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Modified growth hormone

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Title: Modified growth hormone.
Abstract: The invention provides, a nucleic acid molecule encoding a growth hormone (GH) in which the RSP sorting signal has been mutated, such that the GH can be constitutively secreted by the nonregulated secretory pathway (NRSP) in a mammalian cell. The invention also provides a nucleic acid molecule encoding a GH in which the three-dimensional conformation of the RSP sorting signal has been altered such that the GH can be constitutively secreted by the NRSP in a mammalian cell. ...


Browse recent The United States Of America, Represented By, Depa Rtment Of Health patents - Bethesda, MD, US
Inventors: Yoke Peng Loh, Niamh Cawley, Bruce J. Baum, Christopher R. Snell
USPTO Applicaton #: #20110135614 - Class: 424 9321 (USPTO) - 06/09/11 - Class 424 
Drug, Bio-affecting And Body Treating Compositions > Whole Live Micro-organism, Cell, Or Virus Containing >Genetically Modified Micro-organism, Cell, Or Virus (e.g., Transformed, Fused, Hybrid, Etc.) >Eukaryotic Cell

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The Patent Description & Claims data below is from USPTO Patent Application 20110135614, Modified growth hormone.

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TECHNICAL

FIELD OF THE INVENTION

The present invention relates to modified growth hormone and related compositions, nucleic acids, vectors, isolated host cells comprising such vectors, and methods of manufacture and use.

BACKGROUND OF THE INVENTION

Growth hormone acts through binding to membrane receptors that belong to the cytokine receptor superfamily (Finidori, Vitam. Horm. 59: 71-97 (2000)). Ligand binding induces receptor dimerization and activation of the receptor-associated kinase JAK-2, resulting in phosphorylation of the kinase, the receptor and many cellular proteins (Finidori (2000), supra). Activation by growth hormone is very transient and several mechanisms are involved in downregulation, including internalization and degradation of the receptor and recruitment of phosphatases or specific inhibitors of the JAK/Stat pathway, namely the SOCS proteins (Finidori (2000), supra).

There are variant forms of human growth hormone (hGH) which include a disulfide dimer, a glycosylated form (20 kD hGH) and two pituitary peptides made up of portions of 22 kD hGH (Lewis et al., Endocr. J. 47 Suppl: S1-8 (March 2000)). The two pituitary peptides (hGH (1-43) and hGH (44-191)) have, respectively, insulin-potentiating and anti-insulin properties (Lewis et al. (March 2000), supra). The smaller peptide may be useful in decreasing the amount of exogenous insulin required by diabetics, whereas the larger peptide may be involved in diabetic retinopathy (Lewis et al. (March 2000), supra).

The increased availability of growth hormone (GH) in the mid-1980s, due to advances in recombinant DNA technology, has allowed research into the use of this hormone at physiological dosage as replacement therapy for adults and children with GH deficiency (GHD) (see, e.g., Carroll et al., Trends Endocrinol. Metab. 11(6): 231-238 (August 2000)) and at pharmocological dosages as a possible therapeutic agent for a number of disease states (Murray et al., Expert Opin. Pharmacother. 1(5): 975-990 (July 2000); see, also, Wit, Endocr. Regul. 34(1): 28-32 (March 2000)). Such disease states include frailty associated with ageing, osteoporosis, morbid obesity, cardiac failure, major thermal injury, hypoglycemic unawareness in diabetes mellitus (Sonksen et al., U.S. Pat. No. 5,426,096, issued Jun. 20, 1995), various acute and chronic catabolic conditions (Murray et al. (July 2000), supra; see, also, Mehls et al., Growth Horm. IGF Res. 10 Suppl. B: S31-37 (April 2000)) and intoxication with poisonous substances that are degraded in the liver by microsomal enzymes (Jorgensen, U.S. Pat. No. 4,816,439, issued Mar. 28, 1989). In combination with DHEA, its use has been proposed for regenerating an involuted thymus (Fahy, International Patent Application WO 95/32991, published Dec. 7, 1995).

MAD in humans is currently treated by growth hormone injection. The problem with such a treatment method is that injections are required every day or couple of days (MacGillivray et al., J. Clin. Endocrinol. Metab. 81(5):1806-1809 (May 1996)). The present invention seeks to overcome such a problem. This and other objects and advantages, as well as additional inventive features, will become apparent from the detailed description provided herein.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides an isolated and purified GH in which the regulated secretory pathway (RSP) sorting signal has been mutated as well as an isolated and purified GH in which the three-dimensional conformation of the RSP sorting signal has been altered. Also provided is a composition comprising an effective amount of such an isolated and purified GH in an excipient.

Further provided is an isolated and purified nucleic acid molecule encoding GH in which the RSP sorting signal has been mutated such that the GH can be constitutively secreted by the nonregulated secretory pathway (NRSP) in a mammalian cell as well as an isolated and purified nucleic acid molecule encoding GH in which the three-dimensional conformation of the RSP sorting signal has been altered such that the GH can be constitutively secreted by the NRSP in a mammalian cell. Still further provided are a vector comprising such an isolated and purified nucleic acid molecule and an isolated host cell comprising such a vector.

