Nucleic acid molecules that bind prion proteins and...

Details Nucleic acid molecules that bind prion proteins and... Nucleic acid molecules that bind prion proteins and...

1998-10-02

2002-07-30

Ketter, James (Department: 1635)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S320100

Reexamination Certificate

active

06426409

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to a process for the identification and isolation of nucleic acid molecules capable of distinguishing the isoforms PrP
c
and PrP
Sc
of prion proteins as well as to the nucleic acid molecules obtainable by this process. Furthermore, the invention relates to pharmaceutical and diagnostic compositions comprising said nucleic acid molecules.
Proteinaceous infectious particles called prions are thought to be the causative agent of transmissible spongiform encephalopathies (TSEs) such as Scrapie of sheep, bovine spongiform encephalopathy (BSE) of calf, transmissible minc encephalopathy (TME) of mink as well as Kuru, Gerstmann-Sträussler-Scheinker syndrome (GSS), Creutzfeldt-Jakob-Disease (CJD) and fatal familial insomnia (FFI) in the case of humans (Prusiner, 1982). The main component of prions associated in amyloid-like rods (Prusiner et al., 1983; 1984) or scrapie associated fibrils (SAF; Hope et al., 1986) was found to be the prion protein PrP27-30 (Prusiner et al., 1981; Prusiner et al., 1983), an N-terminal truncated, highly protease resistant version of the prion protein PrP
Sc
(Oesch et al., 1994), which is also found to a minor extend in prion preparations (Prusiner et al. 1983). PrP27-30, which is devoid of 67 amino acids at the aminoterminal end, results from PrP
Sc
by proteinase K digestions (Prusiner et al., 1984; Stahl et al., 1993) or by lysosomal protease digestion (Caughey et al., 1991). The distribution of PrP
Sc
and PrP27-30 in prion preparations varies dependent from the absence or presence of proteases.
No specific nucleic acid could be detected so far in prion preparations (Kellings et al., 1992) suggesting that the prion is infectious and can replicate in the absence of any nucleic acid (Prusiner, 1982). According to the protein-only hypothesis (Prusiner, 1982) exogenous PrP
Sc
/PrP27-30 could convert the ubiquitous cellular isoform PrP
c
to PrP
Sc
/PrP27-30. It is assumed that chaperons may be involved in this process (Edenhofer et al., 1996). PrP
Sc
/PrP27-30 could appear as a monomer (Prusiner. 1982) or as a nucleation or crystal seed consisting of a PrP
Sc
/PrP27-30 oligomer (Lansbury and Caughey. 1995). PrP
c
differs from PrP27-30 only with respect to its secondary structure: the &agr;-helical and &bgr;-sheet contents of PrP
c
are 42% and 3%, respectively (Pan et al., 1993). In contrast. the &agr;-helical and &bgr;-sheet contents of PrP27-30 were proven to be 21% and 54%. respectively (Pan et al., 1993). These results indicate that the conversion of PrP
c
to PrP
Sc
/PrP27-30 is most likely concomitant with extreme alterations in the secondary structure of the prion protein. Although a series of experiments employing knock-out mice which no longer express PrP
c
suggest that cellular prion proteins could play a crucial role in a number of cellular processes (Collinge et al., 1994; Sakaguchi et al., 1996: Tobler et al., 1996), a precise physiological role of PrP
c
remains speculative. It is, however, proven that PrP
c
is necessary for the development of transmissible spongiform encephalopathies (Büeler et al., 1993; Brandner et al., 1996).
Translation of the mRNA from Scrapie infected Syrian golden hamster has led to a 254 amino acid protein including a 22 amino acid signal peptide at the NH
2
-terminus and a 23 amino acid signal sequence at the carboxy terminus (Oesch et al., 1985; Basler et al., 1986). The mature protein PrP
c
as well as the Scrapie isoform PrP
Sc
contain amino acids 23 to 231. Only PrP
Sc
can be processed to the proteinase K resistant isoform PrP27-30 (amino acids 90-231) consisting of 142 amino acids (Prusiner et al, 1984).
This property has been used to design a diagnostic assay for diseases in connection with prion proteins in which a probe is treated with proteinase K in order to degrade all PrP
c
