Correction of genetic defects using chemical chaperones

Details Correction of genetic defects using chemical chaperones Correction of genetic defects using chemical chaperones

2001-03-30

2003-04-01

Leary, Louise N. (Department: 1627)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

C435S026000, C435S023000, C435S024000, C435S963000

Reexamination Certificate

active

06541195

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to the treatment of phenotypic defects that are caused by improper protein folding and processing.
When a protein is synthesized, its amino acid side chains interact, causing the polypeptide backbone to fold into thermodynamically preferred three dimensional structures or “conformations.” The biological properties and proper localization (either within the cell or secreted out of the cell) of proteins are contingent on assuming certain biologically significant conformations. Proteins that for whatever reason do not assume “correct” biologically active conformations are inactive and/or misprocessed and/or mislocalized and/or degraded. Failure to assume a proper conformation can lead to disease, and can be fatal. Reviewed in Thomas, P. J. et al.,
TIBS,
20:456-459 (1995).
Abnormalities in protein folding constitute the molecular basis for a number of diseases. Thomas, P. J., et al., 1995,
TIBS
20:456-459; Welch, W. J. et al., 1996,
Cell Stress & Chap.
1:109-115. Oftentimes single point or deletion mutations give rise to subtle folding defects that result in either a loss of protein function, or a failure of the protein to be correctly localized. A number of pathological conditions are reportedly the result of improper folding. For example, in cystic fibrosis, a mutation (e.g., &Dgr;F508) which results in improper folding leads to improper targeting of the cystic fibrosis transmembrane conductance regulator; mutant proteins are retained in the endoplasmic reticulum and not delivered to their normal site of action at the plasma membrane. Cheng, S. H. et al.,
Cell,
63:827 (1990); Denning, G. M. et al.,
Nature,
358:761 (1992); Gregory et al.,
Mol. Cell. Biol.,
11:3886 (1991); Kartner et al.,
Nature Genet.,
1:321 (1992); G. M. Denning, G. M. et al.,
J. Cell Biol.,
118:551 (1992). In emphysema and chronic liver diseases, conformational defects result in the failure to secrete alpha-1 antitrypsin inhibitor, leading to tissue damage. Lomas, D. A. et al.,
Nature,
357:605-607 (1992); Lomas, D. A. et al.,
Am. J. Physiol.,
265:L211-219 (1993). In familial hypercholesterolemia, a mutation within the coding region of low density lipoprotein (LDL) receptor results in a failure of the protein to localize to the plasma membrane, leading to abnormal levels of serum cholesterol and heart disease. Amara, J. F. et al.,
Trends Cell Biol.,
2:145-149 (1992); Hobbs, H. H. et al.,
Annu. Rev. Genet.,
24:133-170 (1990); Yamamoto, T. et al.,
Science,
232:1230-1237 (1986). In Tay Sachs disease, a mutation within the coding region of the alpha subunit of beta-hexosaminidase is not delivered to its normal lysosomal location; the mutant protein is instead retained in the endoplasmic reticulum. Amara, J. F. et al., supra; Lau, M. M. H. et al.,
J. Biol. Chem.,
264:21376-21380 (1989). Other diseased states that are due to improperly folded protein are known in the art. Bychkova, V. E. et al.,
FEBS Let.,
359:6-8 (1995); Thomas, P. J. et al.,
Trends Biol. Sci.,
20:456-459 (1995).
Factors which influence a protein's preferred conformation include amino acid sequence, intra- and intermolecular charge interactions, hydrophobic interactions, steric interactions, Van der Waals forces, and disulphide bond linkages; reversible and irreversible post-translational modification (e.g., phosphorylation or glycosylation); degree of hydration; and nature and composition of the solvent medium.
The primary driving force for assuming a biologically active conformation in vivo is thought to be the amino acid sequence. Certain proteins have been shown to spontaneously fold to assume their proper conformation, even after repeated cycles of denaturation and renaturation. Changes in sequence (e.g., mutations) may result in dramatic conformational alterations.