A method of treating GHD in a mammal is also provided. The method comprises administering to the mammal the aforementioned composition, nucleic acid or vector, wherein the nucleic acid or vector expresses an effective amount of the encoded GH and whereupon GHD in the mammal is treated.

Also provided are a method of making a GH in which the RSP sorting signal is mutated and the GH so produced. The method comprises mutating one or more amino acids in the RSP sorting signal in GH, whereupon a GH in which the RSP sorting signal is mutated is obtained.

Still also provided are a method of making a GH in which the three-dimensional conformation of the RSP sorting signal is altered and the GH so produced. The method comprises mutating one or more amino acids outside of the RSP sorting signal so that the three-dimensional conformation of the amino acids of the RSP sorting signal in GH is altered, whereupon a GH with an RSP having an altered three-dimensional conformation is obtained.

Similarly provided is a method of making a nucleic acid molecule encoding a GH that can be constitutively secreted by the NRSP in a mammalian cell and the nucleic acid molecule so produced. The method comprises mutating one or more codons encoding amino acids in the RSP sorting signal in an isolated and purified nucleic acid molecule encoding GH such that, upon expression in a mammalian cell, the GH can be constitutively secreted by the NRSP in a mammalian cell.

Also similarly provided is a method of making a nucleic acid molecule encoding a GH that can be constitutively secreted by the NRSP in a mammalian cell and the nucleic acid molecule so produced. The method comprises mutating one or more codons encoding amino acids outside of the RSP sorting signal in an isolated and purified nucleic acid molecule encoding GH such that the three-dimensional conformation of the amino acids of the RSP sorting signal in GH is altered and, upon expression in a mammalian cell, the GH can be constitutively secreted by the NRSP in a mammalian cell.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 sets forth nucleic acid (SEQ ID NO: 1) and amino acid (SEQ ID NOS: 2 (lower case letters represent single letter designations of amino acids in accordance with convention) and 3) sequences with respect to GH and modified GH.

DETAILED DESCRIPTION

OF THE INVENTION

The present invention provides an isolated and purified GH in which the RSP sorting signal has been mutated. Any GH can be modified in accordance with the present invention. Preferably, the GH is mammalian. Particularly preferred is hGH. The amino acid sequence of hGH is known (Genbank accession no. A 15072; see also biosynthetic hGH of Dalboge et al., U.S. Pat. No. 5,633,352, issued May 27, 1997, and U.S. Pat. No. 5,635,604, issued Jun. 3, 1997) and is reproduced herein as SEQ ID NO: 2 (see FIG. 1). By “mutated” is meant chemical modification, substitution, deletion or insertion. Methods of chemical modification, substitution, deletion and insertion are known in the art and include in vitro chemical synthesis (e.g., Merrifield synthesis) of the desired mutant GH (see, e.g., Barany et al., in The Peptides, Gross and Meienhofer, eds. Academic Press: New York (1979), Vol. 2, pp. 3-254; and Parkhurst et al., J. Immunol. 157: 2539-2548 (1996)). Substitution is preferred. Preferably, the isolated and purified GH consists essentially of the amino acid sequence of SEQ ID NO: 2 in which the sorting signal comprises glutamic acid at amino acid position 174 (Glu 174), leucine at amino acid position 177 (Leu 177), valine at amino acid position 185 (Val 185) and glutamic acid at amino acid position 186 (Glu 186) and one or more of the aforementioned amino acids is mutated. Preferably, each of Glu 174 and Glu 186 is mutated, preferably by substitution with alanine.

Especially preferred is when the isolated and purified GH consists essentially of the amino acid sequence of SEQ ID NO: 2 in which the sorting signal comprises glutamic acid at amino acid position 174 (Glu 174), leucine at amino acid position 177 (Leu 177), valine at amino acid position 185 (Val 185) and glutamic acid at amino acid position 186 (Glu 186), each of Glu 174 and Glu 186 is substituted with alanine, and Phe 191 is substituted with LLGILQISSTVAAARV (see SEQ ID NO: 3 in FIG. 1). Optionally, Leu 177 and/or Val 185 is/are mutated, such as by substitution.

Also provided is an isolated and purified GH in which the three-dimensional conformation of the RSP sorting signal has been altered. Preferably, the isolated and purified GH consists essentially of the amino acid sequence of SEQ ID NO: 2 in which the sorting signal comprises Glu 174, Leu 177, Val 185 and Glu 186 and in which one or more amino acids outside of the RSP sorting signal is mutated. Preferably, the cysteine at amino acid position 189 (Cys 189) is mutated, preferably by substitution with serine.

While the above-described mutations are preferred, other mutations that either interfere with the charge of the acidic residues or alter the three-dimensional conformation of the sorting signal are possible. For example, a positively charged residue(s), such as arginine or lysine, can be introduced close to or beside either one or both of the glutamic acid residues in order to neutralize the acidic charge of the neighboring glutamic acid residue. Alternatively, a praline residue can be introduced near the sorting signal so as to alter the three-dimensional conformation of the sorting signal region.



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stats Patent Info
Application #
US 20110135614 A1
Publish Date
06/09/2011
Document #
File Date
04/17/2014
USPTO Class
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