Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1998-12-17
2001-11-13
Wortman, Donna C. (Department: 1648)
Chemistry: molecular biology and microbiology
Vector, per se
C435S069700, C530S350000, C530S826000, C536S023400, C536S027700
Reexamination Certificate
active
06316252
ABSTRACT:
CROSS REFERENCES TO RELATED APPLICATIONS
Not applicable.
BACKGROUND OF THE INVENTION
The present invention relates to a transport peptide for delivering therapeutic and reporter proteins into cells, and vectors capable of expressing such proteins. More specifically it relates to the use of a bovine herpesvirus protein as part of a fusion protein that can be used for such purposes.
An important challenge facing the biomedical field is the need to provide effective methods to introduce therapeutic and reporter proteins into cells. While various existing approaches are adequate in some situations, they are usually deficient in others.
In G. Elliott et al., 73 J. Gen. Virol. 723-726 (1992); G. Elliott et al., 88 Cell 223-233 (1997); and A. Phelan et al., 16 Nat. Biotech. 440-443 (May 1998) the authors describe a human herpesvirus protein “HSV-VP22”. They suggest that it may assist the delivery of proteins that are fused to it to surrounding cells. The disclosure of these publications and of all other publications referred to herein are incorporated by reference as if fully set forth herein.
However, there are safety concerns involved in using proteins from human herpesvirus in vivo in a human, and the efficiency of such a system appears to be undesirably low.
Moreover, the HSV-VP22 protein is a relatively large protein. Thus, due to its size, it is projected to be more likely to interfere with the activity of certain linked effectors, and/or may induce an antibody response.
In X. Liang et al., 69 J. Virol. 3863-3867 (1995) and X. Liang et al., 15 Vaccine 1057-1064 (1997) a bovine herpesvirus gene was described. In X. Liang et al., NCBI Entrez, (Accession U21137) (1996) the nucleotide sequence of this bovine gene (SEQ ID NO: 1) and its protein product (SEQ ID NO: 2) were provided. The gene was being studied in connection with vaccine research.
HSV VP22 is a 38 kD protein of 302 amino acids (906 bp of DNA) while the aforesaid bovine protein is a 32 kD protein of 259 amino acids (777 bp of DNA). Further, utilizing a computer program we determined that there was only about 28.7% amino acid identity between the two proteins.
A need therefore exists for an improved delivery system for delivering biotherapeutic proteins of interest to mammalian cells.
BRIEF SUMMARY OF THE INVENTION
In one aspect the invention provides a peptide which is SEQ ID NO: 2 bovine herpesvirus protein linked to a non-bovine protein selected from the group consisting of therapeutic proteins and reporter proteins. Almost any proteinaceous material that can serve a human or animal therapeutic purpose (e.g. drugs such as enzymes, vaccines, transcription factors, and viral inhibitors) can be linked to the bovine protein and then delivered using the fusion protein. It does not appear to be critical which of the two proteins are at the N-terminus.
As an example we selected a known anti-cancer toxin (restrictocin) (see generally B. Lamy et al., 19 Nuc. Acid. Res. 1001-1006 (1991) and B. Lamy et al., NCBI Entrez, (Accession X56176) (1991)(DNA and amino acid sequence) and SEQ ID NO: 3 (which also provides the amino acid sequence of this toxin protein)).
Alternatively, the SEQ ID NO: 2 protein can be linked to an innocuous reporter protein, such as the Aequorea victoria green fluorescent protein described in D. Prasher et al., NCBI Entrez (Accession M62654) (1992) (DNA and amino acid sequence). See also SEQ ID NO: 4 which also provides the amino acid sequence of that protein. Various other reporter proteins (e.g. radioactive; color indicating; and bioluminescent) can be used such as &bgr;-galactosidase or luciferase.
In another aspect, the invention provides a nucleotide sequence coding for SEQ ID NO: 2 bovine herpesvirus protein linked to a non-bovine protein selected from the group consisting of therapeutic proteins and reporter proteins. A preferred coding sequence for the SEQ ID NO: 2 protein is nucleotides 390 through 1161 of SEQ ID NO: 1, with the following insertions made at the end of the bovine gene starting at nucleotide 1162: acc ggt cgc, followed by an insertion of cac c, followed by the atg of the fusion protein.
In another aspect, the invention provides a nucleotide vector having a vector backbone and a nucleotide sequence coding for SEQ ID NO: 2 bovine herpesvirus protein linked to a non-bovine protein selected from the group consisting of therapeutic proteins and reporter proteins. The vector may be any nucleotide vector that a cell will accept and permit to express within the cell (e.g. plasmid vectors, viral vectors). A preferred vector is a mammalian expression plasmid having a promoter and marker.
The invention is primarily intended for delivering therapeutic and/or reporter proteins to mammalian cells, either in vitro or in vivo. It appears to be especially well suited to deliver such proteins to ruminant and primate (e.g. human) cells. It therefore has in vivo and in vitro pharmaceutical, veterinary and research applications.
The objects of the present invention therefore include providing:
(a) fusion proteins of the above kind that are suitable to deliver biotherapeutic proteins and/or reporter genes to cells of interest;
(b) nucleotide sequences of the above kind that can express such proteins in cells; and
(c) recombinant nucleotide vectors capable of delivering such nucleotide sequences to cells. These and still other objects and advantages of the present invention will be apparent from the description which follows. The following description is merely of the preferred embodiments. Thus, the claims should be looked to in order to understand the full scope of the invention.
REFERENCES:
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patent: 6017735 (2000-01-01), O'Hare et al.
patent: 6184038 (2001-02-01), O'Hare et al.
patent: WO 97/05265 (1997-02-01), None
Harms et al., Distinctions between Bovine Herpesvirus 1 and Herpes Simplex Virus Type 1 VP22 Tegument Protein Subcellular Associations. Journal of Virology 74(7):3301-3312, 2000.*
G. Elliott et al., The herpes simplex virus type 1 tegument protein VP22 is encoded by gene UL49, 73 J. Virol. 723-726 (1992).
G. Elliott et al., Intercellular Trafficking and Protein Delivery by a Herpesvirus Structural Protein, 88 Cell 223-233 (1997).
A. Phelan et al., Intercellular delivery of functional p53 by the herpesvirus protein VP22, 16, Nature Biotech. (May, 1998) (Applicant reserves the right to determine whether this is prior art).
X. Liang et al., excerpt from 69 J. Virol. 3863 (1995) in NCBI nucleotide listing—Bovine herpesvirus 1 virion tegument protein gene, (1996).
X. Liang, et al., Characterization of Bovine Herpesvirus 1 UL49 Homolog Gene and Product: Bovine Herpesvirus 1 UL49 Homolog Is Dispensable for Virus Growth, 69 J. Virol. 3863-3867 (1995).
X. Liang et al., Study of immunogenicity and virulence of bovine herpesvirus 1 mutants deficient in the UL49 homolog, UL49.5 homolog and dUTPase genes in cattle, 15 Vaccine 1057-1064 (1997).
B. Lamy et al., Isolation and nucleotide sequence of theAspergillus restrictusgene coding for the ribonucleolytic toxin restrictocin and its expression inAspergillus nidulans. . . , 19 Nuc. Acids Res. 1001-1006 (1991).
B. Lamy et al., excerpt from Exhibit 4—A. restrictus restrictocin gene (1991).
D. Prasher et al., excerpt from 111 Gene 229-233 (1992) in NCBI nucleotide listing—Aequorea victoriagreen-fluorescent protein mRNA, complete cds. (1992).
Harms Jerome S.
Splitter Gary A.
Wisconsin Alumni Research Foundation
Wortman Donna C.
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