adenovirus or herpes virus, as a helper virus for efficient replication and a productive life cycle (for review, see Muzyczka et al., Curr. Topics in
Micro. And Immunol. (1992) 158: 97-129).

AAV is not associated with any disease cause. AAV is not a transforming or oncogenic virus. Integration of AAV into the chromosome of the human cell line does not cause any significant changes in the growth or morphological characteristics of the cells. Due to these properties, AAV
Is recommended as a potentially useful vector for human gene therapy.

AAV is also one of the few viruses that may integrate its DNA into non-dividing cells, such as lung epithelial cells and muscle cells, and exhibits a high frequency of stable integration (eg, Flotte et al. ., (1992) Am. J. Respir. Cell. Mol. 7: 349-356; Samluski.
et al., (1989) L. Virol. 63: 3822-3828; and McLaughlin et al., (1989) J.
Virol. 62: 1963-1973). Vectors containing as little as 300 base pairs of AAV can be packaged and integrated. Space for foreign DNA is limited to about 4.5 kb. AAV vectors, such as those described in Tratschin et al., (1985) Mol. Cell. Biol. 5: 3251-3260, can be used to introduce DNA into cells. Diverse nucleic acids
It has been introduced into different cell types using AAV vectors (eg, Hermonat et al.
., (1984) PNAS USA 81: 6466-6470; Tratschin et al., (1985) Mol. Cell. Bio
l. 4: 2072-2081; Wondisford et al. Mol. Endocrinol. 2: 32-39; Tratschin et.
al., (1984) J. Virol. 51: 611-619; and Flotte et al., (1993) J. Biol. C.
hem. 268: 3781-3790).

AAV-based expression vectors that are typically used are directly transfected using available restriction enzyme sites or after cutting out the transgene with restriction enzymes, blunting the ends,
Flanking both ends of the restriction enzyme site, which can be used for subcloning of the transgene, by appropriate DNA linker ligation, restriction enzyme cleavage and ligation to sites between ITRs
Contains the 145 nucleotide AAV inverted terminal repeat (ITR). The capacity of the AAV vector is about 4.4 kb. The following proteins have been expressed using various AAV-based vectors and various promoters / enhancers: neomycin phosphotransferase, chloramphenicol acetyltransferase, Fanconi anemia gene, cystic fibrosis transmembrane conductance. Regulatory factor and granulocyte macrophage colony stimulating factor (Kotin, RM, Human Gene Therapy 5:79
3-801, 1994, Table 1). Transgenes for introducing the various DNA constructs of the present invention
It can be contained in an AAV-based vector. Recombinant encoding a fusion protein, eg, a fusion protein containing an activation domain or a DNA binding domain
Instead of including a constitutive promoter such as CMV to drive DNA expression, the AAV promoter can be used (ITR itself or AAV p5 (Flotte et al.,
J. Biol. Chem. 268: 3781-3790, 1993)).

[0125] Such vectors can be packaged into AAV virions by methods previously described. For example, a human cell line such as 293 can be transformed with an endogenous AAV-based expression vector.
It can be co-transfected with an AAV promoter or other plasmid containing an open reading frame encoding AAV rep and cap (essential for replication and packaging of the recombinant viral construct) under the control of a heterologous promoter. In the absence of a helper virus, the rep proteins Rep68 and
Rep78 prevents the accumulation of replicative forms, but upon superinfection with adenovirus or herpes virus, these proteins allow replication from the ITR (only present in constructs containing the transgene) and expression of the viral capsid protein. This system results in the packaging of transgene DNA into AAV virions (Ca
rter, BJ, Current Opinion in Biotechnology 3: 533-539, 1992; Kotin, RM
., Human Gene Therapy 5: 793-801, 1994). Typically, three days after transfection, recombinant AAV is harvested from the cells with the adenovirus, and the contaminating adenovirus is then inactivated by heat treatment.

Methods for improving AAV titers can also be used for transgene expression in AAV virions. Such strategies include, but are not limited to: a second plasmid that directs viral packaging after stable expression of the transgene carrying the ITR at both ends in the cell line. Transfection at
Use of a cell line that expresses AAV protein inducibly (eg, temperature sensitive or pharmacologically inducible expression). Alternatively, cells can be placed at the 5'ITR
A first AAV vector containing a 3'ITR, and a DNA sequence that can be induced by an agent (e.g., SV40T antigen) and encodes AAV rep and cap proteins;
It can be transformed with a second AAV vector containing an inducible origin of replication (eg, the SV40 origin of replication). Following drug induction, the second AAV vector may replicate at a high copy number, thereby producing an increased number of infectious AAV particles (see, e.g., Chiorini et al., U.S. Patent No. 5,693,531, 1997). Issued on December 2, 2008). Yet another method for producing large amounts of recombinant AAV uses a plasmid that introduces the Epstein-Barr nuclear antigen (EBNA) gene, the Epstein-Barr virus late replication origin (oriP), and the AAV genome. These plasmids are maintained as multiple copies of extrachromosomal elements in cells such as 293 cells. With the addition of wild-type helper function, these cells will produce large amounts of recombinant AAV (Lebkowsk
i. et al., US Pat. No. 5,691,176, issued Nov. 25, 1997). Another system provides an AAV packaging plasmid that allows expression of the rep gene, where
The p5 promoter controlling rep expression is replaced with a heterologous promoter (Flotte et al., US Pat. No. 5,658,776, issued Aug. 19, 1997). Further, WO 96/39 by Wilson et al.
As described at 530, the efficiency of AAV transduction may be increased by treating cells with an agent that facilitates conversion of the single-stranded form to the double-stranded form.

The AAV stock was prepared as described in Hermonat and Muzyczka (1984) PNAS 81: 6466, and was constructed from pAAV / Ad described by Samulski et al., (1989) J. Virol. 63: 3822. And can be modified using For virus concentration and purification, AA
First report of V vector in vivo expression (Flotte et al., J. Biol. Chem. 268: 37
81-3790, 1993), banding in a cesium chloride gradient, or by chromatographic purification as described in O'Riordan et al., WO 97/08298.

A method for in vitro packaging of AAV vectors is also available and has the advantage of not limiting the size of DNA that can be packed into particles (US Patent No. 5,688,67 to Zhou et al.).
No. 6, issued on November 18, 1997). This operation includes the preparation of a cell-free packaging extract.

AAV useful in the practice of the present invention, including methods and materials for transgene introduction, propagation and purification of recombinant AAV vectors containing the transgene, and their use in transfecting cells and mammals For more detailed guidance on technology, see, for example, Carter et al., US Pat. No. 4,797,368 (Jan. 10, 1989); Muzy.
U.S. Patent No. 5,139,941 to Cczka et al. (August 18, 1992); U.S. Patent No. 5,173,414 to Lebkowski et al. (December 22, 1992); U.S. Patent No. 5,252,479 to Srivastava et al.
U.S. Pat.No. 5,354,678 to Lebkowski et al. (Oct. 11, 1994); S
U.S. Patent No. 5,436,146 to Henk et al. (July 25, 1995); U.S. Patent No. 5,454,935 to Chatterjee et al. (December 12, 1995); WO 93/24641 to Charter et al. (Published December 9, 1993) And U.S. Patent No. 5,622,856 to Natsoulis et al. (April 22, 1997).
Further information on AAV and required adenovirus or herpes helper function can be found in the following article: Berns and Bohensky (1987), "Adeno-Associated Viruses: An Update" Advanced in Virus Research, Academic Press, 3
3: 234-306. The AAV genome is described in Laughlin et al., (1983) "Cloning of the Infectious Adeno-Associated Virus Genome on Bacterial Plasmids", Gene, 23: 65-73. AAV expression is described in Beaton et al., (1989) "Expression from adeno-associated virus p5 and p19 promoters is negatively regulated in trans by the rep protein." J. Virol., 63: 4450-4454. I have. The production of rAAV has been described in a number of publications: Tratschin et al. (1984) "Adeno-associated virus vectors for high frequency integration, expression and recapture of genes in mammalian cells" Mol. Cell. B
iol., 4: 2072-2081; Hermonat and Muzyczka (1984) "Use of adeno-associated virus as a mammalian DNA cloning vector: transduction of neomycin resistance into mammalian tissue culture cells" Proc. Natl. Acad. Sci. USA, 81: 6466-6470; McLaug
hlin et al. (1988) "Adeno-associated virus common transduction vector: analysis of proviral structure", J. Virol., 62: 1963-1973; and Samulski et al. (1989). Helper stock: normal integration does not require viral gene expression. "J. Virol., 63: 3822-3828. Cell lines that can be transformed with rAAV are described in Lebkowski et al. (1988) "Adeno-associated virus: a vector system for efficient transfer and integration of DNA into a variety of mammalian cell types", Mol. Cel.
l. Biol., 8: 3988-3996. The "producer" or "packaging" cell line used for the production of recombinant retroviruses is Doughert
y et al. (1989) J. Virol., 63: 3209-3212; and Markowitz et al. (1988) J.
Virol., 62: 1120-1124.

Hybrid Adenovirus-AAV Vector The hybrid adenovirus-AAV vector comprises a nucleic acid containing a portion of an adenovirus and an AAV-derived 5 'and 3' flanking a selected transgene under the control of a promoter. And the adenovirus capsid containing the ITR sequence. For example,
See Wilson et al., International Patent Application Publication WO 96/13598. This hybrid vector is characterized by the delivery of a high titer transgene to the host cell and the ability to stably integrate the transgene into the host cell chromosome in the presence of the rep gene. The virus is capable of infecting virtually all cell types (conferred by its adenoviral sequence) and stable long-term transgene integration into the host cell genome (conferred by its AAV sequence).

The adenovirus nucleic acid sequence used in this vector can be used to hybridize the product of the deleted gene to the packaging cells by the hybrid virus method from the minimum amount of sequence that would require the use of a helper virus for hybrid virus particle production. It can range from a simple deletion of an adenovirus gene that can be supplied. For example, the hybrid virus may have the adenovirus 5 'and 3' inverted terminal repeat (ITR) sequences, which function as origins of replication. The available left-end sequence (5 ') sequence of the Ad5 genome extends from bp 1 of the normal adenovirus genome to about 360 (also referred to as map units 0-1) and includes the 5'ITR and the packaging / enhancer domain. Hybrid virus
The 3 'adenovirus sequence includes a right terminal 3' ITR sequence of about 580 nucleotides (approximately bp 35,353 to the end of the adenovirus, also referred to as a map unit of about 98.4-100).

AAV sequences useful in hybrid vectors are viral sequences in which sequences encoding the rep and cap polypeptides have been deleted, and are usually cis-acting 5 ′ and 3′ITRs.
Is an array. Thus, the AAV ITR sequence has the selected adenovirus sequence at both ends and the AAV ITR sequence itself is at both ends of the selected transgene. Preparation of hybrid vectors is described in further detail in published PCT application WO 96/13598 (Wilson et al.), Entitled "Hybrid Adenovirus-AAV Virus and Methods of Use Thereof".

It may be useful in practicing the present invention, including methods and materials for transgene introduction, propagation and purification of recombinant viruses containing the transgene, and their use in transfecting cells or mammals. More detailed guidance on adenovirus and hybrid adenovirus-AAV technology is provided by Wilson et al., WO 94/28938,
See WO 96/13597 and WO 96/26285 and the references cited therein.

Retroviruses Retroviruses are a group of single-stranded RNA viruses characterized by their ability to convert RNA to double-stranded DNA in infected cells by the process of reverse transcription (Coffin (1990) Duplicates ”, edited by Fields, Knipe, Virology, New York: Raven
Press). The resulting DNA then stably enters the chromosome of the cell as a provirus, directing the synthesis of viral proteins. This integration results in the retention of the viral gene sequence in the recipient cell and its progeny. Retrovirus genome is 3
Contains two genes, gag, pol, and env, each of which is a capsid protein,
Encodes polymerase enzyme and envelope components. The sequence found upstream of the gag gene is named psi and functions as a signal for packaging the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5 'and 3' ends of the viral genome. They contain strong promoter and enhancer sequences and are also required for integration into the host cell genome (Coffin (1990), supra).

For the construction of a retroviral vector, a nucleic acid of interest is inserted into the viral genome in place of certain viral sequences to create a replication-defective virus. The production of virions creates a packaging cell line that contains the gag, pol and env genes, but lacks the LTR and psi components (Mann et al., (1983) Cell 33: 153). When a recombinant plasmid containing human cDNA is introduced into this cell line, along with the retroviral LTR and psi sequences (e.g., by calcium phosphate precipitation), the psi sequence causes the RNA transcript of the recombinant plasmid to be packed into the virus particle. And then the viral particles are secreted into the medium (Nicolas and Rubenstein (1988) "Retroviral Vectors", edited by Rodriguez and Denhardt, "Vectors: Overview of Molecular Cloning Vectors and Their Use", Stoneham: Butterworth; Temin, (1986) "Retroviral Vectors for Gene Transfer: Efficient Integration into Exogenous DNA and Its Expression in the Vertebrate Cell Genome", Kuncherlapati, eds. "Gene Transfer", New York
: Plenum Press; Mann et al., 1983, supra). The medium containing the recombinant retrovirus is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors can infect a wide variety of cells. However, integration and stable expression require the division of host cells (Paskind et al., (1975) Virology 67: 242).

A major prerequisite for the use of retroviruses is to ensure the safety of their use, particularly with respect to the potential for wild-type spread in cell populations. A specialized cell line that produces only replication-defective retroviruses (referred to as "packaging cells")
) Has increased the usefulness of retroviruses for gene therapy, and defective retroviruses have been characterized sufficiently to be used for gene transfer for gene therapy purposes (for review, Miller, AD (1990) Blood 76: 271). In this way, a part of the retrovirus coding sequence (gag, pol, env) can be replaced with a nucleic acid encoding the fusion protein of the present invention, and a recombinant retrovirus deficient in replication of the retrovirus can be produced. . The replication defective retrovirus is then packaged into virions that can be used to infect target cells with a helper virus by standard methods. Creation of recombinant retrovirus,
Protocols for infecting cells with these viruses in vitro and in vivo are described in Current Protocols in Molecular Biology, Ausubel, FM et al.
, (Eds.) Greene Publishing Associates, (1989), section 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZ1P, pWE and pEM well known to those skilled in the art. A preferred retroviral vector is pSR
MSVtkNeo (Muller et al., (1991) Mol. Cell. Biol. 11: 1785) and pSR MSV
(Xbal) (Sawyers et al., (1995) J. Exp. Med, 181: 307) and derivatives thereof. For example, the unique BamHI site in both these vectors
Cleavage with BamHI, supplemented with Klenow enzyme, replicated, pSMTN2 and pSMTN respectively
It can be removed by making SMTX2 (as described in PCT / US 96/09948 by Clackson et al.). Examples of suitable packaging virus systems for the preparation of allotrophic and amphotropic retrovirus systems include Crip, Cre, 2 and Am.

Retroviruses transfer various genes to many different cell types, such as neurons, epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells in vitro and / or in vivo. (Eg, Eglitis et al.,
(1985) Science 230: 1395-1398, Danos and Mulligan, (1988) PNAS USA 85: 646
0-6464; Wilson et al., (1988) PNAS USA 85: 3014-3018; Armentano et al., (
1990) PNAS USA 87: 6141-6145; Huber et al., (1991) PNAS USA 88: 8039-8043;
Ferry et al., (1991) PNAS USA 88: 8377-8381; Chowdhury et al., (1991) Sc
ience 254: 1802-1805; van Beusechem et al., (1992) PNAS USA 89: 7640-7644;
Kay et al., (1992) Human Gene Therapy 3: 641-647; Dai et al., (1992) PNA
S USA 89: 10892-10895; Hwu et al., (1993) J. Immunol. 150: 4104-4115; US Patent 4,868,116; US Patent 4,980,286; PCT Application WO 89/07136; PCT Application WO 89 /
02468; PCT application WO 89/05345; and PCT application WO 92/07573).

In addition, it has been shown that the infection spectrum of retroviruses, and thus retrovirus-based vectors, can be limited by modifying the viral packaging proteins on the surface of the virus particles (eg, PCT Publication WO 93/25234, WO 94 /
06920 and WO 94/11524). For example, strategies for altering the infection spectrum of retroviral vectors include: binding antibodies specific for cell surface antigens to the viral env protein (Roux et al., (1989) PNAS USA 86: 9079
-9083; Julan et al., (1992) J. Gen Virol 73: 3251-3255; and Groud et al.
, (1983) Virology 163: 251-254), or binding cell surface ligands to the viral env protein (Neda et al., (1991) J. Biol. Chem. 266: 14143-14146).
. The linkage may be in the form of a chemical cross-link with the protein or other (eg, lactose to convert the env protein to asialoglycoprotein) and by forming a fusion protein (eg, a single-chain antibody / env fusion protein). Is also possible.
This method is useful for limiting or directing the infection of certain tissues and can also be used to convert an ecotropic vector to an amphotropic vector.

Other Viral Systems Other viral vector systems applied to gene therapy include herpes virus,
For example, herpes simplex virus (Woo et al., US Patent No. 5,631,236, May 20, 1997
, Vaccinia virus (Ridgeway (1988) Ridgeway "Mammalian Expression Vectors", edited by Rodriguez RL, Denhardt DT, "Vectors: Overview of Molecular Cloning Vectors and Their Use", Stoneham: Butterworth; Baichwal and Sugden (1986)
"Vectors for gene transfer from animal DNA viruses: transient and stable expression of the introduced genes", Kuncherlapati R, "Gene Transfer", New York: Plenum P
ress; Coupar et al. (1988) Gene, 68: 1-10) and several RNA viruses. Preferred viruses include alphavirus, poxvirus, arenavirus, vaccinia virus, poliovirus and the like. In particular, herpesvirus vectors may provide a unique strategy for the maintenance of recombinant genes in cells of the central nervous system and eye tissue (Pepose et al., (1994) Invest Ophthal
mol Vis Sci 35: 2662-2666). They provide several attractive features for various mammalian cells (Friedman (1989) Science, 244: 1275-1281; Ridgeway,
1988, supra; Bichwal and sugden, 1986, supra; Coupar et al., 1988; Horwich
et. (1990) J. Virol., 64: 642-650).

The recent findings of defective hepatitis B virus have provided a new perspective on the structure-function relationship of different viral sequences. In vitro studies have shown that this virus can retain helper-dependent packaging and reverse transcription, despite deletions of up to 80% of its genome (Horwich et al., 1990, supra). ). This suggested that a large portion of this genome could be replaced with foreign genetic material. Liver affinity and persistence (integration) are attractive properties, especially for liver-directed gene transfer. Chang et al. Recently reported that chloramphenicol acetyltransferase (CA
The T) gene was introduced into the duck hepatitis B virus genome in place of the polymerase and pre-surface coding sequences. It was co-transfected into the avian hepatocyte line with the wild-type virus. Medium containing high titers of recombinant virus was used to infect primary duck chick hepatocytes. Stable CAT
Gene expression was detected for at least 24 days after transfection (Chang
et al., (1991) Hepatology, 14: 124A).

