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Title:  Single-chain insulin analog and a polynucleotide sequence encoding the analog

United States Patent:  6,630,348

Issued:  October 7, 2003

Inventors:  Lee; Hyun Chul (Seodaemungu Hongeundong 268, Dongdo-academyhouse A-402, Seoul, KR); Kim; Su-Jin (Dukyanggu Haengsindong 938 Haibit 1819-1304, Goyangsi, KR); Kim; Kyung Sup (Yongdeungpogu Yeoyeedodong Samik, Apt. B-202, Seoul, KR); Shin; Hang-Cheol (Seochogu Wonjidong 401-37, Seoul, KR); Yoon; Ji-Won (206 Edgeview Drive, N.W., Calgary, Alberta, CA T3A 4X5)

Appl. No.:  706690

Filed:  November 7, 2000

Abstract

The subject matter of the invention is directed to a single-chain insulin analog that is used to treat diabetes by gene therapy methods.

SUMMARY OF THE INVENTION

The present invention has met the hereinbefore described need.

A method of introducing at least one gene encoding a product into at least one cell of a mammalian tissue for use in treating a mammalian host is provided in the present invention. This method includes employing recombinant techniques to produce a DNA vector molecule containing the gene coding for the product and introducing the DNA vector molecule containing the gene coding for the product into the tissue cell. The DNA vector molecule can be any DNA molecule capable of being delivered and maintained within the target cell or tissue such that the gene encoding the product of interest can be stably expressed. The DNA vector molecule preferably utilized in the present invention is either a viral or plasmid DNA vector molecule. This method preferably includes introducing the gene encoding the product into the cell of the mammalian tissue for a therapeutic use.

An object of the invention is to provide a single-chain insulin analog compound of formula (I) having the properties of greater insulin receptor binding activity than proinsulin and less insulin receptor binding activity than insulin:

B chain-X-A chain (I)

wherein:

B and A chains are the human insulin chains, respectively, or functional analogs thereof; and

X is a joining peptide of from 5 to 18 amino acids.

In the above compound, preferably, X may be from 6 to 9 amino acids.

Also, in the above compound, when X has the formula Ul --Zn --Ym --Zl --Un, the following limitations may be placed:

U is an arginine or lysine residue;

Z is an amino acid residue;

Y is a peptide;

l is an integer of 2-n;

n is an integer of 0, 1 or 2; and

m is an integer of 2 to 5

In this compound, Z may be glycine; and Y may be glycine-proline-glycine. Furthermore, Z may be glycine; and Y may be alanine-proline-glycine-aspartic acid-valine. Alternatively, Z may be glycine; and Y may be tyrosine-proline-glycine-aspartic acid-valine. Further, Z may be glycine; and Y may be histidine-proline-glycine-aspartic acid-valine.

Another object of the invention is to provide a polynucleotide encoding the single-chain insulin analog described above. Another embodiment of the invention includes a recombinant vector comprising the polynucleotide that encodes the single chain insulin analog described above. The vector may be a plasmid or a virus. If a virus, preferably, it is adeno-associated virus. Moreover, it is preferred that the promoter be inducible. More preferably, the promoter may be regulated by glucose. Even more preferably, the promoter is a pyruvate kinase gene promoter. Most preferably, the promoter is the hepatocyte-specific L-type pyruvate kinase gene promoter.

The invention is also directed to a cell line transformed with the above-described vector.

Another embodiment of the invention is directed to a method for treating a patient suffering from diabetes comprising:

a) generating a recombinant viral or plasmid vector comprising a polynucleotide encoding a single-chain insulin analog operatively linked to a promoter; and

b) introducing said recombinant viral or plasmid vector to said patient, such that expression of said polynucleotide within said patient results in remission of diabetes.

Preferably, the viral vector is adeno-associated virus, and the promoter is an inducible promoter. Preferably, the promoter is regulated by glucose. In the method described above, preferably the dosage of said viral vector is at least about 1011 viral particles. Preferably, the treatment method is accomplished by using a vector that is introduced to the patient through the cell line comprising the single chain insulin analog described above.

The present invention is also directed to a method for treating a patient suffering from diabetes comprising administering the single chain insulin analog compound described above to a patient in need thereof. Preferably, the diabetes is type I diabetes.

These and other objects of the invention will be more fully understood from the following description of the invention, the referenced drawings attached hereto and the claims appended hereto.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "patient" includes members of the animal kingdom including but not limited to human beings.

