Prion propagation inhibition by dominant-negative prion...

Details Prion propagation inhibition by dominant-negative prion... Prion propagation inhibition by dominant-negative prion...

1999-11-03

2003-07-15

Shukla, Ram R. (Department: 1632)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S320100, C435S325000, C435S455000, C536S023500

Reexamination Certificate

active

06593105

ABSTRACT:

The present invention relates to a vector having a nucleic acid insert coding for a mutated prion protein, a mutated prion protein, a DNA coding for such a protein and a process for the preparation thereof. Furthermore, this invention concerns antibodies, a vaccination agent and the use of the above-mentioned subject matters. Moreover, the present invention relates to non-human mammals, e.g. working animals, which are resistant to prion infections.
The prion diseases which have spread in the past few years and include inter alia BSE, Creutzfeldt-Jakob disease and scrapie, represent a group of absolutely fatal neurodegenerative diseases in human beings and animals which are caused by infectious pathogens whose structure is still unclear and which are referred to as “prions” below. For the time being there are no satisfactory possibilities regarding the prophylaxis and/or treatment of these diseases. The development of such prophylaxis/treatment possibilities is made more difficult because the molecular pathogenesis of prion diseases is presently not clarified to a sufficient extent. Only a slight delay of the prion replication has been achieved by means of various drugs so far, but the infection could by no means be brought to a stand-still (Priola et al., Inhibition of scrapie-associated PrP accumulation. Probing the role of glycosaminoglycans in amyloidogenesis, Mol. Neurobiol. 8, pp. 113-120 (1994). Furthermore, such a treatment is accompanied by considerable side-effects, which is due to the fact that these drugs only have a very unspecific effect.
Therefore, it is the object of the present invention to provide a product serving for rendering working animals whose tissues or products are used as foodstuffs, drug constituents or cosmetics for human use resistant to prion propagation and protecting infected human or animal individuals from the onset of the infection and the progression thereof, respectively.
According to the invention this is achieved by the subject matters defined in the claims.
Thus, the subject matter of the present invention relates to a vector containing a nucleic acid insert coding for a mutated prion protein, the mutated prion protein having an insertion, substitution and/or deletion within the H1 region. It should preferably be a vector suitable for gene prophylaxis and gene therapy, respectively.
On the basis of this invention the inventors modified critical regions, e.g. the Helix1 region (H1) (Gasset et al., Predicted alpha-helical regions of the prion protein when synthesized as peptides form amyloid, Proc. Natl. Acad. Sci. USA 89, p. 10940, 1992) in the prion protein by means of genetic technique by carrying out e.g. mutations in the form of insertions, substitutions or deletions, particularly deletions. In this connection, the inventors found out that these modified recombinant proteins lose their capability of being convertible into the amyloid-like, disease-associated form of the prion protein (PrP
Sc
). In addition, it was found that the presence of the mutated proteins has an inhibitory effect on the conversion of the endogenous wild-type prion protein (PrP
c
) which is naturally present in the organism into the disease-associated form (PrP
Sc
) (what is called dominant-negative effect).
The expression “vector suitable for gene therapy” comprises any vector which is suitable for the gene transfer, i.e. the introduction of nucleic acids into cells. The vector may remain episomally within the cells or be integrated into the genome. Furthermore, the vector may be a plasmid vector or viral vector. Examples of viral vectors are retroviral, herpes simples virus, cytomegaly virus (CMV), adenovirus, vaccinia virus or adeno-associated virus (AAV) vectors. pUC18 or pUC19 have to be mentioned as preferred plasmid vectors.
The expression “mutated prion protein” comprises a prion protein which as compared to the wild-type has been modified as regards its function and/or its length by means of suitable measures with which a person skilled in the art is sufficiently familiar. This may be done e.g. by inserting mutations on a nucleic acid level, e.g. by carrying out insertions, substitutions and particularly deletions. Methods of producing the above modifications within the nucleic acid sequence are known to the person skilled in the art and described in standard works of molecular biology, e.g. in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2
nd
edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y. (1989). The mutation is carried out within the H1 region of the prion protein within which an accumulation of identical amino acids occurs (Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala) and which is conserved with all animal species. As to the sequences of the prion proteins of various organisms reference shall be made to Prusiner, S. B., Molecular structure, biology and genetics of prions, Adv. Virus Res. 35, p. 83 et seq. (1988); Schätzl, H. M. et al., Prion protein gene variation among primates, J. Mol. Biol. 245, p. 362 et seq. (1995) and Gabriel, J. M. et al., Molecular cloning of a candidate chicken prion protein, Proc. Natl., Acad. Sci. USA 89, p. 9097 et seq. (1992). In a particularly preferred embodiment, the modification concerns amino acids 108-121 (based on the mouse PrP sequence), preferably 112-121, most preferably 114-121. This modification must usefully cover more than one amino acid, i.e. at least two, since otherwise the described properties such as the non-convertibility into the disease-associated form of the prion protein (PrP
Sc
) and/or the trans-dominant inhibition of the accumulation of wild-type PrP
Sc
cannot be expected. Since as proved the region of amino acids 112 to 121, preferably 114 to 121, contains the strongest amyloidogenic amino acids of the prion protein, a deletion is the preferred route of preparing a modified protein comprising the above-mentioned properties. Following the deletion of at least two of the amyloidogenic amino acids in the above-mentioned region, another route is the substitution by amino acids which are known to have no tendency of forming a &bgr;-pleated sheet structure or can even destroy an existing &bgr;-pleated sheet structure (e.g. proline). The direct insertion of amino acids which counteract the formation of a &bgr;-pleated sheet structure, e.g. proline, is also conceivable. Another advantageous modification of the prion protein is made at the carboxy terminus. The already spontaneously occurring secretion of PrP molecules is enhanced by this modification (Borchelt et al., Release of the cellular prion protein from cultured cells after loss of its glycoinositol phospholipid anchor,
Glycobiology
3, pp. 319-329 (1993). This is achieved according to the invention in that mutated prion proteins are secreted to an increased extent by the producing cells by a lack of glycoinositol phospholipid anchor. Thus, it is possible to reach by diffusion also those cells in spatial neighborhood which do not express the mutated prion protein per se. As a result, the desired dominant negative effect is expanded to a much greater number of target cells.
The expression “nucleic acid insert” comprises any nucleic acid such as DNA or RNA which codes for a mutated prion protein. It is favorable for the nucleic acid insert to be expressible, e.g. when it is controlled by a constitutive or inducible promoter. A nucleic acid insert in the form of a DNA which comprises.the following is preferred:
(a) the DNA of
FIG. 4
or a DNA differing therefrom by one or several base pairs,
(b) a DNA hybridizing with the DNA from (a), or
(c) a DNA related to the DNA from (a) or (b) via the degenerated genetic code.
The expression “hybridizing DNA” refers to a DNA which hybridizes with a DNA from (a) under conventional conditions, particularly at 20° C. below the melting point of the DNA. In this connection, the term “hybridizing” refers to conventional hybridization conditions, preferably to hybridization conditions where 5×SSPE, 1% SDS, 1×Denhardts solution are used as the solution and the hybridization te

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