In general, viral particles are administered in a biocompatible solution or pharmaceutically acceptable delivery vehicle (vehicle), such as sterile (physiological) saline or other aqueous or water-insoluble isotonic sterile Transfer to an injectable solution or suspension (many examples of which are well known to those skilled in the art, for example Ringer's solution, phosphate buffered saline or other similar carriers). Delivery of recombinant viral vectors includes intramuscular injection, intravenous administration, subcutaneous injection,
Intrahepatic administration, catheterization (including cardiac catheterization), intracranial injection, nebulization /
It can be performed by any of several routes of administration, including inhalation or bronchoscopic instillation.

The dosage of DNA or recombinant virus depends on the target cells of the recipient (such as muscle cells, hepatocytes, various tracheal epithelial cells and smooth muscle cells, neurons (neurons), cardiomyocytes, etc.). (Including, but not limited to) transfecting the cells to produce observable ligand-responsive secretion of the target protein.
Preferably, it is at a level that confers a therapeutic effect without undue side effects, and in an amount sufficient to provide a sufficient level of transgene expression to occur.

[0143] The optimal dose of DNA or virus will depend on various factors, as described above, and may therefore vary somewhat from patient to patient. Also, a therapeutically effective dose of virus may range from about 1 × 10 ⁷ to about 1 × 10 ¹⁰ pfu of virus per ml, eg, 1 × 10 ⁸ to 1 × pfu.
It believed to range from about 20 to about 50 ml of (physiological) saline containing concentrations of 10 ⁹ pfu of the virus.

Uses In one use, the cells engineered according to the present invention are used to produce a target protein in vitro. In such uses, the cells are cultured or maintained until the production of the target protein is as desired. In that case, the appropriate ligand is added to the medium in an amount sufficient to produce the desired level of target protein production. The protein thus produced may be recovered from the medium or the cells and, if desired, purified from other components of the cells or the medium.

[0145] Proteins for commercial and research purposes are often produced using mammalian cell lines that have been engineered to express the protein. The use of mammalian cells rather than bacterial, insect or yeast cells is indicated where the proper function of the protein requires post-translational modifications that are generally not performed by non-mammalian cells. Examples of proteins produced commercially by this method include, among others, erythropoietin, BMP-2, tissue plasminogen activator, VIII: c factor, IX
Factors and antibodies.

In other applications, cells in an animal host or human patient are engineered according to the present invention, or the cells so engineered are introduced into an animal or human patient, and in each case the target gene is transferred to the recipient. Provides for ligand-mediated regulation of secretion of. In the case of non-human animals, this can be done as part of veterinary treatment of the animal or for the generation of an animal model for a variety of research purposes. In the case of human patients, this can be done as part of a therapeutic or prophylactic treatment program.

The present invention is applicable to various treatment methods. For example, the target protein to be regulated
(Eg, therapeutic proteins) may be endogenous or heterologous proteins, the secretion of which may be activated by the addition of a ligand.

In some cases, a therapeutic protein is a factor necessary for the growth and / or differentiation of one or more cell types of interest (of interest). For example, it may be desirable to stimulate growth factor and lymphokine secretion in a patient in which at least some of the blood cells have been destroyed (eg, by radiation or chemotherapy). For example, secretion of erythropoietin stimulates the production of red blood cells, secretion of G-CSF stimulates the production of granulocytes,
The secretion of GM-CSF stimulates the production of various leukocytes. Similarly, a disease or condition in which one or more specific cell types have been destroyed during the course of the disease (eg, an autoimmune disease)
In, certain cells can be recruited by stimulating the secretion of one or more factors that stimulate the proliferation of these cells. The method of the present invention may also be used to increase lymphocyte numbers in AIDS patients, for example, by stimulating the secretion of lymphokines (eg, IL-4) that stimulate the proliferation of certain T helper (Th) cells. Can also be used.

In another case, the target protein is a hormone or endorphin that must be quickly and efficiently supplied to its site of action. For example, patients with insulin-dependent diabetes mellitus (IDDM) must artificially maintain the physiological concentration of insulin in the bloodstream. Instead of frequent insulin injections, it would be highly desirable to change to a regulated expression system that allows patients to rapidly produce their own insulin when needed. Current regulated expression systems make use of the transcription machinery, with protein concentrations increasing approximately 12-16 hours after addition of the ligand. In contrast, the present invention will allow insulin to be delivered to a given site within 20-30 minutes of ligand binding. As with insulin, the invention described herein could be used to treat any condition for which a rapid delivery of a therapeutic protein would be beneficial. For example, the present invention may be useful for the supply of any protein that requires a regular or once daily supply for biological reasons. Such proteins may include, inter alia, parathyroid hormone and growth hormone. Other uses include the supply of proteins for inflammatory redness disorders such as rheumatoid arthritis and inflammatory bowel disease. Examples of such treatments include TNF, soluble TNFR
And IL-1RA antibodies. More generally, insulin (above) or other blood glucose management agents, anti-pain peptides; inflammation (above)
Any demand such as, leptin, contraception (eg, LHRH antibodies)
-demand) "or the regulated secretion of a self-medication scenario would benefit the patient.

Methods for Identifying CRDs Methods for identifying, verifying, and improving CRD candidates in each of the three types described above are disclosed below.

[0151] 1. CRDs Comprising Natural Example Proteins Retained in the Secretory Compartment in a Small Molecule-Reversible Way Candidate CRDs of this type include any naturally-occurring protein and its subdomains. Such proteins can typically be identified by the presence of a secretory signal sequence at the start of their coding sequence. The characteristics of such signal sequences are well known, and computer algorithms are available to help identify them. Using such methods, secretory proteins can be identified from searches of sequence databases. Preferred subsets of secreted proteins are those that are known to bind small molecules or are predicted to be so due to their homology to other small molecule binding proteins.
A small molecule may be a ligand or substrate that is temporarily bound to the protein during its normal function, or it may be a cofactor that usually remains permanently bound. In each case, these small molecules provide a starting point for identifying ligands for candidate CRDs. In some cases (one example is rat RPB), small molecule-mediated release of proteins from the secretory compartment has been published in the scientific literature.

To test whether a candidate protein can function as a CRD, the DNA encoding the candidate polypeptide can be obtained from a suitable cellular source, or cDNA or genomic DN.
A Amplify by PCR or RT-PCR using standard methods from appropriate sources, such as genomic DNA isolated from the library or complete or poly A + RNA. The PCR primers are engineered to contain restriction sites that allow for insertion into the vector for expression in mammalian cells, or other eukaryotic cells of interest. Or,
The sequence of interest can be isolated as a restriction fragment. The PCR or restriction fragments are then cloned in frame into the expression vector polylinker.

Preferred vectors are of the type shown in FIG. 10A. Where hCMV is human CMV
Means immediate-early promoter and enhancer, SS means signal sequence,
poly is the polylinker region, FCS is the furin cleavage site, and hGH is the cDNA for human growth factor hormone. The components of this vector can be replaced as appropriate: for example, FCS can be replaced with another TGN protease cleavage site, and hGH can be readily replaced by secreted alkaline phosphatase (SEAP) or erythropoietin (EPO). It can be detected and therefore replaced with other secreted proteins useful as reporter proteins. Optionally, an epitope tag that allows immunochemical detection of the protein (eg, FLAG sequence: IBI / Kodak) can be included in the vector sequence or incorporated via any PCR primer.

To determine whether a candidate polypeptide acts as a CRD, the expression vector is introduced into cells in culture using standard techniques, for example, lipofection. After 24 hours, aliquots of the medium are removed and assayed for the presence of hGH using standard techniques (Rivera et al., 1996). Thereafter, fresh media containing different concentrations of the candidate CRD ligand is added. After an additional 2 to 24 hours, the medium is again taken for assay for the presence of hGH. CRD-like activity of a candidate polypeptide is indicated by low levels of hGH in the medium in the absence of the compound and increasing amounts in the presence of the compound. Suitable candidate CRD ligands to investigate include compounds that are known ligands for the protein under consideration (eg, retinol for RBP) and useful different properties such as cell permeability or effect on protein ER retention Chemically related molecules that may have (eg, various retinoids for RBP). Suitable concentrations to investigate for these ligands are in the range 1 pM to 1 mM.

An important approach to optimizing the effectiveness of CRD candidates is the repetition with multiple copies of their domains in an attempt to amplify any condition-retaining effects. For example, some proteins "lag" in the secretory pathway in the absence of ligand, but are not completely retained-that is, retention is "leaky". This may be desirable in some applications of the present invention. Other applications may require well-repressed protein production in the absence of drug. In such cases, repetition of the CRD may enhance the ability to cause retention of the heterologous protein. That is,
The above experiments were optionally performed with different numbers of concatamerization candidate CRDs: typically 1-8
, Will be repeated in the construct housing the.

Another control that can be implemented to verify the activity of CRD discovered through the above method is to confirm that the protein is retained inside the secretory apparatus,
Includes immunochemical detection of CRD and hGH domains inside cells treated or untreated with CRD ligand. These experiments utilize immunofluorescence after a standard cell fixation procedure. Secreted hGH can also be checked for correct processing from the fusion protein by SDS-PAGE followed by size analysis using an immunoblot with an anti-hGH antibody. For a more rigorous check, hGH can be purified (eg, on an hGH binding protein affinity column) and then analyzed for molecular weight by mass spectrometry and for correct processing by N-terminal sequence analysis after immobilization on PVDF.

[0157] CRD searches will center on naturally secreted proteins and a subset of secreted proteins with further known small molecule binding activities, but will test any polypeptide using the methods described above. can do. That is, a protein that is not naturally secreted but has a known small molecule binding activity can be cloned into an FCS-hGH expression vector and reversed by a small molecule (or related molecule).
Can be tested for CRD behavior. In the broadest sense, any polypeptide-
-Including those that are not normally secreted normally and have no known small molecule binding activity can be tested. In such cases, the candidate CRD-FCS-hGH expression construct is first
hGH retention can be tested. If retention is observed, cells containing the construct may be subjected to additional experiments using a diverse set of candidate small molecules to identify molecules capable of promoting secretion of the retained fusion protein. Can be done. Suitable sets of molecules include natural product collections as well as members of synthetic or semi-synthetic combinatorial libraries. Screening may be facilitated by arranging cells in 96 or 384 well plates to allow for automated high throughput setups and experimental analysis.

[0158] 2. selected for properties which are selectively retained in the absence of a given small molecule, a screening method CRD for such CRD is a mutant of the natural protein, of course, the method According to.
The polypeptide of interest is cloned into the FCS-hGH fusion expression vector described above. Again, preferred polypeptides are those that have a known small molecule binding activity. Individual mutants of the candidate CRD are engineered by standard methods. These mutant constructs are then assayed for (i) retention of hGH and (ii) secretion of hGH after addition of small molecules. The choice of small molecules to be tested and their concentrations are as described above. Assays for multiple mutants can be performed simultaneously by a multi-well plate assay.

[0159] Mutations can be selected to optimize the likelihood of inducing a change in the property of the protein that results in condition retention. Mutations of particular interest are expected to interfere with the efficient folding of proteins: such proteins could also be retained via an ER quality control system. Examples of mutations are gain-of-size mutations in the side chains that make up the hydrophobic core of the protein; and critical for secondary or tertiary structural forms, such as glycine residues in the β-turn motif. And other residue changes that are important. Other amino acids of interest are those that form or approximate small molecule binding sites. Mutants with reduced folding efficiency are preferred because such changes are most likely to be stabilized by small molecule binding, providing a mechanism for selective small molecule mediated release of the retained protein. Thus, knowledge of the three-dimensional structure of the candidate CRD can be very useful in focusing mutagenesis at key locations.

Both single and multiple mutant muteins can be engineered and tested. Often, the best muteins will vary in several positions. Repetitive screening of mutants to identify the best combination of multiple residue changes can be boring and time consuming. Another approach is to utilize a selection procedure that creates a large set of mutants and then goes through a selection step to directly identify the best mutants together. Clackson and Wel
ls (Trends Biotech 1994 12: 173). To provide a means for directly selecting a protein to act as a CRD, the hGH coding sequence should be either CD2 or p75.
The above-mentioned expression vector is changed by exchanging for a DNA encoding a cell surface marker such as a low affinity nerve growth factor receptor. Although the extracellular and transmembrane domains of cell surface markers are included, it is preferred that most intracellular domains be deleted to eliminate the possibility of signaling through the receptor. p75
A suitable expression vector using is shown in FIG. 10B, where ECD and TM are the extracellular and transmembrane domains of p75, respectively.

To select CRDs from a large set of candidates, a gene encoding the candidates is linked to a polylinker to create a library. This library is introduced into mammalian cells by established methods. Ideally, (i) the number of mutants introduced into each cell is small and (i) so that the properties of the mutants can be tested individually.
i) A method that provides for stable transfer of the vector should be selected so that cell growth and selection can be performed multiple times. A preferred approach is therefore to make the library in a retroviral vector followed by retroviral infection of the cells. This approach results in stable integration of one or a few copies of the vector.

The selection of the CRD can be made directly or indirectly. Direct screening is performed in two steps using a fluorescence activated cell sorter. In the first step, cells containing a library of large numbers of CRD candidates are grown in culture and then incubated with a fluorescently labeled antibody against p75 ECD. Cells containing clones for active CRD will not bind because p75 is retained in the secretory apparatus. However, cells containing ineffective CRD will bind because the protein is not retained. Labeled cells are sorted by FACS, and unstained cells are collected by passing through a gate (gating) and grown again in culture. Sorting can optionally be repeated several times with slightly higher gates to isolate cells expressing the lowest levels of p75.

In the second step, one candidate CRD ligand (selected as described above) is added, and the labeling process is repeated. This time, as the retained p75 is released by the CRD ligand, cells with effective CRD will be labeled. FA cells
Sort by CS and separate labeled cells. Also in this case, the selection step can be repeated as desired. Once the appropriate population of cells has been isolated, variants conferring CRD activity can be identified by isolating the genomic DNA of the cells and then performing PCR amplification using primers flanking the vector polylinker. it can. PC
The R product can then be cloned and sequenced. The ability of the identified variants to act as CRDs can be confirmed by cloning them individually into an hGH expression vector and then testing as described above. Indirect screening can also be performed by determining whether the CRD directs the surface localization of membrane proteins, which can subsequently activate signaling pathways.

The introduced mutant can be targeted to the interesting residues shown above,
Or it can be taken in at random. Several suitable methods for engineering large sets of mutants have been described in the literature, including alanine scanning mutagenesis (Cunningham and Wells (1989) Science 244 1081-1085), degenerate primers Vector `` Kunkel '' mutagenesis (e.g., Lawman and Wells, 1993
, J. Mol. Biol. 234: 563-578), PCR misincorporation mutagenesis (e.g., Cadw
ell and Joyce (1992) PCR Meth. Applic. 2, 28-33), and DNA shuffling (Stemmer (1994) Nature 370: 389-391).

[0165] 3. Methods for identifying proteins that interact with one another, CRDs, which are proteins that self-assemble in a small molecule reversible manner, are well known. A conventional technique is the two-hybrid system, in which one partner is fused to a DNA binding domain and the other is fused to a transcription activation domain. When an interaction between partners occurs, transcription factors are reconstituted and transcription of a reporter gene is activated. The reporter gene can be identified by screening (eg, production of β-galactosidase or SEAP) and / or result in cell survival,
Thus, means for selecting interacting partners (e.g., his his in a yeast his strain)
Gene transcription). Two-hybrid assays can be performed on yeast or mammalian cells, and methods are well known in the art.

One preferred embodiment is based on the vectors and cells reported by Rivera et al. (Nature Med 1996 2, 1028-1032). Two types of expression vectors are constructed for the chimeric transcription factor. That is, the candidate CRD fused to the hybrid DNA domain ZFHD1 (one) and the candidate CRD fused to the activation domain of the NF-kB p65 subunit such as amino acids 361-550 (the other). These vectors are transiently or stably transfected into mammalian cells (eg, HT1080 cells) together with a SEAP reporter gene under the control of a ZFHD1 binding site. The collection of candidate CRDs results in the reorganization of the active transcription factor and thus the production of SEAP. Having thus identified the self-assembling protein, the addition of the candidate CRD ligand can be used to test whether the assembly can be dissociated by the ligand. A decrease in SEAP production after ligand addition would indicate this activity. Although any polypeptide can be selected for testing CRD activity in this manner, the preferred proteins to test are those for which small molecule binding activity is already known. In this case, the known binding ligand provides a starting point for selecting compounds that are likely to disaggregate the binding protein.

As noted above, an important additional form to investigate is the concatemer formation of candidate CRDs. The presence of more than one assembling domain may increase the apparent affinity of the assembling interaction by avidity effects.

[0168] Both native and muteins can be tested for CRD activity. Since examples of induced aggregate activity by point mutations are known from the literature (e.g., sickle cell hemoglobin or alpha ₁ antitrypsin previously described),
Mutants of the native protein appear to be good sources of CRD. That is,
Numerous mutants of a large set of candidate proteins can be cloned into two-hybrid vectors as described above and tested for aggregation activity that can be reduced by the addition of small molecules. The criteria that dictate the selection of the position to mutate will often be the same as those described above for screening for CRD directly in the secretory system (2 above). In addition, assembling mutants may be provided by converting polar surface residues to less polar amino residues. Single or multiple mutants can be engineered using methods such as those described above.

A CRD selection scheme can also be devised. In that case, the library of muteins is cloned into a two-hybrid vector and analyzed collectively for CRD activity. Such experiments are easiest to perform in yeast, and methods for two-hybrid selection are well known in the art. For example, the candidate
The expression vector for the CRD mutant is fused to a GAL4 DNA binding domain or activation domain vector, and the his gene carrying the his gene under the control of the GAL4 binding site.
Transform into s-deleted yeast strain. When the library is plated in his deletion medium, only cells containing an interactive CRD on the two chimeric transcription factors will result in growth. These positive cells can then be replica plated on plates with an increased content of candidate CRD ligands to identify CRDs whose interaction can be interrupted by small molecules. Such proteins are candidates for use as CRDs.

A complication of the above selection scheme is that the same mutant must be fused to both the DNA binding domain and the activation domain in order to identify self-assembling proteins. To achieve this, the expression vector of the chimeric protein can be modified so that the mutated gene is linked to both transcription domains at the level of splicing. The domains of interest are encoded in separate exons. An outline of a suitable vector is shown in FIG. 10C. In the figure, CRD cand is a candidate CRD, and a library of candidates (eg, muteins) is inserted here. DBD and
AD is the DNA binding and activation domain of the transcription factor. A and D indicate the splice acceptor and donor sites. stop indicates a translation stop codon. By providing a suboptimal splice acceptor site in the DBD, the CRD exon will be spliced into both the DBD and AD exons. Thus, in each cell, a fusion protein will be expressed in which both AD and DBD are fused to the same candidate CRD.