As used herein, the term "mammalian host" includes members of the animal kingdom including but not limited to human beings.

As used herein, the term "diabetes" is a hormonal disorder. Insulin is needed to control the blood sugar levels.

As used herein, the term "Type I diabetes" means insulin-dependent diabetes mellitus (IDDM).

As used herein, the term "Type II diabetes" means non insulin-dependent diabetes mellitus (NIDDM).

Insulin is composed of two peptide chains referred to as the A chain and B chain. A and B chains are linked together by two disulfide bonds, and an additional disulfide is formed within the A chain. In most species, the A chain consists of 21 amino acids and the B chain of 30 amino acids. Although the amino acid sequence of insulin varies among species, certain segments of the molecule are highly conserved, including the positions of the three disulfide bonds, both ends of the A chain and the C-terminal residues of the B chain. These similarities in the amino acid sequence of insulin lead to a three dimensional conformation of insulin that is very similar among species, and insulin from one animal is very likely biologically active in other species. Indeed, pig insulin has been widely used to treat human patients.

Insulin molecules have a tendency to form dimers in solution due to hydrogen-bonding between the C-termini of B chains. Additionally, in the presence of zinc ions, insulin dimers associate into hexamers.

These interactions have important clinical ramifications. Monomers and dimers readily diffuse into blood, whereas hexamers diffuse very poorly. Hence, absorption of insulin preparations containing a high proportion of hexamers is delayed and slow. This problem, among others, has stimulated development of a number of recombinant insulin analogs. The first of these molecules to be marketed--called insulin lispro--is engineered such that lysine and proline residues on the C-terminal end of the B chain are reversed; this modification does not alter receptor binding, but minimizes the tendency to form dimers and hexamers.

As used herein, the "single-chain insulin analog (SIA)" encompasses a group of structurally-related proteins wherein the A and B chains are covalently linked by a polypeptide linker. SIA has the properties of greater insulin receptor binding activity and/or glucose uptake activity than proinsulin, and less insulin receptor binding activity and glucose uptake activity than insulin. "SIA-1", "SIA-2" and so on belong to the SIA group.

The polypeptide linker connects the C-terminus of the B chain to the N-terminus of the A chain. The linker may be of any length so long as the linker provides the structural conformation necessary for SIA to have a glucose uptake and insulin receptor binding effect. Preferably, the linker is about 5-18 amino acids long. Preferably, it is 6-12 amino acids long. Even more preferably, it is 6-9 amino acids long. Most preferably it is 7 amino acids long. The most preferred sequence for the linker are Gly-Gly-Gly-Pro-Gly-Lys-Arg (SEQ ID NO:1) or Arg-Arg-Gly-Pro-Gly-Gly-Gly (SEQ ID NO:2). However, it should be understood that many variations of this sequence are possible such as in the length (both addition and deletion) and substitutions of amino acids without substantially compromising the effectiveness of the produced SIA in glucose uptake and insulin receptor binding activities. For example, several different amino acid residues may be added or taken off at either end without substantially decreasing the activity of the produced SIA. In addition, the amino acid Gly may be replaced with any amino acid residue.

It is also to be understood that the insulin A and B chains may be modified or fragmented so long as the modified or fragmented form has glucose uptake activity and/or binds to the insulin receptor, wherein a SIA formed from these chains possesses greater insulin receptor binding activity and/or glucose uptake activity than proinsulin, and less insulin receptor binding activity and glucose uptake activity than insulin.

As used herein, a "promoter" can be any sequence of DNA that is active, and controls transcription in an eucaryotic cell. Preferably, the promoter is active in mammalian cells. The promoter may be constitutively expressed or inducible. Preferably, the promoter is inducible. Preferably, the promoter is inducible by an external stimulus. More preferably, the promoter is inducible by hormones or metabolites. Still more preferably, the promoter is regulatable by glucose. Even more preferably, the promoter is a pyruvate kinase gene promoter. Most preferably, the promoter is a hepatocyte-specific L-type pyruvate kinase gene promoter.

Likewise, "enhancer elements", which also control transcription, can be inserted into the DNA vector construct, and used with the construct of the present invention to enhance the expression of the gene of interest.

As used herein viral vectors include any virus that is useful in in vivo or ex vivo gene therapy protocols. Preferably, the virus is non-pathogenic. More preferably, the virus is adeno-associated virus (AAV).