A number of low molecular weight compounds are reportedly effective in stabilizing proteins in vitro against thermally induced denaturation. Germsla, et al., 1972,
Int. J. Pept. Proteins Res.
4:372-378; Back, J. F., et al., 1979,
Biochem.
18:5191-5199; Gekko, K. et al., 1983,
J. Biochem.
94:199-208. Representative compounds include polyols such as glycerol, solvents such as DMSO, and deuterated water (D
2
O). In addition to their effects in vitro some of these compounds appear to influence protein folding and/or stability in vivo. Lin, P. S. et al., 1981,
J. Cell. Physiol.
108:439-448; Henle, K. J. et al., 1983,
Cancer Res.
43:1624-1633; Edington, B. V. et al., 1989,
J. Cell. Physiol.
139:219-228. For example, animal cells incubated in the presence of either deuterated water or glycerol can withstand severe heat shock treatments that would otherwise be lethal to the cells. Here addition of the compounds to the cells helps to reduce the overall extent of thermal denaturation of intracellular proteins. In yeast and bacteria, the addition of glycerol into the growth medium not only protects the cells against thermal treatments, but in some cases also is effective in correcting protein folding abnormalities due to specific mutations. Hawthorne, D. C. et al., 1964,
Genetics
50:829-839. This type of “osmotic remediation” has been shown to be the most effective for those mutant proteins which exhibit a temperature sensitive protein folding defect.
Some proteins appear to require interaction with other molecules in order to fold properly. Substances that aid proteins to assume their biologically active conformations have been identified in a variety of cell types and cell compartments. Fischer, G. et al.,
Biochemistry,
29:2206-2212 (1990); Freedman, R. B.,
Cell,
57:1069-1072 (1989); Ellis et al.,
Annu. Rev. Biochem.,
60:337-347 (1991). Among the best known are a class of proteins called “molecular chaperones” (Dingwall, C. K. et al.,
Seminars in Cell Biol.,
1:11-17 (1990)); (Hemmingsen, S. M. et al.,
Nature,
333:330-334 (1988)), or “polypeptide chain binding proteins” (Rothman, J. E.,
Cell,
59:591-601 (1989)). Chaperones stabilize newly synthesized polypeptides until they are assembled into their proper native structure or until they are transported to another cellular compartment, i.e., for secretion. Sambrook et al.,
Nature,
342:224-225 (1989); U.S. Pat. No. 5,474,914, which issued to R. Spaete on Dec. 12, 1995. They reportedly prevent the formation of undesirable protein aggregates by binding to unfolded or partially denatured polypeptides.
The heat-shock proteins of the hsp70 and hsp60 families are examples of chaperones. Langer, T. et al.,
Curr. Topics in Microbiol. and Immun.,
167:3-30 (1991); Pelham, H. R. B.,
Nature,
332:776-777 (1988); and Hartl, F.,
Seminars in Immunol.
3, (1991). U.S. Ser. No. 07/261,573, filed Oct. 24, 1988, described the folding function of hsp60, isolated from yeast mitochondria. See also McMullin, T. W. et al.,
Molec. Cell. Biol.
8:371-380 (1988); Reading, D. S. et al.,
Nature,
337:655-659 (1989); Cheng, M. Y. et al.,
Nature,
337:620-625 (1989); Ostermann, J. et al.,
Nature,
341:125-130 (1989); and Cheng, M. Y. et al.,
Nature,
348:455-458 (1990)). The essential role in protein folding of the members of the hsp60 family has since been demonstrated in vivo and in vitro. Other chaperones include the rubisco binding protein of chloroplasts, reviewed by Barraclough, R. et al.,
Biochim. Biophys. Acta,
608:19-31 (1980); Musgrove, J. E. et al.,
Eur. J. Biochem.,
163:529-534 (1987); and Gatenby, A. A. et al.,
Rev. Cell Biol.,
6:125-149 (1990), and proteins such as GroEL, isolated from
E. coli
(Georgopoulos, C. et al.,
J. Molec. Biol.,
76:45-60 (1973); Stomborg, N. J.,
Molec. Biol.,
76:25-44 (1973); Hendrix, R. W. J.,
Molec. Biol,
129:375-392 (1979); Bochkareva, E. S. et al.,
Nature,
336:254-257 (1988); Goloubinoff, P. et al.,
Nature,
342:884-889 (1989); Van Dyk, T. K. et al.,
Nature,
342:451-453; Lecker, S. et al.,
EMBO J.,
8:2703-2709 (1989); Laminet, A. A. et al.,
EMBO J.,
9:2315-2319 (1990); Buchner, J. et al.,
Biochemistry,
30:1586-1591 (1991)). U.S. Ser. No. 07/721,974 entitled “Chaperonin-Mediated Pr

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