Another format for selection of self-assembling proteins is the E. coli λ repressor fusion system (Hu et al. 1990 Science 250: 1400-1403; for a review, He 1995 Structure 3: 431-433). ). This technique demonstrates that bacteriophage λ repressor cI binds to DNA as a homodimer and that binding of this homodimer to operator DNA inhibits transcription of phage genes involved in the lytic pathway of the phage life cycle. Use. That is, bacterial cells that express a functional λ repressor become immune to lysis by superinfection with λ bacteriophage. The repressor protein contains an amino-terminal DNA binding domain (amino acids 1-92) connected to a C-terminal dimerization domain by a 40 amino acid flexible linker. The separated N-terminal domain binds very weakly to DNA due to inefficient dimer formation.
Fusion of this domain to a heterodimerization domain such as GCN4 leucine zipper can restore high affinity DNA binding. Thus, a selection system using phage immunity as a means of selecting interacting proteins is possible.

For example, to select CRDs from a library of large numbers of candidates, those candidates are cloned in-frame by the repressor N-terminus and the library is transformed into E. coli.
Transform into. The gene for the assembling protein is isolated from the surviving colonies on plates containing high titers of λ phage. These colonies
The plate can then be re-inoculated on plates containing both phage lambda and candidate CRD ligand. If the ligand dissociates the assembly, E. coli will not grow on these plates this time. The λ repressor selection method has several advantages for the identification of CRDs, including that the system is suitable for screening homodimers and that larger library sizes can be obtained using E. coli. is there.

Another way to directly test whether a protein can act as a CRD in living cells is to fuse its coding sequence to green fluorescent protein (GFP) or a variant thereof. The cells expressing such fusion proteins can then be examined directly by fluorescence microscopy to see if the CRD candidate is likely to give rise to GFP aggregates. Thereafter, candidate CRD ligands can be added to determine if the assembly subsequently dissociates. Once a candidate CRD has been identified by any of these methods, it can be tested for activity as a CRD using the methods outlined in Section 1.

Administering the Pharmaceutical Composition and Its Administration to an Individual Containing the Genetically Engineered Cells The administered ligand can be administered to a human or non-human individual using pharmaceutically acceptable administration materials and methods of administration. The recipient's condition and condition, the desired response, the biological half-life and bioavailability of the ligand, the biological half-life and specific activity of the target protein product, the number and location of genetically engineered cells present, etc. A variety of formulations, routes of administration, doses and dosing schedules can be used for administering the ligand, depending on the factors. The drug can be administered parenterally or, more preferably, orally. For use in the present invention, the most preferred route of administration is that by which a rapid onset of response occurs, such as sublingual, buccal, buccal, dermal patch (paste), and inhalation. No. Dosage and frequency will depend on factors such as those set forth above. The agent can be administered orally, buccally, sublingually, or as an intravenous, intraperitoneal, subcutaneous injection, or other route as a pill, powder, or dispersion. The formulation of the drug can be performed using conventional methods and materials well known in the art for various routes of administration. The exact dose and particular mode of administration will depend on the factors described above and will be determined by the attending physician or other health care provider.

The specific dosage of an agent for a particular use can be determined according to conventional techniques and procedures for monitoring therapeutic dosage. A dose of the drug within a predetermined range is administered and the response of the patient is monitored so that the level of therapeutic response and the time course of protein secretion can be determined. Depending on the expression level and the therapeutic response observed during the monitoring period, the level of the next dose can be adjusted, the resulting expression level changed, or the therapeutic response improved. This process can be repeated many times until the dose is optimized for a therapeutic response. For long-term drug administration, determining the maintenance dose of the drug will ensure that the overall therapeutic response is sustained.
Periodic tracking and monitoring may be performed.

If it is desired to reverse activation by a drug, administration of the drug can be stopped so that the cells return to their basal secretion rate. To achieve a more aggressive reversal of treatment, an antagonist of the drug may be administered. An antagonist binds to the drug or drug binding domain and inhibits the interaction of the drug with the fusion protein;
Thus, compounds that inhibit downstream biological events. That is, if there are side effects or if one wishes to end the therapeutic effect, and if rapid reversal is desired, the antagonist can be administered by any convenient method, especially intravascular or by inhalation / nebulization.

Compositions The agents (ie, ligands) used in the present invention can exist in free form or, if appropriate, in the form of a salt. The preparation of a variety of pharmaceutically acceptable salts is well known to those skilled in the art. Pharmaceutically acceptable salts of the various compounds include conventional non-toxic salts or quaternary ammonium salts of such compounds, for example formed from inorganic or organic acids or bases. Drugs may also form hydrates or solvates. It is known to those skilled in the art that charged compounds form hydrate species when lyophilized with water and form solvate species when concentrated in solution with a suitable organic solvent. I have.

The drug can also be administered as a pharmaceutical composition comprising a therapeutically (or prophylactically) effective amount of the drug and a pharmaceutically acceptable carrier or excipient. As a carrier,
Examples include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof, and are described in more detail below. The compositions can, if desired, also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder (powder). The composition can also be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. The formulation operation may include mixing, granulating and compressing or dissolving the components as appropriate for the desired formulation. The pharmaceutical carrier used is
For example, it may be either solid or liquid.

Examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Solid carriers are flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants
Can contain one or more substances that can also act as compression aids, binders, or disintegrants; this may also be an encapsulating material. In powder,
The carrier is a finely divided solid, which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain up to 99% of the active ingredient. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugar, lactose, dextrin, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidine, low melting waxes, and ion exchange resins.

Examples of liquid carriers include syrup, peanut oil, olive oil, water and the like. Liquid carriers are used in preparing solutions, suspensions, emulsions, syrups, elixirs, and pressurized compositions. The active ingredient is water, an organic solvent, a mixture of both,
Alternatively, it can be dissolved or suspended in a liquid carrier acceptable for a drug such as a fat acceptable for the drug. Liquid carriers include other suitable auxiliaries, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickeners, coloring agents, viscosity modifiers, stabilizers or osmotic pressure regulators. Formulation additives may be included. Suitable examples of liquid carriers for oral and parenteral administration include water (partially containing additives as described above, for example, cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohols [monohydric alcohol and Polyhydric alcohols (eg, glycols) and their derivatives, and oils (eg, fractionated coconut and arachis oils). For parenteral administration, the carrier can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid carders are useful in sterile liquid form compositions for parenteral administration. Liquid carriers for pressurized compositions can be halogenated hydrocarbons or other pharmaceutically acceptable propellants. Liquid pharmaceutical compositions in the form of sterile solutions or suspensions can be utilized by, for example, intramuscular, intraperitoneal or subcutaneous injection. Sterile solutions can also be administered intravenously. The agent can also be administered orally in the form of either a liquid or solid composition.

[0181] Carriers or excipients include slow-release materials well known in the art, such as glyceryl monostearate or glyceryl distearate alone or in combination with waxes, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate, and the like. You may go out. PHOSAL P when prescribed for oral administration
G-50 (phospholipid concentrate with 1,2-propylene glycol, A. Nattermann & Cie. G
0.01% Tween 80 in mbH) can be used as the oral formulation of various agents used in the practice of the present invention.

[0182] A variety of dosage forms can be employed. If a solid carrier is used, the preparation may be tableted or filled into a hard gelatin capsule in powder or pellet form,
Or it can be in the form of a troche or rosindi. The amount of solid carrier will vary widely but will preferably be from about 25 mg to 1 g. If a liquid carrier is used, the preparation will be in the form of a syrup, emulsion, soft gelatin capsule, sterile injectable solution or suspension in an ampoule or vial or non-aqueous liquid suspension.

To obtain a stable water-soluble dosage form, the pharmaceutically acceptable salt of the drug may be dissolved in an aqueous solution of an organic or inorganic acid, such as a 0.3 M aqueous solution of succinic or citric acid. . Alternatively, the acidic derivative can be dissolved in a suitable basic solution. If a soluble salt form is not available, the compound is dissolved in a suitable co-solvent or a combination of two or more. Examples of such suitable co-solvents include, but are not limited to, alcohols, propylene glycol, polyethylene glycol 300, polysorbate 80, glycerin, polyoxyethylated fatty acids, fatty alcohols or glycerin hydroxy fatty acid esters, and the like. And the total volume is 0
Used at concentrations in the range of ~ 60%.

Various supply systems including tablets, capsules, injection solutions, encapsulation in liposomes, fine particles, microcapsules, etc., or various prescription compositions thereof are known, and the drug is administered. Can be used for Preferred routes of administration to the patient are oral, sublingual, transdermal (patch), intranasal, pulmonary, or buccal. Methods of introduction include, but are not limited to, cutaneous, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, epidural, ocular and (as usually preferred) oral routes. Administration of the drug can be by any convenient or other suitable route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.). And may be administered with other biologically active agents.
Administration can be systemic or local. For ex vivo administration, the agent will be supplied to the cell composition as a liquid solution.

In certain embodiments, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. In general, the components may be mixed separately or together in unit dosage form, for example,
It is supplied as a lyophilized powder or anhydrous concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active ingredient. If the composition is to be administered by infusion, it can be supplied using an infusion bottle containing sterile pharmaceutical water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

Also, in some cases, it is envisioned that the compound may be placed inside a device placed above, inside, or below the skin. Such devices include patches, implants, and infusions that release the compound into the skin by a passive or active release mechanism.

The materials and methods for making the various formulation compositions are well known in the art and are applicable to the practice of the present invention. For example, U.S. Patent Nos. 5,182,293 and 4,837,311 (tablets, capsules and other oral and intravenous formulations) and European Patent Application Publication No. 0 649 659 (published April 26, 1995; See, for example, rapamycin formulations for intravenous administration) and No. 0 648 494 (published April 19, 1995; exemplify rapamycin formulations for oral administration).

An effective dose of the agent will typically be from about 0.01 to about 50 mg / kg of mammal weight.
Preferably, it is in the range of about 0.1 to about 10 mg / kg and is administered once or multiple times. Generally, the compound can be administered to a patient in need of such treatment in a daily dose range of about 1 to about 2000 mg / person. In embodiments where the compound is rapamycin or an analog thereof with some residual immunosuppressive effect, it is preferred that the dosage administered be less than the dose with excessive immunosuppression.

The amount of an agent that is effective in treating or preventing a particular disease or condition will depend, in part, on the severity of the disease or condition, and can be determined by standard clinical techniques. It may also be useful to determine optimal dosage ranges in vitro or in vivo.
Assays may optionally be employed. Effective doses may be extrapolated from dose-response curves obtained from in vitro or animal model test systems. The exact dosage level should be determined by the attending physician or other health care provider, and should include the route of administration,
And the age, weight, gender, and general health of the individual (patient); including the nature, severity, and clinical stage of the disease; concomitant use of concurrent treatment; and the nature and extent of genetic engineering of cells within the patient. Will vary depending on known factors.

The medicament is also provided in the form of a medicament pack or kit comprising one or more containers containing one or more components of the medicament composition of the present invention. Such containers may optionally be accompanied by a notice in the form specified by the governmental authority overseeing the manufacture, use or sale of the pharmaceutical or biological product, the notice notifying the user of the product for human administration. Reflects regulatory approval of manufacturing, use or sale.

The entire contents of all references cited herein, including references from scientific literature, issued patents and published patent applications, are expressly incorporated herein by reference. The following examples include important information, illustrations, and guidelines that can be adapted for the practice of various aspects of the present invention and their equivalents. The examples are provided for illustrative purposes only and are not to be understood as limiting in any way. As mentioned throughout this document, the present invention is widely applicable and allows a wide range of design choices to be made by the practitioner.

The practice of the present invention will, unless otherwise indicated, be within the skill of the art in cell biology, cell culture, molecular biology, transformation biology, microbiology, recombinant DNA, immunology, virology. Conventional techniques of pharmacology, chemistry and pharmaceutical prescribing and administration will be employed. Such techniques are explained fully in the literature. For example, Molecular Cloning, A Labor
atory Manual, 2nd edition, edited by Sambrook, Fritsch & Maniatis (Cold Spring Harbor
Laboratory Press: 1989); DNA Cloning Volume I-II (Edited by DN Glover, 1985);
Oligonucleotide Synthesis (Ed.MJ Gait, 1984); Mullis et al. U.S. Patent No.
No. 4,683,195; Nucleic Acid Hybridization (BD Hames & SJ Higgins Edition 1
984); Transcription And Translation (BD Hames & SJ Higgins ed. 1984);
Culture Of Animal Cells (RI Freshney, Alan R. Liss, Inc., 1987); Immo
bilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practial Guide
To Molecular Cloning (1984); the treatise, Methods In Enzymology (Acade
mic Press, Inc., NY); Gene Transfer Vectors For Mammalian Cells (JH
Miller and MP Calos eds., 1987, Cold Spring Harbor Laboratory); Methods
In Enzymology, Vols. 154 and 155 (Edited by Wu et al.), Immunochemical Methods
In Cell And Molecular Biology (ed by Mayer & Walker, Academic Press, London
, 1987); Handbook Of Experimental Immunology, Volumes I-IV (DM Weir & CC
Blackwell, 1986); Manipulating the Mouse Embryo (Cold Spring Harbor L
aboratory Press, Cold Spring Harbor, NY, 1986).

[0193]

【Example】

Example 1 Formation of Domain and Vector Used for Expression of F (36M) Fusion Protein A. Expression Vector The vector for producing expression of the fusion protein is a mammalian expression vector pC.
Derived from GNN (Attar and Gilman, MCB 12: 2432-2443, 1992). XbaI to pCGNN
The insert cloned as a -BamHI fragment was transcribed under the control of a human CMV promoter and enhancer sequence (nucleotides -522 to +32 relative to the cap site) and the N-terminal nuclear localization sequence (NLS; SV40T It is expressed together with the antigen (from the antigen) and an epitope tag (the 16 amino acid portion of the H. influenzae hemagglutinin gene).

PCGNN was modified by site-directed mutagenesis using oligonucleotides VR65, VR119 and VR120 to create pC4EN. The resulting plasmid has a unique restriction site upstream of the CMV enhancer / promoter region (MluI) and between the promoter and the protein coding region (EcoRI).

VR65: TCCCGCACCTCTTCGGCCAGCGaaTTccAGAAGCGCGTAT VR119: GACTCACTATAGGaCGcgTTCGAGCTCGCCCC VR120: CATCATTTTGGCAAAGgATTCACTCCTCAGG The individual components of the fusion protein are an XbaI site immediately upstream of the first codon, a frame immediately downstream of the last codon, and a Spein immediately downstream of the last codon, an XbaI site immediately downstream of the last codon It was generally produced as a fragment containing a BamHI site. Chimeric proteins containing multiple (multiple) components were assembled by stepwise insertion of the XbaI-BamHI fragment into the SpeI-BamHI open-chain vector or insertion of the XbaI-SpeI fragment into the XbaI or SpeI open-chain vector.

B. Generation of F (36M) in which the phenylalanine at amino acid 36 has been changed to methionine at F (36M) domain is based on a single FK cloned into pCGNN with upstream XbaI and downstream SpeI and BamHI.
This was done by mutagenizing the BP domain (Rivera et al., Nat. Med 2: 1028-1032, 1996) with oligo VR1 to generate pCGNN-F (36M). SpeI-Bam
Stepwise insertion of the XbaI-BamHI fragment into the HI open vector produced 2, 3, 4 and 6 tandem copies of F (36M).

VR1: GATGGAAAGAAAatgGATTCCTCCCGG CF (36M) fusion protein (FIG. 3) (a) EGFP fusion The EGFP coding sequence was amplified from pEGFP-1 (Clontech) using oligos VR2 and VR3. The resulting fragment containing the upstream XbaI and downstream SpeI sites was inserted into pCGN, a derivative of pCGNN lacking the SV40 nuclear localization sequence, to create pCGN-EGFP.

VR2: tctagaGTGAGCAAGGGCGAGGAG VR3: ggatccttaTTAACTAGTCTTGTACAGCTCGTCCATG XbaI-SpeI fragment containing 3, 4 or 6 copies of F (36M) was converted to XbaI of pCGN-EGFP.
Inserted into the site, pCGN-F (36M) 3-EGFP, pCGN-F (36M) 4-EGFP, and pCGN-F (36M) 6
An F (36) M-EGFD fusion was prepared by preparing -EGFP.

(B) hGH fusions RT-RNA using oligos VR109 and VR110 of RNA expressed from a cell line containing the genomic hGH gene (Rivera et al., Nat. Med. 2: 1028-1032, 1996). Amplify the region from 40 bp upstream of ATG to 60 bp after the stop codon by PCR amplification.
NA (506-81) was obtained. The obtained HindIII to EcoRI fragment was ligated with the SEAP gene and SV40.
ZHWTx12-IL2-SEAP in which the early intron and polyadenylation signal have been replaced by a polylinker and SV40 late polyadenylation signal (Rivera et al., Nat.
d. 2: 1028-1032, 1996), Z12I-PL-2.

VR109: aagcttACCACTCAGGGTCCTGTGG VR110: gaattcGTGGCAACTTCCA To construct the hGH fusion protein, Z12I-hGH-2 was mutagenized with oligos VR185, VR186, and VR187 to: i) EcoRI site 32 bp upstream of ATG, ii ) Create an XbaI site immediately after the last amino acid in the signal sequence, and iii) a SpeI site immediately after the last amino acid in hGH.

VR185: cacaggaccctGAATTCtaagcttgtggc VR186: ATAAGGGAATGGTtctagaGGCACTGCCCT VR187: atgccacccgggactagtGAAGCCACAGCTG When the resulting EcoRI-SpeI fragment is cloned into pC4EN, the hGH that expresses hGH from the CMV enhancer is 1-hGH from the CMV enhancer. Next, the XbaI-BamHI fragment of pC4S1-hGH was replaced with an XbaI-SpeI fragment containing 2, 3, 4 or 6 copies of F (36M) and a SpeI-BamHI fragment encoding the furin cleavage site-hGH fusion. Then pC4S1-F (36M) -F
A CS-hGH fusion was generated.

[0202] The SpeI-BamHI fragment encoding the FCS-hGH fusion protein was constructed using the oligo VR of the hGH cDNA.
4 and formed by amplification with VR5. VR4: actagtGCTAGAAACCGTCAGAAGAGATTCCCAACCATTCCCTTAAGC VR5: ggatcccgggCTAGAAGCCACAGCTGCCCTC Downstream neo-resistance gene of encephalomyocarditis virus internal ribosome entry sequence (IRES
/ Neo; Amara et al., PNAS 94: 10618-23, 1997).
Insert into the appropriate SpeI-BamHI open chain vector, pC4S1-F (36M) -FCS-hGH / neo and pC4S1-F (36M) -FCS-hGH / neo
Form 4S1-hGH / neo vector.