As used herein, the term "DC-chol" means a cationic liposome containing cationic cholesterol derivative. The "DC-chol" molecule includes a tertiary amino group, a medium length spacer arm (two atoms) and a carbamoyl linker bond (Gao et al., Biochem. Biophys. Res, Commun., 179:280-285, 1991).

As used herein, "SF-chol" is defined as a type of cationic liposome.

As used herein, the term "biologically active" used in relation to liposomes denotes the ability to introduce functional DNA and/or proteins into the target cell.

As used herein, the term "biologically active" in reference to a nucleic acid, protein, protein fragment or derivative thereof is defined as an ability of the nucleic acid or amino acid sequence to mimic a known biological function elicited by the wild type form of the nucleic acid or protein.

As used herein, the term "maintenance", when used in the context of liposome delivery, denotes the ability of the introduced DNA to remain present in the cell. When used in other contexts, it means the ability of targeted DNA to remain present in the targeted cell or tissue so as to impart a therapeutic effect.

The present invention discloses in vivo techniques for delivery of a DNA sequence of interest to the tissue cells of the mammalian host. The in vivo technique involves directly administering a DNA vector containing a DNA sequence of interest or other delivery vehicle of interest into the tissue cells, to the target area of the mammalian host, so as to effect in vivo expression of the gene product of interest. The vector is preferably a viral vector.

Alternatively, the present invention discloses ex vivo and in vivo techniques for delivery of a DNA sequence of interest to the tissue cells of the mammalian host. The ex vivo technique involves culturing target tissue cells, in vitro transfecting a DNA vector containing a DNA sequence of interest or other delivery vehicle of interest into the tissue cells, followed by transplantation of the modified tissue cells to the target area of the mammalian host, so as to effect in vivo expression of the gene product of interest.

As an alternative to the in vitro manipulation of cells, the gene encoding the product of interest is introduced into liposomes and injected directly into the target area, where the liposomes fuse with the tissue cells, resulting in an in vivo gene expression of SIA.

As an additional alternative to the in vitro manipulation of tissue cells, the gene encoding the product of interest is introduced into the target area as naked DNA. The naked DNA enters the tissue cell, resulting in an in vivo gene expression of SIA.

One ex vivo method of treating diabetes comprises initially generating a recombinant viral or plasmid vector which contains a DNA sequence encoding single-chain insulin analog or biologically active fragment thereof. This recombinant vector is then used to infect or transfect a population of in vitro cultured tissue cells, resulting in a population of cells containing the vector. Expression of this DNA sequence of interest is useful in substantially reducing at least one deleterious pathology associated with diabetes.

More specifically, this method includes employing SIA, or a biologically active derivative or fragment thereof or a biologically active derivative or fragment thereof.

A further embodiment of the present invention includes employing SIA or a biologically active derivative or fragment thereof, and employing as the DNA plasmid vector any DNA plasmid vector known to one of ordinary skill in the art capable of stable maintenance within the targeted cell or tissue upon delivery, regardless of the method of delivery utilized.

One such method is the direct delivery of the DNA vector molecule, whether it be a viral or plasmid DNA vector molecule, to the target cell or tissue. This method also includes employing SIA or biologically active derivative or fragment thereof.

Another embodiment of this invention provides a method for introducing at least one gene encoding a product into at least one cell of a target tissue for use in treating the mammalian host. This method includes employing non-viral means for introducing the gene coding for the product into the tissue cell. More specifically, this method includes a liposome encapsulation, calcium phosphate coprecipitation, electroporation, or DEAE-dextran mediation, and includes employing as the gene a gene capable of encoding a member of transforming growth factor superfamily or biologically active derivative or fragment thereof, or biologically active derivative or fragment thereof.

Another embodiment of this invention provides an additional method for introducing at least one gene encoding a product into at least one cell of a tissue for use in treating the mammalian host. This additional method includes employing the biologic means of utilizing a virus to deliver the DNA vector molecule to the target cell or tissue. Preferably, the virus is a pseudo-virus, the genome having been altered such that the pseudo-virus is capable only of delivery and stable maintenance within the target cell, but not retaining an ability to replicate within the target cell or tissue. The altered viral genome is further manipulated by recombinant DNA techniques such that the viral genome acts as a DNA vector molecule which contains the heterologous gene of interest to be expressed within the target cell or tissue. Preferably, the viral vector is adeno-associated virus.