and then reacted with an antibody directed against prion proteins (Groschup et al., 1994). However, this assay has the disadvantage that sensitivity might be hampered by the fact that the proteinase K digestion of PrP
c
is not complete, thereby leading to false positive results. Furthermore, the additional step of proteinase K digestion is time consuming. In order to be able to directly assay for the presence or absence of PrP
c
and/or PrP
Sc
one would need antibodies which could distinguish between these two isoforms.
However, so far attempts to provide antibodies that can distinguish between the cellular isoform PrP
c
and the isoforms PrP
Sc
, as well as the truncated version PrP27-30, have failed (Groschup et al.. 1994 and ref. therein). Thus, up to now it was not possible to distinguish the isoforms PrP
c
and PrP
Sc
of prion proteins by immunological or other means which would be the prerequisite for a simple and reliable method of diagnosing a transmissible spongiform encephalopathy.
Therefore, the technical problem underlying the present invention is to provide a process for the identification and isolation of molecules which are capable of distinguishing between the isoforms PrP
c
and PrP
Sc
or PrP27-30 of prion proteins and which are useful tools for diagnosis and therapy of transmissible spongiform encephalopathies.
The solution to said technical problem is achieved by the provision of the embodiments characterized by the patent claims.
SUMMARY OF THE INVENTION
Thus, the present invention relates to a process for the identification and isolation of nucleic acid molecules which are capable of distinguishing between the isoforms PrP
c
and PrP
Sc
or PrP27-30 of prion proteins associated with transmissible spongiform encephalopathies comprising the steps of
(i) incubating a prion protein isoform or peptide fragment or derivative of this prion protein isoform with a pool of nucleic acid molecules comprising different sequences;
(ii) selecting and isolating those nucleic acid molecules which are capable of binding to said prion protein isoform or fragment or derivative thereof;
(iii) optionally, amplifying the isolated nucleic acid molecules and repeating steps (i) and (ii); and
(iv) determining the binding specificity of the isolated nucleic acid molecules for the PrP
c
and PrP
Sc
or PrP27-30 isoforms of prion proteins.
DETAILED DESCRIPTION OF THE INVENTION
The process according to the invention is based on a method called “in vitro selection”. This method allows for the identification of nucleic acid molecules (RNA, modified RNA. ssDNA or dsDNA) which bind with high affinity to a defined molecular target from a large randomized population of nucleic acid molecules (Tuerk and Gold, 1990; Famulok and Szostak, 1992). Using this method it has been possible to isolate nucleic acids specifically recognizing a variety of protein targets including HIV-1 reverse transcriptase (Tuerk et al., 1992), HIV-1 Integrase (Allen et al., 1995), human &agr;-thrombin (Kubik et al., 1994) and Drosphila sex-lethal protein (Sakashita and Sakamoto, 1994). However, up to now it has not been possible to provide by this method nucleic acid molecules being capable of distinguishing the two isoforms of prion proteins, PrP
c
and PrP
Sc
. In the scope of the present invention the term PrP
c
comprises the cellular isoform of the prion protein as well as fragments and derivatives thereof irrespective of the source organism. The term PrP
Sc
comprises the isoform of the prion protein associated with various transmissible spongiform encephalopathies. This term also comprises fragments of this prion protein isoform such as the truncated version of the isoform PrP
Sc
, the prion protein PrP27-30, which is the main component of prions. In particular, this term also includes PrP
Sc
proteins of the various Scrapie strains including those adapted to hamster, mouse or other vertebrates. Also included are derivatives of the prion protein isoform PrP
Sc
.
The term derivatives includes chemically modified versions of the prion protein isoforms PrP
c
and PrP
Sc
as well as mutants of these proteins, namely proteins which differ from the naturally occurring prion protein isoforms at one or more

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