(C) RT-PCR amplification of human pancreatic poly A + RNA (Clontech) using insulin fusion oligos VR220 and VR221 from 9 bp upstream of ATG (EcoRI) to 13 bp after stop codon (BamHI) Was amplified to obtain human insulin cDNA. The resulting EcoRI-BamHI fragment was cloned into pC4EN to form pC4-hIn.

VR220: cGAATTCttctgccATGGCCCTGTGGATGCGC VR221: cGGATCCgcaggctgcgtCTAGTTGCAGTAG The SpeI-BamHI fragment encoding the furin cleavage sequence-insulin fusion protein is
Formed by RT-PCR amplification with oligos VR222 and VR221.

VR222: cACTAGTGCTAGAAACCGTCAGAAGAGATTTGTGAACCAACACCTGTGCGGC VR221: cGGATCCgcaggctgcgtCTAGTTGCAGTAG Wild-type insulin gene and FCS-insulin fusion oligo VR223, VR224
, VR225, respectively, i) amino acid B10 was changed to Asp, ii) FCS was prepared for BC bond, and iii) FCS was prepared for CA bond.

VR223: CCTGTGCGGCTCAgACCTGGTGGAAGC VR224: CTTCTACACACCCAgGACCaagCGGGAGGCAGAGG VR225: CCCTGGAGGGGTCCCgGCAGAAGCGTGGC pC4-hIn-m3 produced a mutation in pC4-hIn. The mutated FCS-insulin fusion was used to replace the FCS-hGH portion of the pC4S1-F (36M) -FCS-hGH fusion,
An F (36M) -FCS-hIn-m3 fusion was made.

(D) LNGFR fusion Human low affinity nerve growth factor receptor (LNGFR; Clackson et al., PNAS 95: 10437-
42, 1998) and the SpeI-BamHI fragment containing 3, 4, or 6 copies of F (36M) containing amino acids 1-274 were cloned into pC4EN and cloned into pC4LNGFR-F.
(36M) A fusion was formed.

(E) Transcription factor fusions A fusion protein of pCGNN-ZFHD1-F (36M) and pCGNN-F (36M) -p65 was reported against a wild-type FKBP fusion (Amara et al., PNAS 94: 10618-23, 1997).

The XbaI-SpeI fragment containing 6 copies of F (36M) was ligated to the XbaI of pCGNN-ZFHD1-p65 or
Insert into SpeI site, pCGNN-F (36M) 6-ZFHD1-p65 and pCGNN-ZFHD1-p65-F (36M) 6
Was formed.

An expression vector that directs the expression of the ZFHD1 coding sequence in pCGNNZFHD1 mammalian cells was prepared as follows. The Zif268 sequence was amplified from the cDNA clone by PCR using primers 5'Xba / Zif and 3'Zif + G. Primer 5'Not Oct HD and Spe /
The Oct1 homeodomain sequence was amplified from the cDNA clone by PCR using Bam 3'Oct. The Zif268 PCR fragment was cut with XbaI and NotI. OctI PCR fragment was transferred to NotI and BamHI
And cut with. Both fragments were ligated with the XbaI site of pCGNN (Attar and Gilman, 1992) and the BamHI
The cDNA insert was under the transcriptional control of the human CMV promoter and enhancer sequences, and was ligated with the SV40 T
PCGNNZFHD1 binding to a nuclear localization sequence from the antigen was generated. Plasmid pCGN
N also contains a gene for ampicillin resistance that can act as a selectable marker.

An expression vector that directs the expression in a mammalian cell of a chimeric transcription factor containing the pCGNNZFHD1-p65 composite DNA binding domain ZFHD1 and a transcription activation domain derived from p65 (human),
It was prepared as follows. Contains activation domain (amino acid residues 450-550)
The sequences encoding the C-terminal region of p65 were ligated with the primers p65 5'Xba and p65 3'Spe /
Amplified from pCGN-p65 using Bam. Digest the PCR fragment with XbaI and BamHI, pCGN
Ligation between the SpeI and BamHI sites of NZFHD1 created pCGNNZFHD-p65AD.

[0212] p65 transcriptional activation sequence contains a linear sequence below: CTGGGGGCCTTGCTTGGCAACAGCACAGACCCAGCTGTGTTCACAGACCTGGCATC CGTCGACAACTCCGAGTTTCAGCAGCTGCTGAACCAGGGCATACCTGTGGCCCCCC ACACAACTGAGCCCATGCTGATGGAGTACCCTGAGGCTATAACTCGCCTAGTGAC AGGGGCCCAGAGGCCCCCCGACCCAGCTCCTGCTCCACTGGGGGCCCCGGGGCTCC CCAATGGCCTCCTTTCAGGAGATGAAGACTTCTCCTCCATTGCGGACATGGACTTC TCAGCCCTGCTGAGTCAGATCAGCTCC.

Example 2 Identification and Synthesis of Ligands for the Condition Retention Domain F36M FKBP AP 21998 and AP 22542 bind with high affinity to F36M-FKBP but only slightly to the wild-type protein, It is a particularly useful FKBP ligand for CAD applications as it is expected to have minimal interaction with endogenous proteins during in vivo applications. The design and assay of this type of "bumped" ligand has been reported to target the hole created by truncating FKBP residue Phe36 (Clac
Natl. Acad. Sci. USA 95: 10437-10442, 1998).

AP 21998 is based on the previously reported acid AP 1867 (Clackson et al., Proc. Natl. Acad. Sci. U.
The compound was prepared from compound 5S) of SA 95: 10437-10442, 1998 via DCC / DMAP-mediated coupling using commercially available N, N-dimethyl-1,3-propanediamine (Scheme 1).

AP 22542 was also synthesized by DCC / DMAP mediated coupling of the acid AP 17362 with alcohol 3.
(Reaction formula 2). Carbinol 3 itself was prepared in a three step reaction as outlined in Scheme 2. Claisen-Schmidt condensation of 3,4-dimethoxybenzaldehyde and 3-acetylpyridine provided unsaturated ketone 1 as a crystalline solid in 68% yield. Transfer hydrogenation of 1 using ammonium formate as the hydrogen source provided the propanone adduct 2 as a crystalline solid in 50% isolated yield. Finally, the enantioselective reduction of the aryl ketone moiety of 2 to the desired R-configuration carbinol 3 is carried out using (+)-b-chlorodiisopinocamphenylborane (DIP-chloride ^™ ).
Reduction (Chandrasekharan et al., J. Org. Chem. 50: 5446, 1985) with a yield of 86%.

The synthesis of the acid component AP 17362 was prepared as described in Scheme 3. Commercially available 3,
The alkylation of 4,5-trimethoxyphenylacetic acid at 0 ° C. with the dianion of NaHMDS of 3,4,5-trimethoxyphenylacetic acid in THF with iodoethane yields the racemic 2-arylbutane derivative 4. Conversion was 83%. The acid was resolved by repeated crystallization of the (-)-cinchonidine salt to yield an optically enhanced 4S in 24% yield.
(48% of theory) and 91% ee. This split acid is then
Chloro-1-methylpyridinium iodide (Mukayama reagent) was used to bind to methyl-L-pipecolate hydrochloride. The resulting coupled product was hydrolyzed without isolation to give the desired crystalline acid, AP 17362, in 42% overall yield and> 99% de. X-ray structural analysis confirmed the absolute stereochemistry (configuration) of the resolved 2-arylbutane center as the S configuration.

[0219]

Embedded image

Scheme 1 AP 21998: A solution of AP 1867 (5.0 g, 7.21 mmol) in CH ₂ Cl ₂ (5.0 mL) at 0 ° C.
After treatment with DCC (178 mg, 0.79 mmol), 30 minutes later, it was treated with N, N-dimethyl-1,3-propanediamine (880 mg, 8.65 mmol) and DMAP (5 mg). After warming the reaction mixture to room temperature and stirring for 5 hours, the reaction mixture was diluted with EtOAc (50 mL), filtered, and the filtrate was extracted with a 5% aqueous citric acid solution (3 × 20 mL). Acid extract then solid
NaHCO ₃ was added to basify and extracted with EtOAc (3 × 50 mL). Na ₂ S
Dried O _4, and filtered to give the crude material by evaporation. This was flash chromatographed on silica gel (5% then 15% MeOH / CH ₂ Cl ₂ ) to give the product (2.2 g, 39%) as a colorless foam.

[0219] IR (neat) 2940, 1735, 1650, 1510, 1460, 1240, 1130cm -1 1 H NMR (CDCl 3, 300 MHz) 7.78 (br t, J = 5.1 Hz, 1H), 7.19 (t, J = 8.6 Hz,
1H), 6.92-6.65 (m, 6H), 6.42 (s, 2H), 5.63 (dd, J = 8.0, 5.5 Hz, 1H), 5.45
(d, J = 4.1 Hz, 1H), 4.49 (s, 2H), 3.86-3.70 (m, 16H), 3.60 (t, J = 7.0 Hz,
1H), 3.47-3.41 (m, 2H), 2.82 (td, J = 13.2, 2.4 Hz, 1H), 2.62-2.29 (m, 12
H), 2.16-1.23 (m, 10H), 0.90 (t, J = 7.3 Hz, 3H); ¹³ C NMR (CDCl ₃ , 75 MHz) 172.7, 170.6, 168.5, 157.5, 153.2, 148.9, 147.
4, 142.3, 136.7, 135.3, 133.4, 129.8, 120.2, 119.6, 113.9, 112.8, 111.8,
111.4, 105.1, 75.7, 67.3, 60.8, 56.3, 56.0, 52.1, 50.7, 44.3, 43.5, 38.
3, 37.4, 31.3, 28.3, 26.8, 25.5, 25.4, 20.9, 12.5; LRMS (ES +): (M + H) + 778; HRMS (FAB): (M + H) + calcd: 778.4278, measured value : 778.4299.

[0220]

Embedded image

Reaction Scheme 2 (E) -3- (3,4-Dimethoxyphenyl) -1-pyridin-3-ylpropenone (1): 3,4-Dimethoxybenzaldehyde (53.7 g, 323) in EtOH (400 mL) mmol) and 3-acetylpyridine (39.1 g, 323 mmol) in piperidine (4.75 mL, 4
8 mmol) and heated at reflux for 4 days. The reaction was then evaporated to a slurry and treated with water (400 mL). The precipitated solid was separated by filtration, air-dried, and recrystallized from EtOAc / hexane to give the product (59.2 g, 68%) as a yellow solid.

Mp 111-112.5 ° C .; TLC (EtOAc) Rf = 0.30; ¹ H NMR (CDCl ₃ , 300 MHz) 9.23 (d, J = 1.8 Hz, 1H), 8.79 (dd, J = 4.8, 1.7 H)
z, 1H), 8.28 (dt, J = 7.9, 1.9 Hz, 1H), 7.79 (d, J = 15.6 Hz, 1H), 7.46-7.42
(m, 1H), 7.35 (d, J = 15.6 Hz, 1H), 7.24 (dd, J = 8.3, 1.9 Hz, 1H), 7.68 (d
, J = 1.9 Hz, 1H), 6.91 (d, J = 8.3 Hz, 1H), 3.95 (s, 3H), 3.93 (s, 3H); ¹³ C NMR (CDCl ₃ , 75 MHz) 189.0, 152.9, 151.9 , 149.7, 149.4, 146.1, 135.
8, 133.8, 127.5, 123.6, 119.4, 111.2, 110.2, 56.0; LRMS (ES ⁺ ) (M + H) ⁺ 270; Elemental analysis C ₁₆ H ₁₅ NO ₃ Calculated: C, 71.36; H, 5.61; N , 5.20; Found: C, 71.13; H, 5.70; N, 4.95.

3- (3,4-Dimethoxyphenyl) -1-pyridin-3-ylpropan-1-one (2): Olefin 1 (20.0 g, 74.2 mmol) in MeOH (400 mL), wet 10% Pd / C (2.0
g) and ammonium formate (14.0 g, 222 mmol) were heated at reflux for 30 minutes.
Filtered hot through a Celite pad. The filtrate was gradually cooled, and the precipitated solid was separated by filtration and air-dried to obtain a product (10.0 g, 50%) as a colorless solid.

Mp 91.5-92.5 ° C .; TLC (EtOAc) Rf = 0.55; ¹ H NMR (CDCl ₃ , 300 MHz) 9.16 (d, J = 2.0 Hz, 1H), 8.76 (dd, J = 4.8, 1.7 H
z, 1H), 8.21 (dt, J = 8.0, 1.9 Hz, 1H), 7.40 (dd, J = 7.9, 4.8 Hz, 1H), 6.83
-6.77 (m, 3H), 3.87 (s, 3H), 3.85 (s, 3H), 3.30 (d, J = 7.3 Hz, 2H), 3.03
(d, J = 7.7 Hz, 2H); ¹³ C NMR (CDCl ₃ , 75 MHz) 198.2, 153.5, 149.6, 149.0, 147.6, 135.3, 133.
4, 132.1, 123.6, 120.2, 111.9, 111.5, 56.0 (2), 40.9, 29.5; Elemental analysis: C ₁₆ H ₁₇ NO ₃ Calculated: C, 70.83; H, 6.32; N, 5.16; Actual: C, 70.63; H, 6.42; N, 5.05.

(R) -3- (3,4-Dimethoxyphenyl) -1-pyridin-3-ylpropan-1-ol (3): (+)-DIP in THF (10 mL) at −25 ° C. - chloride ^TM (7.09 g, 22.1 mmol) ketone a solution of 2 (2.0 g, 7.37 mmol) was treated with. The resulting mixture was allowed to stand at −20 ° C. for 2 hours, then placed in a −10 ° C. freezer for 48 hours, and then the mixture was concentrated with diethyl ether (50 mL), and then with diethanolamine (4.24 mL, 44.2 mmol). Processed.
The viscous mixture was stirred at room temperature for 6 hours and then Celite using diethyl ether.
And filtered through a pad of. The filtrate was concentrated and the crude obtained was flash chromatographed (EtOAc then 10% MeOH / EtOAc) to give the product. This product was redissolved in diethyl ether (50 mL) and again treated again with diethanolamine (2.12 mL, 22.1 mmol) as described above to give the product (1.74 g, 86%).
Was obtained as a colorless transparent oil (Chiralpak AD HPLC, 96% ee with 15% EtOH / hexane, retention time: 6.1 minutes for S-enantiomer, 19.4 minutes for target R-enantiomer).

TLC (EtOAc) Rf = 0.25; IR (neat) 3210, 2935, 1590, 1515, 1465, 1420, 1260, 1155, 1070, 1030,
1030 cm ^-1 ; ¹ H NMR (CDCl ₃ , 300 MHz) 8.50 (d, J = 1.7 Hz, 1H), 8.44 (dd, J = 4.7, 1.5 H
z, 1H), 7.71 (dt, J = 7.8, 1.7 Hz, 1H), 7.28-7.24 (m, 1H), 6.80-6.70 (m, 1
H), 4.72 (dd, J = 7.9, 5.2 Hz, 1H), 3.85 (s, 6H), 3.21 (brs, 1H), 2.77-2.9
(m, 2H), 2.18-1.96 (m, 2H); ¹³ C NMR (CDCl ₃ , 75 MHz) 149.0, 148.6, 147.7, 147.4, 140.3, 134.0, 133.
8, 123.6, 120.2, 111.8, 111.4, 71.3, 56.0, 55.8, 40.7, 31.5; LRMS (ES ⁺ ) (M + H) ⁺ 274; HRMS (ES ⁺ ) (M + H) ⁺ Calculated: 274.1462, measured Value: 274.1443.

1- [2 (S)-(3,4,5-trimethoxyphenyl) -butyryl] -piperidine-2 (S) -carboxylic acid, 3- (3,4-dimethoxyphenyl) -1-pyridine -3-yl-propane-1 (R) -ol ester (AP 22542): alcohol 3 (600 mg, 2.20 mmol) in CH ₂ Cl ₂ (2.5 mL) at −10 ° C., acid AP 173
62 (882 mg, 2.42 mmol) and a solution of DMAP (2.41 mg, 1.98 mmol) in DCC (498
mg, 2.42 mmol). The mixture was heated to about 5 ° C. in one hour,
For another 16 hours. The reaction mixture was then diluted with EtOAc (3 mL), filtered, evaporated and the crude material was purified by flash chromatography (75% then 100%
EtOAc / Hexanes) gave the product (1.15 g, 85%) as a colorless foam.

TLC (EtOAc) Rf = 0.40; IR (neat) 2940, 1740, 1645, 1590, 1515, 1455, 1420, 1240, 1130, 1030 c
m ^-1 ; ¹ H NMR (CDCl ₃ , 300 MHz) 8.50 (dd, J = 4.6, 1.5 Hz, 1H), 8.42 (d, J = 1.7 H
z, 1H), 7.27 (d, J = 8.6 Hz, 1H), 7.19 (dd, J = 7.7, 4.7 Hz, 1H), 6.78 (d, J
= 7.7 Hz, 1H), 6.66-6.64 (m, 2H), 6.46 (s, 2H), 5.69 (dd, J = 7.7, 6.0 Hz,
1H), 5.47 (d, J = 4.3 Hz, 1H), 3.86-3.73 (m, 16H), 3.59 (t, J = 7.1 Hz, 1H),
2.72 (td, J = 13.2, 2.6 Hz, 1H), 2.60-2.38 (m, 2H), 2.30 (d, J = 12.4 Hz, 1
H), 2.16-2.02 (m, 2H), 1.99-1.90 (m, 1H), 1.79-1.57 (m, 4H), 1.46-1.37 (
m, 1H), 1.32-1.19 (m, 1H), 0.90 (t, J = 7.3 Hz, 3H); ¹³ C NMR (CDCl ₃ , 75 MHz) 172.6, 170.5, 153.3, 149.5, 149.0, 148.3, 147.
5, 136.9, 135.6, 135.3, 133.8, 1323.0, 123.6, 120.2, 111.7, 111.5, 105.1
, 73.6, 60.9, 56.1, 56.0, 52.0, 50.7, 43.5, 37.9, 31.1, 28.3, 26.7, 25.3
LRMS (ES ⁺ ) (M + H) ⁺ 621; HRMS (FAB) (M + H) ⁺ calculated: 621.3176, found: 621.3178.

[0229]

Embedded image

Reaction Formula 3 (R / S) -2- (3,4,5-trimethoxyphenyl) butyric acid: 3,4,5-trimethoxyphenylacetic acid (40.0 g) in THF (125 mL) at 0 ° C. , 176.8 mm
ol) in a 2N THF solution of sodium bis (trimethylsilyl) amide (181
mL, 362 mmol, Lancaster) was treated dropwise over 1 hour while maintaining the internal reaction temperature below 8 ° C. After 15 minutes, iodomethane (14.9 mL, 185.7 mmol) was added portionwise over 30 minutes while maintaining the internal reaction temperature at 6-8 ° C., and the resulting solution was allowed to warm to room temperature. After 2 hours, the mixture was poured into EtOAc (700 mL) and a 2.0N HCl solution was added.
(325 mL) was added in portions to acidify. The organic component was further washed with saturated sodium bisulfite solution (50 mL) and then with brine (2 × 50 mL), then dried over anhydrous Na ₂ SO ₄ and concentrated to give a waxy residue (43.8 g). Obtained. This crude product is treated with hot EtOAc / hexane
(30 mL / 30 mL) to give the product (37.1 g, 83%): mp 103-104 ° C
TLC (AcOH / EtOAc / hexane, 2:49:49) Rf = 0.50.

