Drug – bio-affecting and body treating compositions – In vivo diagnosis or in vivo testing
Reexamination Certificate
1999-11-02
2003-03-18
Swartz, Rodney P (Department: 1641)
Drug, bio-affecting and body treating compositions
In vivo diagnosis or in vivo testing
C424S009200, C424S184100, C424S185100, C424S278100, C435S325000, C530S300000, C530S350000
Reexamination Certificate
active
06534036
ABSTRACT:
The present invention relates to prion proteins.
Prions are infectious pathogens that differ from bacteria, fungi, parasites, viroids, and viruses, both with respect to their structure and with respect to the diseases that they cause. Molecular biological and structural studies of prions promise to open new vistas into fundamental mechanisms of cellular regulation and homeostasis not previously appreciated. Kuru, Creutzfeldt-Jakob disease (CJD), fatal familial insomnia (FFI) and Gerstmann-Sträussler-Scheinker syndrome (GSS) are all human neurodegenerative diseases that are caused by prions and are frequently transmissible to laboratory animals. Familial CJD and GSS are also genetic disorders. No effective therapy exists to prevent these fatal disorders
2
.
In addition to the prion diseases of humans, disorders of animals are included in the group of known prion diseases. Scrapie of sheep and goats is the most studied of the prion diseases. Bovine spongiform encephalopathy (BSE) is thought to result from abnormal feeding practices. BSE threatens the beef industry of Great Britain and possibly other countries; the production of pharmaceuticals involving cattle is also of concern. Control of sheep scrapie in many countries is a persistent and vexing problem
2
.
Since 1986, more than 170,000 cattle have developed BSE in Great Britain. Many investigators contend that BSE, often referred to as “mad cow disease”, resulted from the feeding of dietary protein supplements derived from rendered sheep offal infected with scrapie to cattle, a practice banned since 1988. It is thought that BSE will disappear with the cessation of feeding rendered meat and bone meal, as has been the case in kuru of humans, confined to the Fore region of New Guinea and once the most common cause of death among women and children. Kuru has almost disappeared with the cessation of ritualistic cannibalism.
Prion diseases are associated with the accumulation of a conformational isomer (PrP
Sc
) of host-derived prion protein (PrP
c
) with an increase in its &bgr;-sheet content
1
. According to the protein-only hypothesis, PrP
Sc
is the principal or sole component of transmissible prions
2
. Although the structure of PrP
c
has been determined
3
and has been found to consist predominantly of &agr;-helices, the insolubility of PrP
Sc
, which is isolated from tissue in a highly aggregated state and which has a high &bgr;-sheet content, has precluded high-resolution structural analysis. Various workers have attempted to make forms of PrP which are intermediate between the normal (PrP
c
) form and the abnormal, pathogenic form (PrP
Sc
), having a predominantly &bgr;-sheet form therefore termed the &bgr;-form.
Hornemann & Glockshuber
PNAS
95, 6010-6014 (1998)
8
describe a &bgr;-intermediate which is an unfolding intermediate of mouse PrP and contains predominantly &bgr;-sheet elements of secondary structure as opposed to &agr;-helix. Swietnicki et al (1997)
J. Biol. Chem
. 272:44, October 31 pp27517-27520 describe an identical folding intermediate derived from human PrP
90-231
. The mouse &bgr;-intermediate is derived from oxidised PrP which contains the native disulphide bond. The mouse PrP intermediate required urea (a denaturant) for stabilisation. The reference on page 6011 “Results” states that the mouse &bgr;-intermediate exhibits stability at pH 4.0 in the absence of denaturant; however this is based upon an equilibrium calculation. The free energy of folding (Table 1, page 6012) is approximated from a fit of the equation described in Materials and Methods (page 6011) to the data in FIG.