A preferred method of the present invention involves direct in vivo delivery of a SIA gene to the target tissue of a mammalian host through use of an adeno-associated virus (AAV) vector. In other words, a DNA sequence of interest encoding a functional SIA protein or SIA fragment is subcloned into the respective viral vector. The SIA containing viral vector is then grown to adequate titer and directed into the targeted area, preferably by injection into the portal vein.

Direct injection of a DNA molecule containing the gene of interest into the joint results in transfection of the recipient tissue cells and hence bypasses the requirement of removal, in vitro culturing, transfection, selection, as well as transplanting the DNA vector containing-cell to promote stable expression of the heterologous gene of interest.

Methods of presenting the DNA molecule to the target tissue includes, but is not limited to, encapsulation of the DNA molecule into cationic liposomes, subcloning the DNA sequence of interest in a retroviral or plasmid vector, or the direct injection of the DNA molecule itself into the targeted area. The DNA molecule is preferably presented as a DNA vector molecule, either as recombinant viral DNA vector molecule or a recombinant DNA plasmid vector molecule. Expression of the heterologous gene of interest is ensured by inserting a promoter fragment active in eukaryotic cells directly upstream of the coding region of the heterologous gene. One of ordinary skill in the art may utilize known strategies and techniques of vector construction to ensure appropriate levels of expression subsequent to entry of the DNA molecule into the targeted tissue.

In a preferred embodiment, tissue cells recovered from the patient are cultured in vitro for subsequent utilization as a delivery system for gene therapy. It will be apparent that Applicants are not limited to the use of the specific tissue type or source disclosed. It would be possible to utilize other tissue sources for in vitro culture techniques. The method of using the gene of this invention may be employed both prophylactically and in the therapeutic treatment of diabetes.

In another embodiment of this invention, a compound for parenteral administration to a patient in a therapeutically effective amount is provided that contains a gene encoding a SIA protein and a suitable pharmaceutical carrier.

Another embodiment of this invention provides for a compound for parenteral administration to a patient in a prophylactically effective amount that includes a gene encoding a SIA protein and a suitable pharmaceutical carrier.

A further embodiment of this invention includes the method as hereinbefore described including introducing the gene into the cell in vitro. This method also includes subsequently transplanting the infected cell into the mammalian host. This method includes after effecting the transfecting of the tissue cell but before the transplanting of the infected cell into the mammalian host, storing the transfected tissue cell. It will be appreciated by those skilled in the art that the infected tissue cell may be stored frozen in 10 percent DMSO in liquid nitrogen. This method includes employing a method to substantially prevent the development of diabetes in a mammalian host having a high susceptibility of developing diabetes.

Another embodiment of this invention includes a method of introducing at least one gene encoding a product into at least one cell of a target tissue of a mammalian host for use in treating the mammalian host as hereinbefore described including effecting in vivo the infection of the cell by introducing the viral vector containing the gene coding for the product directly into the mammalian host. Preferably, this method includes effecting the direct introduction into the mammalian host by injection through the portal vein. This method includes employing the method to substantially prevent the development of diabetes in a mammalian host having a high susceptibility of developing diabetes. This method also includes employing the method on a diabetic mammalian host for therapeutic use.

In a preferred embodiment, the inventors generated a single chain insulin analog (SIA), which possesses biologically active insulin activity without processing and produced a recombinant adeno-associated virus expressing SIA (rAAV-LPK-SIA) under the control of hepatocyte-specific L-type pyruvate kinase gene promoter which regulates SIA expression depending on the level of blood glucose. When the inventors administered rAAV-LPK-SIA through the portal vein of streptozotocin (STZ)-induced diabetic rats, the blood glucose levels decreased, reaching the level of normoglycemia in 1 week and maintained a normoglycemic state for more than 6 months without hypoglycemia or any apparent side effects. In addition, the treatment of diabetic NOD mice with rAAV-LPK-SIA resulted in the complete remission of autoimmune diabetes as seen in STZ-induced diabetes. This novel SIA gene therapy is believed to have therapeutic value for the cure of autoimmune diabetes in humans.

Claim 1 of 11 Claims

What is claimed is:

1. A single-chain insulin analog compound of formula (I) having the properties of greater insulin receptor binding activity than proinsulin and less insulin receptor binding activity than insulin:

B chain-X-A chain (I)

wherein:

B and A chains are the human insulin chains, respectively; and

X is a joining peptide of about 5 to 18 amino acids comprising the following sequence:

Gly-Gly-Gly-Pro-Gly-Lys-Arg (SEQ ID NO: 1).




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