(S) -2- (3,4,5-Trimethoxyphenyl) butyric acid (4S): A solution of compound 4 (3.09 g, 12.15 mmol) in CH ₃ CN (130 mL) was added to (-)- Cinchonidine
(3.58 g, 12.15 mmol) and the mixture was heated at reflux. The resulting homogeneous solution was slowly cooled to room temperature, accompanied by salt precipitation. After 1 hour at room temperature, the solution was cooled to 0 ° C. for 30 minutes, then the solution was filtered to give 4.05 g of a chalky colorless solid. The operation of recrystallization of this solid was repeated four more times using about 20 mL of CH ₃ CN per gram of salt. 5
The diastereomeric salt (1.64 g) isolated by the second crystallization was added to EtOAc (100 mL).
And treated with 10% aqueous HCl (10 mL). Then the organic phase was washed with water (2 × 15 m
L), then washed with brine (10 mL), dried over anhydrous MgSO _4, and concentrated to the product (0.
75 g, 24%) was converted to a colorless solid (Chiralcel OD HPLC, 1: 5: 94 formic acid / i-PrOH / hexane, 91% ee, retention time: 19.6 min for R-enantiomer, desired S-enantiomer) Body
22.1 min).

Mp 84-85 ° C. (99.1% ee material); [a] 22D + 54.8 (c = 1.07, MeOH, 30 min, 99.1% ee material); UV (MeOH) 1max 270 (e 895), 232 ( e 7,440), 207 (e 40,994) nm; ¹ H NMR (DMSO-d6, 300 MHz) 6.34 (s, 2H), 3.52 (s, 6H), 3.40 (s, 3H), 3.
11 (t, J = 7.6 Hz, 1H), 1.76-1.64 (m, 1H), 1.46-1.36 (m, 1H), 0.60 (t, J = 7
.3 Hz, 3H); ¹ H NMR (CD ₃ OD, 300 MHz) 6.78 (s, 2H), 4.00 (s, 6H), 3.90 (s, 3H), 3.55
(t, J = 7.7 Hz, 1H), 2.24-2.12 (m, 1H), 1.97-1.83 (m, 1H), 1.07 (t, J = 7.3
Hz, 3H); ¹³ C NMR (DMSO-d6, 75 MHz) 175.1, 153.1, 136.9, 135.8, 105.4, 60.3, 56.
2, 53.1, 26.7, 12.4; ¹³ C NMR (CD ₃ OD, 75 MHz) 178.1, 154.9, 138.7, 137.4, 106.8, 61.5, 57.0,
55.3, 28.3, 12.9; HRMS ( FAB): (MH) - Calculated: 253.1076, Found: 253.1063; Elemental analysis: C ₁₃ H ₁₈ O ₅ Calculated: C, 61.41; H, 7.13; Found: C, 61.47; H,
7.20.

[S- (R ^* , R ^* )]-1- [1-oxo-2- (3,4,5-trimethoxyphenyl) butyl] -2-piperidinecarboxylic acid (AP 17362): CH ₂ A solution of compound 5 (0.75 g, 2.95 mmol) in Cl ₂ (15 mL) was treated with methyl-L-pipecolate hydrochloride (0.539 g, 3.00 mmol) followed by 2-chloro-1-methylpyridinium iodide ( 0.958 g, 3.75 mmol) and triethylamine (1.25 mL, 8.95 mm
ol). After stirring the reaction mixture for 3.5 h, the solution was diluted with EtOAc (100 mL), water (15 mL), 5% aqueous citric acid (25 mL), saturated Na ₂ CO ₃ solution (10 mL), water
(15 mL), and finally washed with brine (15 mL). Drying the organic phase over MgSO ₄ ,
Concentration gave a yellow oil, which was dissolved in MeOH (14 mL). This methanol solution was treated with water (1 mL) and then with lithium hydroxide monohydrate (0.620 g, 14.78 mmol). After 4 hours, the mixture was diluted with EtOAc (100 mL) and washed with saturated NaHCO ₃ solution (3 × 40 mL), then with water (20 mL). Combine the aqueous portions and carefully add 10% aqueous HCl
Acidified to about pH 3. After the resulting suspension was extracted with EtOAc (2 × 75 mL), the extract was washed with water (2 × 25 mL), brine (20 mL), dried over MgSO ₄ and concentrated to dryness . The obtained solid was dissolved in a refluxing EtOAc (75 mL) solution and slowly cooled to room temperature. The precipitated crystalline substance was separated by filtration and air-dried. de,
The retention time was 40.0 minutes for the SR-diastereomer and 43 for the target SS-diastereomer.
0.0 min, RR-diastereomer 46.5 min, RS-diastereomer 67.5 min).

Mp 173.5-174 ° C .; [a] 22D +10.9 (c = 1.01, DMSO, 30 min); UV (MeOH) 1max 270 (e 990), 232 (e 11,161), 207 (e 49,079) nm; ¹ H NMR (DMSO-d6, 300 MHz) 6.55 (s, 2H), 5.13 (d, J = 4.4 Hz, 1H), 3.85-3
.64 (m, 11H), 2.77-2.70 (m, 1H), 2.12 (d, J = 13.4 Hz, 1H), 1.99-1.85 (m,
1H), 1.65-1.55 (m, 4H), 1.38-1.18 (m, 2H), 0.84 (t, J = 7.2 Hz, 3H); ¹ H NMR (CD ₃ OD, 300 MHz) 6.74 (s, 2H) , 5.43 (d, J = 4.0 Hz, 1H), 4.13-3.8
3 (m, 11H), 3.03 (td, J = 13.5, 3.0 Hz, 1H), 2.44 (d, J = 13.8 Hz, 1H), 2.24
-2.14 (m, 1H), 1.90-1.40 (m, 6H), 1.09 (t, J = 7.3 Hz, 3H); ¹³ C NMR (DMSO-d6, 75 MHz) 172.9, 172.2, 153.0, 136.2, 105.4, 60.2, 56.
2, 56.0, 51.8, 49.4, 43.1, 28.5, 26.8, 25.3, 21.0, 12.8; ¹³ C NMR (CD ₃ OD, 75 MHz) 175.4, 174.5, 154.9, 137.5, 106.8, 61.5, 57.1,
53.9, 52.1, 45.2, 29.9, 28.2, 26.8, 22.3, 13.2; HRMS (FAB): (MH) - Calculated: 364.1760, Found: 364.1774; Elemental analysis: C ₁₉ H ₂₇ O ₆ N Calculated: C, , 62.45; H, 7.45; N, 3.83; Found: C, 62.32; H, 7.61; N, 3.88.

Example 3 Condition Preserving Domain F (36M) FKBP; Study with hGH To test whether F (36M) can function as a condition preserving domain that allows for regulated secretion of fusion heterologous proteins. A fusion protein having the design shown in FIG. 2B was prepared. This fusion protein contains a signal sequence from the human growth hormone (hGH) gene, four copies of the F (36M) domain, a furin cleavage sequence from human stromelysin 3, and a coding sequence from the mature hGH protein. . The resulting fusion protein essentially simply contains multiple F (36M) domains and a furin cleavage signal inserted at the cleavage site between the signal sequence and the mature hGH peptide sequence. Since the furin recognition sequence is N-terminal to the cleavage site, the recognition sequence is constructed such that appropriate cleavage produces the same hGH amino acid sequence as would be produced by natural cleavage of its own signal sequence. Can be located.

A vector driving the expression of this fusion protein under the control of a potent constitutive enhancer from CMV was transiently transfected into HT1080 cells of a human fibrosarcoma cell line. After incubating the cells overnight in the absence of ligand,
The medium was rinsed and fresh medium with or without ligand was added.
Two hours later, the amount of hGH secreted into the medium was determined by radioimmunoassay. As shown in FIG. 3A, the amount of hGH secreted was low in the absence of ligand. In contrast, hGH secretion was several hundred times greater in the presence of ligand. this is,
It demonstrates that F (36M) can act as a condition-retaining domain when fused to a heterologous protein.

Next, the F (36M) -hGH expression vector was stably transfected into HT1080 cells to generate a cell line. For comparison, the native hGH gene driven by the same CMV enhancer was also stably transfected into cells. Expression of a selectable marker from the same transcript as the wt hGH or F (36M) -hGH fusion protein by using an internal ribosome transduction signal to allow an initial assessment of any potential toxic effects of the retained fusion protein . Equal numbers of clones were obtained. This suggests that there was no toxic effect of the fusion protein.

A pool of clones stably transfected with the F (36M) -hGH fusion protein (HT88 pool) was analyzed as described for transiently transfected cells. As shown in FIG. 3B, again hGH secretion is very low in the absence of ligand, and is induced hundreds of times by incubation for 2 hours with ligand. To determine the constitutive hGH secretion rate, the amount of hGH secreted in the presence of ligand was measured from cells previously exposed to ligand for 15 hours. As shown in column 3, the constitutive secretion rate from the HT88 cell line was very similar to the secretion rate from the HT89 cell line stably transfected with the wild-type hGH protein (column 4). That is, in the presence of ligand, the F (36M) domain has no detectable "retention" activity. In addition, this indicates that in the absence of ligand, the fusion protein accumulates at levels about 10-fold higher than those seen when secretion is not disrupted. The steady-state levels of this stored F (36M) fusion protein can persist for months in cell lines without apparent toxic effects.

Example 4: Localization / cleavage of the fusion protein To analyze the localization of the fusion protein, the EGFP coding sequence was incorporated into the fusion protein, as shown in Figure 2C. In cells stably transfected with this fusion protein in the absence of ligand, the fusion protein appeared as a large green spot concentrated at multiple points in the perinuclear space. Colocalization experiments showed that this fusion protein was as expected (J. Rothman, data not shown),
Demonstrate that they are assembled and retained inside the ER. Upon addition of the ligand, the assemblage disperses over the next 15-60 minutes. This disassembly occurs simultaneously with the appearance of the hGH protein in the cell supernatant. As described for the HT88 cell line, the ligand induces a hundred-fold increase in hGH (data not shown).

To further analyze the status of the fusion protein, cell lysates and supernatants were prepared from HT88 cells incubated for 2 hours in the presence or absence of ligand. These samples were then immunoblotted with anti-hGH and anti-FKBP antibodies. As shown in FIG. 4, in the absence of ligand, a band of about 75 kDa size
(This corresponds to the expected size of the F (36M) -hGH fusion protein) is detected in the lysate (column 1), but in the supernatant of unstimulated cells for both anti-hGH and anti-FKBP antibodies. Not detected (column 3). In cells stimulated with the ligand for 2 hours, very little fusion protein is detected in the cell lysate, but instead the cleaved protein is detected in the supernatant. Anti-hGH blots co-migrate with purified recombinant hGH (row 7).
) Indicates the presence of a 222 kDa size protein (row 6). The anti-FKBP blot shows the presence of a 53 kDa protein that is around the expected size of the rest of the fusion protein (F (36M) -FCS). Together, these results indicate that the F (36M) -hGH fusion protein is actually retained in the ER in the absence of ligand, released by interaction with the ligand, and subsequently cleaved at the appropriate position, It shows that secretion of the F (36M) -FCS portion and the complete hGH protein occurs.

Example 5 Dose Response and Kinetics of hGH Secretion The amount of hGH secreted from HT88 cells in response to a ligand is dose dependent (FIG. 5A). Peak levels of secretion occur at about 2-3 μM AP21998, and half-maximal secretion occurs at 600 nM.

To determine the kinetics of secretion, after stimulating the cells with the ligand, an aliquot of medium is taken at various time points and the accumulation of hGH in the supernatant is measured. Following the addition of saturating levels of ligand, low levels of hGH are detected in the supernatant within minutes, with a peak rate of secretion occurring between 20-30 minutes (FIG. 5B). This corresponds to the amount of time required for the newly synthesized protein to be secreted. After massive release of the stored fusion protein, the secretion rate decreases rapidly.

To further examine the kinetics of secretion in response to ligand, media was harvested at hourly intervals after cells were incubated overnight in the presence or absence of ligand. Cells were washed extensively between time points and the media was replaced with media with or without ligand as indicated. FIG. 6A (Group A) shows the constitutive secretion rate from cells. In group B, a large release is observed since the cells were not previously exposed to the ligand. However, once the ligand has been washed away, the hGH secretion rate sharply decreases and returns to a low basal rate within 2 hours. Group C shows that when cells are exposed to ligand after massive release, the secretion rate within 2 hours is increased by constitutively producing cells (A
Group) speed.

The constitutive hGH production rate was only 75 ng / 10 ⁶ cells / hr, whereas 1250 ng / 10 ⁶ cells were released in the first hour after emptying the reservoir, Refilling things should take some time. As shown in FIG. 6B, the stored hG
When H is released by incubation with the highest concentration of ligand, the magnitude of the second burst is comparable to that of the first (or more, since the cell number has increased in that time). 8-24 to refill the stock
Takes time. Therefore, in order to achieve consistent rapid and pulsatile secretions, the reservoir must not be completely empty. As shown in FIG. 6C, when a ligand concentration lower than the maximum concentration is added (eg, 250 or 500 nM), an equivalent amount of hGH can be secreted after 4 hours.

The degree of aggregation increases as the number of F (36M) domains increases. To test whether the degree of retention can be manipulated, constructs containing 2, 3 or 6 F (36M) domains were fused to hGH, creating a stable cell line and And hGH secreted in the absence. As shown in FIG. 7, basal secretion in the absence of ligand increases as the number of F (36M) domains decreases. This probably reflects a reduction in the size of the assembly that displaces the monomeric fusion protein from retention. Increased “leakyness” of fusion protein secretion is also reflected as a decrease in the amount of stored fusion protein, and thus a reduced amount of released protein following ligand addition. This could be exploited to provide a transient high level evoked secretion against a relatively high constitutive basal secretion backdrop. Such a situation may be particularly desirable in the case of insulin production for the treatment of type 1 diabetes.

Example 6: To test whether the regulated insulin secretory condition retention domain, F (36M), can also be used to enable regulated secretion of insulin, the design shown in FIG. A fusion protein was made. This fusion protein has the F (36M) -hGH described in Example X except that the mature hGH coding sequence has been replaced with a coding sequence from the adult human insulin gene.
Similar to a fusion protein. Normally, in (Langerhans) islet cells, proinsulin is processed exclusively by endopeptidases expressed in neuroendocrine cells into mature active A and B chain complexes. Therefore, in order to be able to properly process insulin in non-endocrine cells, mutations were introduced into the BC and CA bonds which would allow the treatment of ubiquitous proteases with furin (Groskreutz et al. ., J. Biol. Chem. 269: 6241-6245, 1994). Also,
A third mutation, in which amino acid 10 (histidine) of the B chain was mutated to aspartic acid, was also introduced to increase protein stability (Groskreutz et al., J. Bi.
ol. Chem. 269: 6241-6245, 1994).

The vector driving expression of this F (36M) -insulin fusion protein (F (36M) 4-hIn-m3) was transiently transfected into HT1080 cells. For comparison, vectors that drive expression of the insulin protein alone, with or without the three mutations (hIn-m3) (hIn-wt), were also transfected. After overnight incubation of the cells in the absence of ligand, the medium is washed away and the monomeric ligand
Fresh medium with or without increasing concentrations of AP21998 was added. After 3 hours, the amount of insulin secreted into the medium was determined by ELISA using an assay that recognizes the epitope within the C-peptide (ALPCO). As shown in FIG. 8, fusion of the F (36M) domain to insulin results in suppression of its secretion in the absence of monomeric ligand. Also, in the presence of monomer, secretion is induced in a dose-dependent manner.

Example 7: Regulated expression of membrane-tethered proteins To determine whether the CRD F (36M) can be used to regulate the surface expression of membrane-tethered proteins , Four or six copies of F (36M) were fused to the extracellular and transmembrane portion of the low affinity nerve growth factor receptor (LNGFR; FIG. 3E). Unlike the hGH and insulin fusions described in Examples 3 and 6, in which the F (36M) domain is initially expressed as part of a soluble protein localized in the lumen of the ER, these fusion proteins , The F (36M) domain should be localized in the cytoplasm and tethered to the plasma membrane. Surface expression was measured using anti-LNGFR antibody (Chromaprobe, Mountain View
, CA) was evaluated by FACS analysis.

As shown in FIG. 9, transfection into HT1080 cells detects two peaks corresponding to low and high levels of LNGFR surface expression in each fusion protein in the absence of monomer . Relatively high levels of surface expression in the absence of monomers indicate that the retention activity of the F (36M) domain is in solution when the fusion protein is membrane-tethered. In comparison, it suggests that it is not as powerful. This may reflect the presence of physical constraints that prevent the formation of higher oligomers. However, these results indicate that as the number of F (36M) domains increases, the retention activity of F (36M) domains clearly increases. Furthermore, in the presence of monomeric ligands, surface expression is significantly increased in all cases. Thus, the F (36M) domain can also be used to conditionally elicit surface expression of membrane-tethered proteins.

Example 8 Construction and Testing of a Construct for Conditional Secretion of hGH Using Rat Retinol-Binding Protein as CRD ER (Melhus et al., J. Biol. Chem., 1992, vol.
267, 12036-12041) and thus are suitable candidates for use as CRD. We have constructed a construct to test whether rRBP can be used to obtain conditional secretion of the target protein human growth hormone (hGH) in response to retinoid ligands. The overall structure of this construct is shown below.

[0251]

Embedded image

This construct comprises an rRBP cDNA containing an authentic signal sequence (SS), followed by a furin cleavage site (FCS) from stromelysin E (amino acid sequence: SARNRQKR), followed by the mature 191 amino acids of hGH. Includes an in-frame stop codon after the cDNA coding sequence (deleting the signal sequence). The stromelysin E cleavage site was chosen because it is of human origin (and is therefore expected to have minimal immunogenicity in future human therapeutic applications) and the P1 ′ residue-cleavage The residue following the site-is known to be recognized by furin in the context of fusion to a protein that is Phe as well as hGH (for review, see Denault and Leduc, FEBS Lett. 1996
379: 113-116). With the exception of an additional threonine codon between rRBP and FCS to accommodate the SpeI site, all bonds between the various sequence motifs and domains are direct bonds and do not contain additional sequences. This expression cassette is called pC4EN
And placed the expression under the control of the strong hCMV immediate-early promoter and enhancer.