1
A. From this an equilibrium constant can be calculated which describes the small proportion of molecules that will exist as the &bgr;-intermediate in the absence of denaturant. The proportion of molecules in this state is low (around 0.2%) and nothing can be said about their solubility in the absence of denaturant as they are not detectable. Indeed one would argue they are extremely unlikely to be soluble in the absence of denaturant because folding intermediates are structural states that are populated during rearrangement of a polypeptide chain from a random structure to a defined native conformation, or vice versa. They are characterised as having native-like secondary structure, few tertiary interactions, increased molecular volume, increased side chain mobility and exposed hydrophobic residues. These properties combined make them prone to aggregation and, as such, are generally insoluble in the absence of denaturants. Several references describe these properties in detail
18-23
.
Moreover, the above calculation is dependent upon the transition being a genuine equilibrium, ie. fully reversible. If the transition is not reversible this analysis is invalid. We have performed similar experiments and have found that full reversibility is abolished at protein concentrations in excess of 1 mg/ml, with refolding yields <100%.
Zhang et al (1997)
Biochem
36:12, 3543-3553 describe a &bgr;-sheet form of recombinant Syrian hamster PrP containing residues 90-231 which is formed by a method involving refolding at a pH of 6.5. It is clear from page 3548, second column and FIG. 7, that the &bgr;-form described is neither monomeric nor soluble in aqueous solution.
According to a first aspect the invention provides a method of making a &bgr;-form of a prion protein which has more &bgr;-sheet than &agr;-helix structure, can exist as a monomer and can retain solubility in aqueous solution in the s absence of a denaturant, the method comprising:
providing a reduced prion protein which does not include a disulphide bond and causing the conformation of the protein to change so that it adopts the &bgr;-form.
Preferably, the change in conformation is caused by exposure to conditions of acidic pH, preferably a pH of 5.5 or less, more preferably a pH of 4.8 or less and most preferably a pH of 4.0.
Skilled persons will appreciate that the &bgr;-sheet and &agr;-helix structure can be shown by circular dichroism spectropolarimetry as described herein. While the native prion protein state is characterised by a strong &agr;-helical signal, the &bgr;-form of the invention shows a shift to a conformation dominated by &bgr;-sheet. By “dominated” in this context we include the meaning that there is more &bgr;-sheet structure of the prion protein than &agr;-helix structure.
By “exist as a monomer” we include the meaning that the &bgr;-form of the prion protein does not exist as an aggregate of two or more &bgr;-form prion proteins. Skilled persons will appreciate that analytical sedimentation studies can be used to determine whether or not a protein exists in solution as a monomer or as an aggregate of two or more proteins. A suitable technique is described in Zhang et al (1997) Biochem, 36:12, 3542-3553 (see page 3545-3546 passage entitled Analytical Sedimentation). The technique involves the use of an analytical ultracentrifuge (Beckman Optimat XL-A) equipped with a six channel cell, using ultraviolet absorption between 220 and 280 nm.
By “can retain solubility in the absence of a denaturant” we include the meaning that a significant proportion eg around 30% or more of the &bgr;-form remains in solution as a monomer after centrifugation at 100,000 g for 1 hour and preferably 150,000 g for 8-16 hours, most preferably at 200,000 g for 8-16 hours. The centrifugation may be carried out on a 2 mg/ml aqueous solution of the &bgr;-form prion protein comprising Na Acetate+10 mM Tris. HCl+pH 4.0 at 25° C. The structural characteristics of the remaining protein in solution can be determined by circular dichroism spectropolarimetry, for example.
Preferably, the &bgr;-form remains soluble without denaturant to a concentration of more than 1 mg/ml, more preferably at least 12 mg/ml, and especially more than 20 mg/ml.
It will of course be appreciated that the above requirement for the &bgr;-form to be capable of retaining solubility in the absence of the denaturant in no way limits the invention to methods or compositions which do not include a dena
Clarke Anthony R.
Collinge John
Jackson Graham S.
D. Gen Limited
Mersereau C. G.
Nikolai & Mersereau , P.A.
Swartz Rodney P
LandOfFree
Biological materials and methods useful in the diagnosis and... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Biological materials and methods useful in the diagnosis and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Biological materials and methods useful in the diagnosis and... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3006237