The DNA fragment encompassing the rRBP cDNA was purified from rat liver poly A + RNA using Clontech First Strand Kit with random primers (obtained from Clontech, catalog # 67).
RT-PCR from 10-1) followed by PCR under conventional amplification conditions using primers RBP-5 '(263) and RBP-3' (264). Purify the PCR product, EcoRI and SpeI
Digested. A second DNA fragment encoding FCS and the mature hGH coding sequence was obtained by PCR amplification from the hGH cDNA expression vector Z12IHB. The PCR primers used were FCS-hGH-5 '(265) and hGH-3' (266). Primer FCS-hGH-
5 '(265) contains an additional sequence encoding FCS. The PCR product was purified and digested with SpeI and BamHI. The two types of DNA fragments are then ligated three-way
ligation) into the EcoRI-BamHI open-chain pC4EN
pC4EN-rRBP-hGH was produced. Positive clones were completely sequenced to check that no errors were incorporated during cloning.

This construct contains restriction sites that can be used to add additional modules to the expressed fusion protein. Therefore, by excising the existing SpeI-AflII fragment and cloning in an appropriate SpeI-AflII compatible oligonucleotide pair,
The stromelysin E FCS can be replaced with another cleavage site. Epitope tags can be added to rRBP upstream of the FCS to allow immunochemical tracking of the rRBP module inside the cell. Another target protein can be cloned as a SpeI-XmaI fragment (the presence of another BamHI site in the rRBP coding sequence eliminates the use of a 3 ′ BamHI site). Another CRD can be cloned as an EcoRI-SpeI fragment instead of rRBP.

An additional construct of particular interest is one that incorporates multiple repeated copies of rRBP. These re-amplify pC4EN-rRBP-hGH using primers 5′-RBP-Xba and 3′-RBP-Spe, and mature rRBP sequence flanked by SpeI compatible 5′XbaI and 3′SpeI sites ( (Without a signal sequence). Purify the PCR product,
Digested with XbaI and SpeI, cloned into SpeI open chain pC4EN-rRBP-hGH, pC4E
Generates N- (rRBPx2) -hGH. Similar techniques can be used to prepare constructs encoding higher order rRBP concatemers.

PCR Primer RBP-5 '(263) 5'CGTACgaattcCAGAAGCGCGTATGGAGTGGGTGTGGGCGCTCGTGCTG RBP-3' (264) 5'GCATGactagtCAAACTGTTTCTTGAGGGTCTGCTTTGACAG FCS-hGH-5 '(265)
hGH-3 '(266) 5'GCTCAggatccCGGGCTAGAAGCCACAGCTGCCCTCCACAGAGCG 5'-RBP-Xba 5'TCAGCtctagaGAGCGCGACTGCAGGGTGAGC 3'-RBP-Spe 5'GAAGCactagtCAAACTGTTTCTTGAGGGTCTG Expression cassette sequence is as follows.

[0257]

Embedded image

[0258]

Embedded image

[0259]

Embedded image

[0260]

Embedded image

The sequence of the encoded protein is as follows: MEWVWALVLLAALGGGSAERDCRVSSFRVKENFDKARFSGLWYAIAKKDPEGLFLQDNIIAEFSVDEKGHMS
ATAKGRVRLLSNWEVCADMVGTFTDTEDPAKFKMKYWGVASFLQRGNDDHWIIDTDYDTFALQYSCRLQNLD
GTCADSYSFVFSRDPNGLTPETRRLVRQRQEELCLERQYRWIEHNGYCQSRPSRNSLTSARNRQKRFPTIPL
SRPFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRI
SLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNS
The nucleotide sequence of the HNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF expression cassette is as follows: ATGGAGTGGGTGTGGGCGCTCGTGCTGCTGGCGGCTCTGGGAGGCGGCAGCGCCGAGCGCGACTGCAGGGTG
AGCAGCTTCAGAGTCAAGGAGAACTTCGACAAGGCTCGTTTCTCTGGGCTCTGGTATGCCATCGCCAAAAAG
GATCCCGAGGGTCTCTTTTTGCAAGACAACATCATCGCTGAGTTTTCTGTCGACGAGAAGGGTCATATGAGC
GCTACAGCCAAGGGACGAGTCCGTCTTCTGAGCAACTGGGAAGTGTGTGCAGACATGGTGGGCACTTTCACA
GATACAGAAGATCCTGCCAAGTTCAAGATGAAGTACTGGGGTGTAGCCTCCTTTCTCCAGCGAGGAAACGAT
GACCACTGGATCATCGATACGGACTACGACACCTTCGCTCTGCAGTATTCCTGCCGCCTGCAGAATCTGGAT
GGCACCTGTGCAGACAGCTACTCCTTTGTGTTTTCTCGTGACCCCAATGGCCTGACCCCGGAGACACGGAGG
CTGGTGAGGCAGCGACAGGAGGAGCTGTGCCTAGAGAGGCAGTACAGATGGATCGAGCACAATGGTTACTGT
CAAAGCAGACCCTCAAGAAACAGTTTGACTAGTGCTAGAAACCGTCAGAAGAGATTCCCAACCATTCCCTTA
AGCAGGCCTTTTGACAACGCTATGCTCCGCGCCCATCGTCTGCACCAGCTGGCCTTTGACACCTACCAGGAG
TTTGAAGAAGCCTATATCCCAAAGGAACAGAAGTATTCATTCCTGCAGAACCCCCAGACCTCCCTCTGTTTC
TCAGAGTCTATTCCGACACCCTCCAACAGGGAGGAAACACAACAGAAATCCAACCTAGAGCTGCTCCGCATC
TCCCTGCTGCTCATCCAGTCGTGGCTGGAGCCCGTGCAGTTCCTCAGGAGTGTCTTCGCCAACAGCCTGGTG
TACGGCGCCTCTGACAGCAACGTCTATGACCTCCTAAAGGACCTAGAGGAAGGCATCCAAACGCTGATGGGG
AGGCTGGAAGATGGCAGCCCCCGGACTGGGCAGATCTTCAAGCAGACCTACAGCAAGTTCGACACAAACTCA
CACAACGATGACGCACTACTCAAGAACTACGGGCTGCTCTACTGCTTCAGGAAGGACATGGACAAGGTCGAG
ACATTCCTGCGCATCGTGCAGTGCCGCTCTGTGGAGGGCAGCTGTGGCTTCTAG To test whether fusion to rRBP can result in ligand-dependent hGH secretion prevention, pC4EN-rRBP-hGH, pC4EN- (rRBPx2) -hGH and their derivatives in the human fibrosarcoma cell line HT1080. Are transfected temporarily using standard methods (e.g., Rivera et al., Nature Med. 2: 1028-1032, 1996; Amara et a.
l., PNAS 94: 10618-10623, 1997). After overnight incubation, the medium is removed and fresh medium without drug or containing retinol at various concentrations is added. After further incubation for 2 to 24 hours, the amount of hGH secreted into the medium is determined by radioimmunoassay (Rivera et al., Nature
Med. 2: 1028-1032, 1996).

As described by Melhus et al. (J. Biol. Chem. 1992, vol. 267, 12036-12041), an important feature of these experiments is the use of defatted serum in the medium. This is because untreated serum contains significant amounts of retinoids that may cause rRBP secretion in the absence of externally added retinol. Methods for preparing defatted serum are known (Rothblat et al., In Vitro 1976 vol. 12, 554-557).

The increase in hGH secretion when retinol was added at increasing concentrations was
This would indicate that rRBP acts as a CRD and retains hGH in the secretory compartment until retinol is added. Experiments to identify the best form of this system include engineering the multimers of rRBP in an attempt to increase the retention effect and testing a variety of different retinol analogs for activity. Further experiments confirming the subcellular location of the rRBP fusion include immunocytochemical subcellular localization of the components of the construct before and after the addition of retinoids, for example, using anti-hGH or anti-rRBP antibodies. Including.

Example 9 Physiological Effects of In Vivo Modulated Insulin Secretion To test whether the system of the present invention can be used to regulate in vivo insulin secretion and achieve changes in serum glucose levels, × 10e7 HT101-10p cells were implanted intramuscularly into male nu / nu mice. HT101-10p cells were generated by stably transfecting HT1080 cells with a vector that drives expression of the F (36M) 4-hIn-m3 fusion protein. Mice were treated with 300 mg / kg streptozotocin (STZ) two days prior to hyperglycemia.

As shown in FIG. 11a, STZ treatment increases serum glucose levels from about 100 mg / dl found in mice not treated with STZ to about 350 mg / dl. Approximately 1 hour after transplanting the cells, the animals are dosed with vehicle or the indicated dose of intravenous AP22542.
(An analog of AP21998). Two hours later, a serum sample was collected and insulin
(Ultrasensitive human insulin specific RIA, Linco) and glucose (Sigma). As shown in FIG. 11a, vehicle or low dose of AP22542 (1 mg / kg)
Does not increase serum insulin levels above the lower limit of detection and there is no change in serum glucose levels. In contrast, 10 mg /
In animals treated with kg AP22542, serum insulin levels increase to about 200 pM and serum glucose levels drop to about 75 mg / dl.

To examine the kinetics of this ligand-induced decrease in serum glucose concentration, 2 ×
STeZ treated mice implanted with 10e7 HT101-10p cells received 30 mg / kg AP22542
Was administered only once intravenously. Serum glucose concentrations were measured at various times from 5 minutes to 10 hours. As shown in FIG. 11b, at 5 minutes and 15 minutes after administration of AP22542, serum glucose levels are indistinguishable from animals treated with vehicle. However, within 30 minutes there is a significant drop in serum glucose concentration and by 2 hours the serum glucose concentration has decreased from an initial concentration close to 500 mg / dl to 50 mg / dl. This effect was temporary, with serum glucose levels rising to 350 mg / dl within 5 hours and 6-
After 10 hours it returns to basal level. Since insulin secretion is dependent on the presence of the drug, administration of smaller amounts of AP22542 or shorter half-life ligands should result in even more transient production of secreted proteins and consequent physiological effects. . Conversely, administration of higher amounts of AP22542 or longer half-life ligands should result in longer secreted proteins.

[Brief description of the drawings]

FIG. 1. Overall design of the fusion protein used in the present invention: from the amino-terminus to the carboxy-terminus, including a secretory signal sequence, a “condition retention domain”, a protease cleavage site, and a secretory target protein of interest.

FIG. 2: Constructs used to make CRD-containing fusion proteins: FIG. 2A: F36M-EGFP fusion protein; FIG. 2B: F36M-hGH fusion protein; FIG. 2C: EGFP-F36M-hGH fusion protein; FIG. 2D: F36M- Insulin fusion protein; FIG. 2E: LNGFR-F36M fusion protein.

FIG. 3. Ligand-dependent secretion of hGH. Levels of hGH secreted into the medium of transiently transfected (FIG. 3A) or stably transfected (FIG. 3B) HT1080 cells in the absence or presence of ligand.

FIG. 4. Immunoblot of cell lysates and supernatants prepared from HT88 cells incubated for 2 hours in the presence or absence of ligand. Anti-hGH and anti-sample
Immunoblotting was performed with the FKBP antibody.

FIG. 5. Dose dependence of hGH secretion from HT88 cells in response to ligand (FIG. 5A). Time course of accumulation of secreted hGH in the medium (FIG. 5B).

FIG. 6. Rate of secretion in response to ligand. FIG. 6A, group A: Constitutive secretion rate from cells.
Group B: secretion from cells not previously exposed to ligand. Group C: cells exposed to ligand after massive release of hGH. FIG. 6B shows the amount of hGH released by incubation with the highest concentration of ligand. FIG. 6C shows the amount of hGH secreted after the addition of a sub-maximal concentration of ligand.

FIG. 7 shows the effect of changes in the number of CRDs on hGH secretion. HGH secretion was measured after addition of ligand in cell lines expressing fusion proteins containing different numbers of CRDs.

FIG. 8. Regulated secretion of insulin. The level of insulin secretion was measured in transiently transfected HT1080 cells treated with different concentrations of AP21998.

FIG. 9. Regulated expression of membrane-tethered proteins. Three, four or six copies of F (36M) were fused to the extracellular and transmembrane portion of the low affinity nerve growth factor receptor (LNGFR; FIG. 3E). Surface expression was assessed by FACS analysis using an anti-LNGFR antibody.

FIG. 10. Constructs useful for screening new CRDs. A. The candidate DNA sequence is
Can be cloned into a polylinker to identify the CRD that triggers ligand-dependent secretion of the CRD. B. Candidate DNA sequences can be cloned into a polylinker to identify CRDs that have triggered ligand-dependent localization of p75.
C. A construct used in a "two-hybrid" type assay in which a fusion protein containing a CRD causes association of a DNA binding domain with a transcription activation domain to induce transcription.

FIG. 11. Ligand-mediated regulation of insulin and glucose concentrations in vivo. (A) FKBP (F36
Insulin and glucose concentrations were measured in mice implanted with the construct containing (M) -insulin before and after administration of AP22542. (B) Serum glucose levels were measured at various times after AP22542 administration in mice transplanted with a construct containing FKBP (F36M) -insulin.

[Procedure amendment]

[Submission date] April 26, 2002 (2002. 4.26)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0006

[Correction method] Change

[Contents of correction]

In addition, the fusion protein further includes an enzyme cleavage site so that the portion of the fusion protein containing the CRD can be cleaved from the portion of the fusion protein containing a peptide sequence heterologous to the CRD. Is preferred in many embodiments. Preferably, the enzyme cleavage site comprises a peptide sequence that is recognized by a trans Golgi-specific endoprotease, such as furin. For example, the cleavage site for furin is given by the peptide sequence SARNRQKR (SEQ ID NO: 1) .

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0077

[Correction method] Change

[Contents of correction]

A typical cleavage enzyme is furin, also known as PACE. Furin is a member of the KEX2 / subtilisin family of proprotein convertases, enzymes that convert proproteins and prohormones into their active forms (Nakayama Kazuhisa, Biochem. J. (1997) 327: 625-635). It is a protein present in the trans-Golgi apparatus, but, like many Golgi proteins such as TGN38, circulates constitutively between the cell surface and the TGN (trans-Golgi network). Furin has a ubiquitous (ubiquitous) tissue distribution and its substrates are numerous and diverse. However, almost all share a common cleavage sequence, RX (K / R) R ( SEQ ID NO: 2) . The following proteins are substrates for furin:
Human proneurotrophin-3 (MSMRVRR, SEQ ID NO: 3) , human proinsulin-like growth factor I (KPAKSAR, SEQ ID NO: 4 ), human proparathyroid hormone (KSVK)
KR, SEQ ID NO: 5 ), human stromelysin-3 (ARNRQKR, SEQ ID NO: 6 ). Furin is also a human insulin proreceptor (RPSRKRR, SEQ ID NO: 7 ) and human
It can also cleave membrane-bound substrates such as hepatocyte growth factor pro-receptor (TEKRKKR, SEQ ID NO: 8 ). Cleavage sites from any furin substrate can be used in the fusion proteins of the invention. In some cases, the cleavage site will be a non-natural peptide sequence that includes a common furin cleavage sequence. One particular advantage of utilizing furin as a cleavage enzyme is that its recognition sequence is located exclusively N-terminal to its cleavage site. This allows the portion of the protein that encodes the target protein to be released from cells that are not modified by the presence of additional amino acids.

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] 0079

[Correction method] Change

[Contents of correction]

In addition, any mammalian protease with a specific cleavage sequence, such as subtilisin, could be used to cleave a fusion protein of the invention once it was targeted to a desired location in the cell. For example, subtilisin could be targeted to TGN by fusing it to a localization sequence from a resident Golgi protein such as TGN38. Alternatively, YKGL (SEQ ID NO: 9) and Se
Motifs known to target furin to TGN, including the r-containing cluster SDSEEDE (SEQ ID NO: 10) , may be sufficient to target cytoplasmic proteases to the Golgi apparatus. The cells may be genetically engineered to express enzymes tailored to cleave sequences present only in the CRD-containing fusion protein.
For example, Ballinger et al. Report a mutant form of subtilisin in which the subtilisin enzyme has been engineered to acquire furin specificity (Ballinger MD, et al.
al. Biochemistry. 1996/10/22; 35 (42): 13579-85), Ballinger MD, et al. Bi
ochemistry. 1995/10/17; 34 (41): 13312-9).

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0088

[Correction method] Change

[Contents of correction]

[0088] Any of the lysosomal targeting signals described above can be used to target a CRD of the invention for disposal. For soluble proteins, the preferred method is to fuse a resident lysosomal protein containing a mannose-6-phosphate signal to the CRD. Examples of such proteins are the cysteine proteases of the cathepsin family: cathepsins B, D, H, L, S, C and K. Other lysosomal enzymes that can be used include carboxypeptidase, prolyl carboxypeptidase and deamidase (cathepsin A). In the case of a membrane-bound CRD, a preferred targeting sequence would be that present on a lysosomal membrane protein, such as a tyrosine-based internalization motif. These motifs are short linear extensions of several amino acids in the cytoplasmic region of the protein to be targeted.
Tyrosine-based motifs are critical in the sequence NPXY (SEQ ID NO: 11) or YXXO (SEQ ID NO: 12), where X is any amino acid and O is an amino acid with a bulky hydrophobic group. Mainly tyrosine residues. In many proteins, glycine in front of the tyrosine of the YXXO-type signal enhances the targeting of these proteins to the lysosome. The sequence used for some applications of the invention is Lamp-1, L
It may be from a protein such as AP (lysosomal acid phosphatase), CD63, Lamp-2 or CD3-γ, all of which are normally targeted to the lysosome. For further information on tyrosine-based sorting motifs, see, for example, Marks et al., Trends in Cell Biology, 7: 124-128, 1997.

[Procedure amendment 5]

[Document name to be amended] Statement

[Correction target item name]

[Correction method] Change

[Contents of correction]

VR65: TCCCGCACCTCTTCGGCCAGCGaaTTccAGAAGCGCGTAT (SEQ ID NO : 13) VR119: GACTCACTATAGGaCGcgTTCGAGCTCGCCCC (SEQ ID NO : 14) VR120: CATCATTTTGGCAAAGgATTCACTCCTCAGG (SEQ ID NO : 15) Was generally generated as a fragment containing a SpeI site, an in-frame stop codon, and a BamHI site. Chimeric proteins containing multiple (multiple) components were assembled by stepwise insertion of the XbaI-BamHI fragment into the SpeI-BamHI open-chain vector or insertion of the XbaI-SpeI fragment into the XbaI or SpeI open-chain vector.

[Procedure amendment 6]

[Document name to be amended] Statement

[Correction target item name]

[Correction method] Change

[Contents of correction]

VR1: GATGGAAAGAAAatgGATTCCTCCCGG (SEQ ID NO : 16) CF (36M) fusion protein (FIG. 3) (a) EGFP fusion The EGFP coding sequence was amplified from pEGFP-1 (Clontech) using oligos VR2 and VR3. The resulting fragment containing the upstream XbaI and downstream SpeI sites was inserted into pCGN, a derivative of pCGNN lacking the SV40 nuclear localization sequence, to create pCGN-EGFP.

[Procedure amendment 7]

[Document name to be amended] Statement

[Correction target item name]

[Correction method] Change

[Contents of correction]

VR2: tctagaGTGAGCAAGGGCGAGGAG (SEQ ID NO : 17) VR3: ggatccttaTTAACTAGTCTTGTACAGCTCGTCCATG (SEQ ID NO : 18) XbaI-SpeI fragment containing 3, 4 or 6 copies of F (36M) was converted to XbaI of pCGN-EGFP.
Inserted into the site, pCGN-F (36M) 3-EGFP, pCGN-F (36M) 4-EGFP, and pCGN-F (36M) 6
An F (36) M-EGFD fusion was prepared by preparing -EGFP.

[Procedure amendment 8]

[Document name to be amended] Statement

[Correction target item name] 0200

[Correction method] Change

[Contents of correction]

VR109: aagcttACCACTCAGGGTCCTGTGG (SEQ ID NO : 19) VR110: gaattcGTGGCAACTTCCA (SEQ ID NO : 20) To construct the hGH fusion protein, Z12I-hGH-2 was mutagenized with oligos VR185, VR186, and VR187 and i) Create an EcoRI site 32 bp upstream of ATG, ii) an XbaI site immediately following the last amino acid of the signal sequence, and iii) a SpeI site immediately following the last amino acid of hGH.

[Procedure amendment 9]

[Document name to be amended] Statement

[Correction target item name] 0201

[Correction method] Change

[Contents of correction]

VR185: cacaggaccctGAATTCtaagcttgtggc (SEQ ID NO : 21) VR186: ATAAGGGAATGGTtctagaGGCACTGCCCT (SEQ ID NO : 22) VR187: atgccacccgggactagtGAAGCCACAGCTG (SEQ ID NO : 23) hGH generated. Next, the XbaI-BamHI fragment of pC4S1-hGH was replaced with an XbaI-SpeI fragment containing 2, 3, 4 or 6 copies of F (36M) and a SpeI-BamHI fragment encoding the furin cleavage site-hGH fusion. Then pC4S1-F (36M) -F
A CS-hGH fusion was generated.

[Procedure amendment 10]

[Document name to be amended] Statement

[Correction target item name] 0202

[Correction method] Change

[Contents of correction]

[0202] The SpeI-BamHI fragment encoding the FCS-hGH fusion protein was constructed using the oligo VR of the hGH cDNA.
4 and formed by amplification with VR5. VR4: actagtGCTAGAAACCGTCAGAAGAGATTCCCAACCATTCCCTTAAGC (SEQ ID NO : 24) VR5: ggatcccgggCTAGAAGCCACAGCTGCCCTC (SEQ ID NO : 25) Downstream neo-resistance gene of encephalomyocarditis virus internal ribosome entry sequence (IRES
/ Neo; Amara et al., PNAS 94: 10618-23, 1997).
Insert into the appropriate SpeI-BamHI open chain vector, pC4S1-F (36M) -FCS-hGH / neo and pC4S1-F (36M) -FCS-hGH / neo
Form 4S1-hGH / neo vector.

[Procedure amendment 11]

[Document name to be amended] Statement

[Correction target item name] 0204

[Correction method] Change

[Contents of correction]

VR220: cGAATTCttctgccATGGCCCTGTGGATGCGC (SEQ ID NO : 26) VR221: cGGATCCgcaggctgcgtCTAGTTGCAGTAG (SEQ ID NO : 27) The SpeI-BamHI fragment encoding the furin cleavage sequence-insulin fusion protein is
Formed by RT-PCR amplification with oligos VR222 and VR221.

[Procedure amendment 12]

[Document name to be amended] Statement

[Correction target item name] 0205

[Correction method] Change

[Contents of correction]

VR222: cACTAGTGCTAGAAACCGTCAGAAGAGATTTGTGAACCAACACCTGTGCGGC (SEQ ID NO : 28) VR221: cGGATCCgcaggctgcgtCTAGTTGCAGTAG (SEQ ID NO : 27) Oligo VR223, VR224
, VR225, respectively, i) amino acid B10 was changed to Asp, ii) FCS was prepared for BC bond, and iii) FCS was prepared for CA bond.

[Procedure amendment 13]

[Document name to be amended] Statement

[Correction target item name] 0206

[Correction method] Change

[Contents of correction]

VR223: CCTGTGCGGCTCAgACCTGGTGGAAGC (SEQ ID NO : 29) VR224: CTTCTACACACCCAgGACCaagCGGGAGGCAGAGG (SEQ ID NO : 30) VR225: CCCTGGAGGGGTCCCgGCAGAAGCGTGTGGC (SEQ ID NO : 31) Mutation of pC4-hIn generated pC4-hIn-m3. The mutated FCS-insulin fusion was used to replace the FCS-hGH portion of the pC4S1-F (36M) -FCS-hGH fusion,
An F (36M) -FCS-hIn-m3 fusion was made.

[Procedure amendment 14]

[Document name to be amended] Statement

[Correction target item name] 0212

[Correction method] Change

[Contents of correction]

[0212] p65 transcriptional activation sequence contains a linear sequence below: CTGGGGGCCTTGCTTGGCAACAGCACAGACCCAGCTGTGTTCACAGACCTGGCATC CGTCGACAACTCCGAGTTTCAGCAGCTGCTGAACCAGGGCATACCTGTGGCCCCCC ACACAACTGAGCCCATGCTGATGGAGTACCCTGAGGCTATAACTCGCCTAGTGAC AGGGGCCCAGAGGCCCCCCGACCCAGCTCCTGCTCCACTGGGGGCCCCGGGGCTCC CCAATGGCCTCCTTTCAGGAGATGAAGACTTCTCCTCCATTGCGGACATGGACTTC TCAGCCCTGCTGAGTCAGATCAGCTCC. (SEQ ID NO: 32)

[Procedure amendment]

[Submission date] April 26, 2002 (2002. 4.26)

[Procedure amendment 15]

[Document name to be amended] Statement

[Correction target item name] 0252

[Correction method] Change

[Contents of correction]

This construct comprises a rRBP cDNA containing an intrinsic signal sequence (SS) followed by a furin cleavage site (FCS) from stromelysin E (amino acid sequence: SARNRQKR) (SEQ ID NO: 33).
Followed by a mature 191 amino acid cDNA coding sequence for hGH (deleting the signal sequence) followed by an in-frame stop codon. The stromelysin E cleavage site was selected because it is of human origin (and is therefore expected to have minimal immunogenicity in future human therapeutic applications) and has a P1 ′ residue-cleavage site. The residue following the site is known to be recognized by furin in the context of fusion to a protein that is Phe as well as hGH (for review, see Denault and Leduc, FEB
S Lett. 1996, 379: 113-116). With the exception of an additional threonine codon between rRBP and FCS to accommodate the SpeI site, all bonds between the various sequence motifs and domains are direct bonds and do not contain additional sequences. This expression cassette was cloned into the expression vector of pC4EN and placed under the control of the strong hCMV immediate early promoter and enhancer.

[Procedure amendment 16]

[Document name to be amended] Statement

[Correction target item name] 0256

[Correction method] Change

[Contents of correction]

PCR Primer RBP-5 ′ (263) 5′CGTACgaattcCAGAAGCGCGTATGGAGTGGGTGTGGGCGCTCGTGCTG (SEQ ID NO: 34)
RBP-3 '(264) 5' GCATGactagtCAAACTGTTTCTTGAGGGTCTGCTTTGACAG (SEQ ID NO: 35) FCS-h GH-5 '(2 6 5) 5' GCAACactagtGCTAGAAACCGTCAGAAGAGATTCCCAACCATTCCCTTAAGCAGGCCTTTTGACAACGC (SEQ ID NO: 36)
hGH-3 '(266) 5'GCTCAggatccCGGGCTAGAAGCCACAGCTGCCCTCCACAGAGCG (SEQ ID NO: 37)
5'-RBP-Xba 5'TCAGCtctagaGAGCGCGACTGCAGGGTGAGC (SEQ ID NO: 38) 3'-RBP-Spe 5'GAAGCactagtCAAACTGTTTCTTGAGGGTCTG (SEQ ID NO: 39) The sequence of the expression cassette is as follows (underlined parts indicate important restriction sites).

[Procedure amendment 17]

[Document name to be amended] Statement

[Correction target item name] 0260

[Correction method] Change

[Contents of correction]

[0260]

Embedded image

[Procedure amendment 18]

[Document name to be amended] Statement

[Correction target item name] 0261

[Correction method] Change

[Contents of correction]

The sequence of the encoded protein is as follows: MEWVWALVLLAALGGGSAERDCRVSSFRVKENFDKARFSGLWYAIAKKDPEGLFLQDNIIAEFSVDEKGHMS
ATAKGRVRLLSNWEVCADMVGTFTDTEDPAKFKMKYWGVASFLQRGNDDHWIIDTDYDTFALQYSCRLQNLD
GTCADSYSFVFSRDPNGLTPETRRLVRQRQEELCLERQYRWIEHNGYCQSRPSRNSLTSARNRQKRFPTIPL
SRPFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRI
SLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNS
The nucleotide sequence of the HNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF (SEQ ID NO: 41) expression cassette is as follows: ATGGAGTGGGTGTGGGCGCTCGTGCTGCTGGCGGCTCTGGGAGGCGGCAGCGCCGAGCGCGACTGCAGGGTG
AGCAGCTTCAGAGTCAAGGAGAACTTCGACAAGGCTCGTTTCTCTGGGCTCTGGTATGCCATCGCCAAAAAG
GATCCCGAGGGTCTCTTTTTGCAAGACAACATCATCGCTGAGTTTTCTGTCGACGAGAAGGGTCATATGAGC
GCTACAGCCAAGGGACGAGTCCGTCTTCTGAGCAACTGGGAAGTGTGTGCAGACATGGTGGGCACTTTCACA
GATACAGAAGATCCTGCCAAGTTCAAGATGAAGTACTGGGGTGTAGCCTCCTTTCTCCAGCGAGGAAACGAT
GACCACTGGATCATCGATACGGACTACGACACCTTCGCTCTGCAGTATTCCTGCCGCCTGCAGAATCTGGAT
GGCACCTGTGCAGACAGCTACTCCTTTGTGTTTTCTCGTGACCCCAATGGCCTGACCCCGGAGACACGGAGG
CTGGTGAGGCAGCGACAGGAGGAGCTGTGCCTAGAGAGGCAGTACAGATGGATCGAGCACAATGGTTACTGT
CAAAGCAGACCCTCAAGAAACAGTTTGACTAGTGCTAGAAACCGTCAGAAGAGATTCCCAACCATTCCCTTA
AGCAGGCCTTTTGACAACGCTATGCTCCGCGCCCATCGTCTGCACCAGCTGGCCTTTGACACCTACCAGGAG
TTTGAAGAAGCCTATATCCCAAAGGAACAGAAGTATTCATTCCTGCAGAACCCCCAGACCTCCCTCTGTTTC
TCAGAGTCTATTCCGACACCCTCCAACAGGGAGGAAACACAACAGAAATCCAACCTAGAGCTGCTCCGCATC
TCCCTGCTGCTCATCCAGTCGTGGCTGGAGCCCGTGCAGTTCCTCAGGAGTGTCTTCGCCAACAGCCTGGTG
TACGGCGCCTCTGACAGCAACGTCTATGACCTCCTAAAGGACCTAGAGGAAGGCATCCAAACGCTGATGGGG
AGGCTGGAAGATGGCAGCCCCCGGACTGGGCAGATCTTCAAGCAGACCTACAGCAAGTTCGACACAAACTCA
CACAACGATGACGCACTACTCAAGAACTACGGGCTGCTCTACTGCTTCAGGAAGGACATGGACAAGGTCGAG
ACATTCCTGCGCATCGTGCAGTGCCGCTCTGTGGAGGGCAGCTGTGGCTTCTAG (SEQ ID NO: 42) Transiently transfected into sarcoma cell line HT1080 using standard methods (e.g., Rivera et al., Nature Med. 2: 1028-1032, 1996; Amara et a.
l., PNAS 94: 10618-10623, 1997). After overnight incubation, the medium is removed and fresh medium without drug or containing retinol at various concentrations is added. After further incubation for 2 to 24 hours, the amount of hGH secreted into the medium is determined by radioimmunoassay (Rivera et al., Nature
Med. 2: 1028-1032, 1996).

[Procedure amendment 19]

[Document name to be amended] Statement

[Correction target item name] 0266

[Correction method] Change

[Contents of correction]

To examine the kinetics of this ligand-induced decrease in serum glucose concentration, 2 ×
STeZ treated mice implanted with 10e7 HT101-10p cells received 30 mg / kg AP22542
Was administered only once intravenously. Serum glucose concentrations were measured at various times from 5 minutes to 10 hours. As shown in FIG. 11b, at 5 minutes and 15 minutes after administration of AP22542, serum glucose levels are indistinguishable from animals treated with vehicle. However, within 30 minutes there is a significant drop in serum glucose concentration and by 2 hours the serum glucose concentration has decreased from an initial concentration close to 500 mg / dl to 50 mg / dl. This effect was temporary, with serum glucose levels rising to 350 mg / dl within 5 hours and 6-
After 10 hours it returns to basal level. Since insulin secretion is dependent on the presence of the drug, administration of smaller amounts of AP22542 or shorter half-life ligands should result in even more transient production of secreted proteins and consequent physiological effects. . Conversely, administration of higher amounts of AP22542 or longer half-life ligands should result in longer secreted proteins.

[Sequence List] SEQUENCE LISTING <110> ARIAD Gene Therapeutics, Inc. <120> Materials and Methods Involving Conditional Retention Domains <130> ARIAD 383B JP <140> JP2000-577309 <141> 1999-10-19 <160> 42 <170> PatentIn version 3.0 <210> 1 <211> 8 <212> PRT <213> homo sapiens <220><221> DOMAIN <222> (1) .. (8) <223> enzymatic cleavage site <400> 1 Ser Ala Arg Asn Arg Gln Lys Arg 1 5 <210> 2 <211> 4 <212> PRT <213> homo sapiens <220><221> DOMAIN <222> (1) .. (4) <223> Consensus cleavage site. Where the first Xaa is any amino acid.Where the second Xaa is K (lysine) or R (arginine). <400> 2 Arg Xaa Xaa Arg 1 <210> 3 <211> 7 <212> PRT <213> homo sapiens <220><221> DOMAIN <222> (1) .. (7) <223> pro-neurotropin-3 cleavage site <400> 3 Met Ser Met Arg Val Arg Arg 1 5 <210> 4 <211> 7 <212> PRT <213> homo sapiens <220><221> DOMAIN <222> (1) .. (7) <223> pro-insulin like growth factor I cleavage site <400> 4 Lys Pro Ala Lys Ser Ala Arg 1 5 <210> 5 <211> 6 <212> PRT <213> homo sapiens <220><221> DOMA IN <222> (1) .. (6) <223> pro-parathyroid hormone cleavage site <400> 5 Lys Ser Val Lys Lys Arg 1 5 <210> 6 <211> 7 <212> PRT <213> homo sapiens <220><221> DOMAIN <222> (1) .. (7) <223> stromelysin-3 cleavage site <400> 6 Ala Arg Asn Arg Gln Lys Arg 1 5 <210> 7 <211> 7 <212> PRT <213> homo sapiens <220><221> DOMAIN <222> (1) .. (7) <223> insulin pro-receptor cleavage site <400> 7 Arg Pro Ser Arg Lys Arg Arg 1 5 <210> 8 <211> 7 <212> PRT <213> homo sapiens <220><221> DOMAIN <222> (1) .. (7) <223> hepatocyte growth factor pro-receptor cleavage site <400> 8 Thr Glu Lys Arg Lys Lys Arg 1 5 <210> 9 <211> 4 <212> PRT <213> homo sapiens <220><221> DOMAIN <222> (1) .. (4) <223> furin targeting motif <400> 9 Tyr Lys Gly Leu 1 <210> 10 <211> 7 <212> PRT <213> homo sapiens <220><221> DOMAIN <222> (1) .. (7) <223> Ser-furin targeting motif <400 > 10 Ser Asp Ser Glu Glu Asp Glu 1 5 <210> 11 <211> 4 <212> PRT <213> homo sapiens <220><221> DOMAIN <222> (1) .. (4) <223> Tyrosine -based lysosomal targeting signa l. Where X is any amino acid. <400> 11 Asn Pro Xaa Tyr 1 <210> 12 <211> 4 <212> PRT <213> homo sapiens <220><221> DOMAIN <222> (1) .. (4) <223> Tyrosine-based lysosomal targeting signal.Where the first and second Xaa are any amino acid.Where the third Xaa is an amino acid with a bulky hydrophobic gro up <400> 12 Tyr Xaa Xaa Xaa 1 <210> 13 <211> 40 <212> DNA <213> Artificial sequence <220><221> misc_feature <222> (1) .. (40) <223> pC4EN plasmid-VR65 <400> 13 tcccgcacct cttcggccag cgaattccag aagcgcgtat 40 <210> 14 <211> 32 <212> DNA <213> Artificial sequence <220><221> misc_feature <222> (1) .. (32) <223> pC4EN plasmid-VR119 <400> 14 gactcactat aggacgcgtt cgagctcgcc cc 32 <210> 15 <211> 31 <212> DNA <213> Artificial sequence <220><221> misc_feature <222> (1) .. (31) <223> pC4EN plasmid-VR120 <400> 15 catcattttg gcaaaggatt cactcctcag g 31 <210> 16 <211> 27 <212> DNA <213> Artificial sequence <220><221> misc_feature <222> (1) .. (27) <223> cloning oligo-VR1 <400> 16 gatggaaaga aaatgg attc ctcccgg 27 <210> 17 <211> 24 <212> DNA <213> Artificial sequence <220><221> misc_feature <222> (1) .. (24) <223> cloning oligo-VR2 <400> 17 tctagagtga gcaagggcga ggag 24 <210> 18 <211> 37 <212> DNA <213> Artificial sequence <220><221> misc_feature <222> (1) .. (37) <223> cloning oligo-VR3 <400> 18 ggatccttat taactagtct tgtacagctc gtccatg 37
<210> 19 <211> 25 <212> DNA <213> Artificial sequence <220><221> misc_feature <222> (1) .. (25) <223> cloning oligo VR109 <400> 19 aagcttacca ctcagggtcc tgtgg 25 <210> 20 <211> 19 <212> DNA <213> Artificial sequence <220><221> misc_feature <222> (1) .. (19) <223> cloning oligo-VR110 <400> 20 gaattcgtgg caacttcca 19 <210 > 21 <211> 29 <212> DNA <213> Artificial sequence <220><221> misc_feature <222> (1) .. (29) <223> cloning oligo-VR185 <400> 21 cacaggaccc tgaattctaa gcttgtggc 29 <210 > 22 <211> 30 <212> DNA <213> Artificial sequence <220><221> misc_feature <222> (1) .. (30) <223> cloning oligo-VR186 <400> 22 ataagggaat ggttctagag gcactgccct 30 <210 > 23 <211> 31 <212> DNA <213> Artificial sequence <220><221> misc_feature <222> (1) .. (31) <223> cloning oligo-VR187 <400> 23 atgccacccg ggactagtga agccacagct g 31 <210> 24 <211> 48 <212> DNA <213> Artificial sequence <220><221> misc_feature <222> (1) .. (48) <223> cloning oligo-VR4 <400> 24 actagtgcta gaaaccgtca gaagagattc ccaaccattc ccttaagc 48 <2 10> 25 <211> 31 <212> DNA <213> Artificial sequence <220><221> misc_feature <222> (1) .. (31) <223> cloning oligo-VR5 <400> 25 ggatcccggg ctagaagcca cagctgccct c 31 <210> 26 <211> 32 <212> DNA <213> Artificial sequence <220><221> misc_feature <222> (1) .. (32) <223> cloning oligo-VR220 <400> 26 cgaattcttc tgccatggcc ctgtggatgc gc 32 <210> 27 <211> 31 <212> DNA <213> Artificial sequence <220><221> misc_feature <222> (1) .. (31) <223> cloning oligo-VR221 <400> 27 cggatccgca ggctgcgtct agttgcagta g 31 <210> 28 <211> 52 <212> DNA <213> Artificial sequence <220><221> misc_feature <222> (1) .. (52) <223> cloning oligo-VR222 <400> 28 cactagtgct agaaaccgtc agaagagatt tgtgaaccaa cacctgtgcg gc 52 <210> 29 <211> 27 <212> DNA <213> Artificial sequence <220><221> misc_feature <222> (1) .. (27) <223> cloning oligo-VR223 <400> 29 cctgtgcggc tcagacctgg tggaagc 27 <210> 30 <211> 35 <212> DNA <213> Artificial sequence <220><221> misc_feature <222> (1) .. (35) <223> cloning oligo-VR224 <400> 30 cttctacaca ccca ggacca agcgggaggc agagg 35 <210> 31 <211> 29 <212> DNA <213> Artificial sequence <220><221> misc_feature <222> (1) .. (29) <223> cloning oligo-VR225 <400> 31 ccctggaggg gtcccggcag aagcgtggc 29 <210> 32 <211> 306 <212> DNA <213> homo sapiens <220><221> misc_feature <222> (1) .. (306) <223> p65 activation domain <400> 32 ctgggggcct tgcttggcaa cagcacagac ccagctgtgt tcacagacct ggcatccgtc 60 gacaactccg agtttcagca gctgctgaac cagggcatac ctgtggcccc ccacacaact 120 gagcccatgc tgatggagta ccctgaggct ataactcgcc tagtgacagg ggcccagagg 180 ccccccgacc cagctcctgc tccactgggg gccccggggc tccccaatgg cctcctttca 240 ggagatgaag acttctcctc cattgcggac atggacttct cagccctgct gagtcagatc 300 agctcc 306 <210> 33 <211> 8 <212> PRT <213 > homo sapiens <220><221> DOMAIN <222> (1) .. (8) <223> stromelysin furin cleavage site <400> 33 Ser Ala Arg Asn Arg Gln Lys Arg 1 5 <210> 34 <211> 49 <212> DNA <213> Artificial sequence <220><221> misc_feature <222> (1) .. (49) <223> RBP-5 'cloning oligo <400> 34 cgtacgaatt ccagaagcgc gtatg gagtg ggtgtgggcg ctcgtgctg 49 <210> 35 <211> 42 <212> DNA <213> Artificial sequence <220><221> misc_feature <222> (1) .. (42) <223> RBP-3 'cloning oligo <400 > 35 gcatgactag tcaaactgtt tcttgagggt ctgctttgac ag 42 <210> 36 <211> 70 <212> DNA <213> Artificial sequence <220><221> misc_feature <222> (1) .. (70) <223> FCS-hGH5 ' cloning oligo <400> 36 gcaacactag tgctagaaac cgtcagaaga gattcccaac cattccctta agcaggcctt 60 ttgacaacgc 70 <210> 37 <211> 45 <212> DNA <213> Artificial sequence <220><221> misc_feature <222> (1) .. (45) <223> hGH3 'cloning oligo <400> 37 gctcaggatc ccgggctaga agccacagct gccctccaca gagcg 45 <210> 38 <211> 32 <212> DNA <213> Artificial sequence <220><221> misc_feature <222>. (1). 32) <223> RBP-Xba cloning oligo <400> 38 tcagctctag agagcgcgac tgcagggtga gc 32 <210> 39 <211> 33 <212> DNA <213> Artificial sequence <220><221> misc_feature <222> (1). . (33) <223> RBP-Spe cloning oligo <400> 39 gaagcactag tcaaactgtt tcttgagggt ctg 33 <210> 40 <211> 1275 <212> DNA <213> Rattus rat tus <220><221> misc_feature <222> (1) .. (1275) <223> rat RBP expression cassette <400> 40 gaattccaga agcgcgtatg gagtgggtgt gggcgctcgt gctgctggcg gctctgggag 60 gcggcagcgc cgagcgcgac tgcagggtga gcagcttcag agtcaaggag aacttcgaca 120 aggctcgttt ctctgggctc tggtatgcca tcgccaaaaa ggatcccgag ggtctctttt 180 tgcaagacaa catcatcgct gagttttctg tcgacgagaa gggtcatatg agcgctacag 240 ccaagggacg agtccgtctt ctgagcaact gggaagtgtg tgcagacatg gtgggcactt 300 tcacagatac agaagatcct gccaagttca agatgaagta ctggggtgta gcctcctttc 360 tccagcgagg aaacgatgac cactggatca tcgatacgga ctacgacacc ttcgctctgc 420 agtattcctg ccgcctgcag aatctggatg gcacctgtgc agacagctac tcctttgtgt 480 tttctcgtga ccccaatggc ctgaccccgg agacacggag gctggtgagg cagcgacagg 540 aggagctgtg cctagagagg cagtacagat ggatcgagca caatggttac tgtcaaagca 600 gaccctcaag aaacagtttg actagtgcta gaaaccgtca gaagagattc ccaaccattc 660 ccttaagcag gccttttgac aacgctatgc tccgcgccca tcgtctgcac cagctggcct 720 ttgacaccta ccaggagttt gaagagcattatatagcc tatatagcc gcagaaccc ccagacctcc ctctgtttct cagagtctat tccgacaccc tccaacaggg 840 aggaaacaca acagaaatcc aacctagagc tgctccgcat ctccctgctg ctcatccagt 900 cgtggctgga gcccgtgcag ttcctcagga gtgtcttcgc caacagcctg gtgtacggcg 960 cctctgacag caacgtctat gacctcctaa aggacctaga ggaaggcatc caaacgctga 1020 tggggaggct ggaagatggc agcccccgga ctgggcagat cttcaagcag acctacagca 1080
agttcgacac aaactcacac aacgatgacg cactactcaa gaactacggg ctgctctact 1140 gcttcaggaa ggacatggac aaggtcgaga cattcctgcg catcgtgcag tgccgctctg 1200 tggagggcag ctgtggcttc tagcccggga tcctgagaac ttcagggtga gtttggggac 1260 ccttgattgt tcttt 1275 <210> 41 <211> 401 <212> PRT <213> Rattus rattus <220><221> PEPTIDE <222 > (1) .. (401) <223> encoded rat RBP protein <400> 41 Met Glu Trp Val Trp Ala Leu Val Leu Leu Ala Ala Leu Gly Gly Gly 1 5 10 15 Ser Ala Glu Arg Asp Cys Arg Val Ser Ser Phe Arg Val Lys Glu Asn 20 25 30 Phe Asp Lys Ala Arg Phe Ser Gly Leu Trp Tyr Ala Ile Ala Lys Lys 35 40 45 Asp Pro Glu Gly Leu Phe Leu Gln Asp Asn Ile Ile Ala Glu Phe Ser 50 55 60 Val Asp Glu Lys Gly His Met Ser Ala Thr Ala Lys Gly Arg Val Arg 65 70 75 80 Leu Leu Ser Asn Trp Glu Val Cys Ala Asp Met Val Gly Thr Phe Thr 85 90 95 Asp Thr Glu Asp Pro Ala Lys Phe Lys Met Lys Tyr Trp Gly Val Ala 100 105 110 Ser Phe Leu Gln Arg Gly Asn Asp Asp His Trp Ile Ile Asp Thr Asp 115 120 125 Tyr Asp Thr Phe Ala Leu Gln Tyr Ser Cys Arg Le u Gln Asn Leu Asp 130 135 140 Gly Thr Cys Ala Asp Ser Tyr Ser Phe Val Phe Ser Arg Asp Pro Asn 145 150 155 160 Gly Leu Thr Pro Glu Thr Arg Arg Arg Leu Val Arg Gln Arg Gln Glu Glu 165 170 175 Leu Cys Leu Glu Arg Gln Tyr Arg Trp Ile Glu His Asn Gly Tyr Cys 180 185 190 Gln Ser Arg Pro Ser Arg Asn Ser Leu Thr Ser Ala Arg Asn Arg Gln 195 200 205 Lys Arg Phe Pro ThrIle Pro Leu Ser Arg Pro Phe Asp Asn Ala Met 210 215 220 Leu Arg Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu 225 230 235 240 Phe Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln 245 250 255 Asn Pro Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser 260 265 270 Asn Arg Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile 275 280 285 Ser Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg 290 295 300 Ser Val Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val 305 310 315 320 Tyr Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly 325 330 335 Arg Leu Glu Asp Gly Ser Pro Arg Thr Gly Gln Il e Phe Lys Gln Thr 340 345 350 Tyr Ser Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys 355 360 365 Asn Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu 370 375 380 Thr Phe Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly 385 390 395 400 Phe <210> 42 <211> 1206 <212> DNA <213> rat <220><221> misc_feature <222> (1) .. (1206) ) <223> rat RBP expression cassette <400> 42 atggagtggg tgtgggcgct cgtgctgctg gcggctctgg gaggcggcag cgccgagcgc 60 gactgcaggg tgagcagctt cagagtcaag gagaacttcg acaaggctcg tttctctggg 120
ctctggtatg ccatcgccaa aaaggatccc gagggtctct ttttgcaaga caacatcatc 180 gctgagtttt ctgtcgacga gaagggtcat atgagcgcta cagccaaggg acgagtccgt 240 cttctgagca actgggaagt gtgtgcagac atggtgggca ctttcacaga tacagaagat 300 cctgccaagt tcaagatgaa gtactggggt gtagcctcct ttctccagcg aggaaacgat 360 gaccactgga tcatcgatac ggactacgac accttcgctc tgcagtattc ctgccgcctg 420 cagaatctgg atggcacctg tgcagacagc tactcctttg tgttttctcg tgaccccaat 480 ggcctgaccc cggagacacg gaggctggtg aggcagcgac aggaggagct gtgcctagag 540 aggcagtaca gatggatcga gcacaatggt tactgtcaaa gcagaccctc aagaaacagt 600 ttgactagtg ctagaaaccg tcagaagaga ttcccaacca ttcccttaag caggcctttt 660 gacaacgcta tgctccgcgc ccatcgtctg caccagctgg cctttgacac ctaccaggag 720 tttgaagaag cctatatccc aaaggaacag aagtattcat tcctgcagaa cccccagacc 780 tccctctgtt tctcagagtc tattccgaca ccctccaaca gggaggaaac acaacagaaa 840 tccaacctag agctgctccg catctccctg ctgctcatcc agtcgtggct ggagcccgtg 900 cagttcctca ggagtgtctt cgccaacagc ctggtgtacg gcgcctctga cagcaacgtc 960 tatgacctcc taaaggacc t agaggaaggc atccaaacgc tgatggggag gctggaagat 1020 ggcagccccc ggactgggca gatcttcaag cagacctaca gcaagttcga cacaaactca 1080 cacaacgatg acgcactact caagaactac gggctgc cagactag gagcatcg cgg gagagcatc gagagcatc gagagcatc gagagcatc gagagcatgc

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) C12P 21/08 G01N 33/15 Z 4H045 C12Q 1/02 33/50 Z G01N 33/15 33/68 33 / 50 C12N 15/00 ZNAA 33/68 5/00 B (31) Priority claim number 60 / 137,787 (32) Priority date June 2, 1999 (1999.6.2) (33) Priority claim Country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV , MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR, TT, UA, UG, US, UZ, VN, Y U (72) Inventor Crackson, Timothy United States, Massachusetts 02140, Cambridge, Cogswell Avenue 47-12 (72) Inventor Rossman, James United States of America, 10021, New York, New York, Ys・ Sixtyforth Street 402 F-term (reference) 2G045 AA40 CB01 DA12 DA13 DA14 DA36 DA54 DA77 FB07 4B024 AA01 AA11 BA01 BA07 BA21 BA31 BA41 CA04 DA01 DA02 DA05 DA11 EA04 FA02 FA17 HA01 4B063 QA01 QA13 QRA72 QQ80 QR55 AG27 AG31 CA19 CC24 4B065 AA01X AA57X AA87X AA93Y AB01 AC14 CA24 CA41 CA44 CA46 4H045 AA10 AA11 BA10 CA40 DA01 DA30 DA75 EA20 EA50

Claims (38)

[Claims] 1. A recombinant nucleic acid encoding a fusion protein comprising at least one condition retaining domain and at least one additional domain heterologous thereto. 2. A condition-retaining domain (CRD) comprising a retinol-binding protein,
FKBP, is derived from IgM or alpha ₁ antitrypsin, claim 1 recombinant nucleic acid according. 3. The recombinant nucleic acid according to claim 2, wherein the CRD contains an FKBP domain having an amino acid substitution at F36 or W59. 4. The recombinant nucleic acid according to claim 3, wherein the CRD comprises an FKBP domain containing an F36M or W59V mutation. 5. The recombinant nucleic acid of claim 1, wherein said heterologous domain comprises a target protein. 6. The recombinant nucleic acid according to claim 5, wherein the target protein is a hormone, endorphin, antibody or immunogen. 7. The recombinant nucleic acid according to claim 6, wherein the target protein is selected from the group consisting of insulin, parathyroid hormone and β-endorphin. 8. The recombinant nucleic acid of claim 1, further comprising a nucleic acid sequence encoding an enzyme cleavage site. 9. The recombinant nucleic acid according to claim 8, wherein the cleavage site is a furin cleavage site. 10. The recombinant nucleic acid according to claim 9, wherein the furin cleavage site comprises the amino acid sequence of SARNRQKR. 11. The recombinant nucleic acid according to claim 8, further comprising a signal sequence. 12. The recombinant nucleic acid according to claim 11, wherein the signal sequence is a signal sequence derived from a human growth hormone gene. 13. The recombinant nucleic acid of claim 11, comprising a nucleic acid sequence encoding a signal sequence, a condition retaining domain, a furin cleavage site, and a target protein. 14. A signal sequence derived from human growth hormone, three F36M FKBPs.
14. The recombinant nucleic acid of claim 13, comprising a domain, a human stromelysin-3 furin cleavage site, and a nucleic acid sequence encoding a target protein. 15. The recombinant nucleic acid of claim 13, further comprising a nucleic acid sequence encoding a lysosomal targeting signal. A fusion protein encoded by the recombinant nucleic acid according to any one of claims 1 to 15. A vector comprising the recombinant nucleic acid according to any one of claims 1 to 15. 18. The vector according to claim 17, wherein the vector is a viral vector. 19. The vector according to claim 18, wherein the viral vector is selected from the group consisting of adenovirus, AAV, retrovirus, and adeno-AAV hybrid. 20. A cell containing the vector according to claim 17. 21. The cell according to claim 20, wherein the cell is a mammalian cell. 22. The cell according to claim 21, wherein the cell is derived from a human. 23. The cell according to claim 22, wherein the cell is a primary cell. 24. An animal containing the cell according to claim 20. 25. An animal containing the cell according to claim 21. 26. A method of genetically engineering a cell to enable regulated secretion of a target protein, comprising introducing the recombinant nucleic acid of any one of claims 13 to 15 into the cell. 27. A method of inducing a cell to secrete a target protein, comprising treating the cell of claim 20 with a ligand that binds to a CRD at a concentration sufficient to induce secretion of the target protein. . 28. A genetically engineered host cell comprising: (a) a reporter gene linked to a regulatable expression control element; (b) the first recombinant nucleic acid sequence encodes a DNA binding domain; A recombinant nucleic acid comprising a polylinker linked to the first and second recombinant nucleic acid sequences, wherein the second recombinant nucleic acid sequence encodes a transcription activation domain, wherein the DNA binding domain and the transcriptional activity Association with the activation domain activates expression of the reporter gene. 29. A genetically engineered host cell comprising: (a) a reporter gene linked to a regulatable expression control element; (b) a transcription activation domain linked to a candidate condition-carrying domain. A first recombinant nucleic acid encoding a fusion protein; (c) a second recombinant nucleic acid encoding a fusion protein comprising a DNA binding domain linked to the candidate condition-retaining domain; Protein association activates reporter gene expression. 30. A method for identifying a ligand capable of binding to a condition-maintaining domain, comprising the steps of: (a) under the appropriate conditions that allow gene expression, the genetically engineered cell of claim 28; Is contacted with a candidate ligand, (b) observing the presence and / or amount of reporter gene expression, and (c) determining the presence and / or amount of reporter gene expression by contacting the cells with one or more candidate ligands. Correlate. 31. A recombinant nucleic acid encoding a fusion protein comprising a signal sequence, a polylinker, an enzyme cleavage site, and a target protein. 32. The recombinant nucleic acid according to claim 31, wherein the members of the DNA library are introduced into a polylinker. 33. A genetically engineered cell containing the recombinant nucleic acid of claim 32. 34. A method for identifying CRD, comprising the steps of: (a) treating the cell of claim 33 with a ligand that binds CRD; and (b) inhibiting the ligand-dependent secretion of a target protein into a medium. Assess presence and (c) correlate secretion of the target protein with one or more individual members of the DNA library. 35. A recombinant nucleic acid encoding a fusion protein comprising a signal sequence, a polylinker, an enzyme cleavage site, and an extracellular and transmembrane domain of a membrane protein. 36. The recombinant nucleic acid according to claim 35, wherein the members of the DNA library are introduced into a polylinker. 37. A genetically engineered cell containing the recombinant nucleic acid of claim 36. 38. A method for identifying CRD, comprising the steps of: (a) treating the cell of claim 37 with a ligand that binds CRD; and (b) ligand-dependent membrane protein on the surface of the cell. Assess the presence of sexual localization,
And (c) correlating the surface localization of the membrane protein with one or more individual members of the DNA library.