Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-06-14
2001-01-09
Elliott, George C. (Department: 1635)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S006120, C435S091100, C435S320100, C435S325000, C435S375000, C536S024100, C514S04400A
Reexamination Certificate
active
06171821
ABSTRACT:
FIELD OF THE INVENTION
The field of the invention is regulation of protein translation.
BACKGROUND OF THE INVENTION
Programmed cell death plays a critical role in regulating cell turnover during embryogenesis, metamorphosis, tissue homeostasis, viral infections, and cancer. Previously, we identified and cloned three mammalian genes encoding inhibitor of apoptosis proteins (IAPs): HIAP1, HIAP2, and XIAP (Farahani, R., et al,
Genomics,
42:514-8, 1997; Liston, P., ct al.,
Genomics,
46:495-503, 1997a; Liston, P., et al.,
Nature,
379:349-53, 1996). While the IAP genes were initially discovered in baculoviruses, their homologues have since been identified in other viruses, insects, birds, and mammals, suggesting a common evolutionary origin.
X-linked IAP (XIAP) is a member of the mammalian IAP gene family. The anti-apoptotic function of XIAP is executed, at least in part, by inhibition of caspase-3 and caspase-7, two principal effectors of apoptosis. Interestingly, XIAP mRNAs are present in all human and murine fetal and adult tissues examined.
Most eukaryotic mRNAs are translated primarily by ribosome scanning. First, the 40S ribosomal subunit with its associated initiation factors binds to the 5′7-methylguanosine (m
7
G)-cap structure of the mRNA to be translated. The complex then scans in the 3′ direction until an initiation codon in a favorable context is encountered, at which point protein translation is initiated. According to this model, the presence of a 5′ untranslated region (UTR) with strong secondary structure and numerous initiation codons would present a significant obstacle, leading to inefficient translation by ribosome scanning. Ribosome reinitiation, shunting, and internal ribosome binding are secondary mechanisms of translation initiation that alleviate the requirement for ribosome scanning and allow translation to proceed in a cap-independent manner.
Internal ribosome entry site (IRES) elements, which were first identified in picornaviruses, are considered the paradigm for cap-independent translation. The 5′ UTRs of all picornaviruses are long and mediate translational initiation by directly recruiting and binding ribosomes, thereby circumventing the initial cap-binding step.
Although IRES elements are frequently found in viral mRNAs, they are rarely found in non-viral mRNAs. To date, the non-viral mRNAs shown to contain functional IRES elements in their respective 5′ UTRs include those encoding immunoglobulin heavy chain binding protein (BiP) (Macejak, D. G., et al.
Nature,
35390-4, 1991); Drosophila Antennapedia (Oh, S. K., et al.,
Genes Dev,
6:1643-53, 1992) and Ultrabithorax (Ye, X., et al.,
Mol. Cell Biol.,
17:1714-21, 1997); fibroblast growth factor 2 (Vagner, S., et al.,
Mol. Cell Biol.,
15:35-44, 1995); initiation factor eIF4G (Gan, et al.,
J. Biol. Chem.,
273:5006-12, 1998); proto-oncogene c-myc (Nanbru, et al.,
J. Biol. Chem.,
272:32061-6, 1995; Stoneley, M.,
Oncogene,
16:423-8, 1998); and vascular endothelial growth factor (VEGF) (Stein, I., et al.,
Mol. Cell Biol.,
18:3112-9, 1998).
Cellular IRES elements have no obvious sequence or structural similarity to picomavirus IRES sequences, or to each other. Moreover, the mechanism for the regulation of IRES-directed translation is not understood. An understanding of the mechanism by which IRES elements direct cap-independent translation of cellular mRNAs and characterization of novel IRES sequences will provide new approaches for regulating the intracellular levels of both endogenously- and exogenously-encoded proteins.
SUMMARY OF THE INVENTION
XIAP protein plays a critical role in regulating programmed cell death by suppressing activation of downstream caspase-3 and caspase-7. We have identified an IRES that mediates XIAP translation. The XIAP IRES element is located within a 265 nucleotide (nt) region of the XIAP 5′ untranslated region (UTR).
IRES-directed translation of XIAP is resistant to the repression of protein synthesis during serum deprivation-induced apoptosis. Furthermore, IRES-mediated translation of XIAP offers enhanced protection against apoptosis induced by serum deprivation in cultured HeLa cells. These studies demonstrate that the presence of an IRES element in mRNA allows a linked protein-encoding sequence to be selectively translated following the repression of cap-dependent translation. The XIAP IRES may be included in a recombinant transcription unit (e.g., a vector) to regulate the level of recombinant protein in a cell, particularly a cell under environmental stress. Furthermore, XIAP IRES antisense nucleic acid may be used to decrease a cell's resistance to apoptosis (e.g., a cancer cell). The XIAP IRES also may be used to identify compounds that modulate cap-independent protein translation.
In a first aspect, the invention features a purified nucleic acid comprising or encoding a XIAP IRES, wherein, if nucleotides are present 5′ or 3′ to the XIAP IRES, the nucleic acid comprises at least one variant nucleotide within a 500 nucleotide region 5′ or 3′ to the XIAP IRES. The variant nucleotide is a nucleotide that is not present at the position of the variant nucleotide in a naturally occurring XIAP gene or XIAP mRNA, relative to the position of the XIAP IRES, and the XIAP IRES increases cap-independent translation of a cistron when the XIAP IRES is located upstream from the cistron within a messenger RNA molecule. In a preferred embodiment of the first aspect of the invention, the XIAP IRES increases stress-induced cap-independent translation. The nucleic acid may be in an expression vector.
In a second, related aspect, the invention features purified nucleic acid comprising or encoding a XIAP IRES, the IRES being 5′ to a coding sequence that encodes a polypeptide that is not XIAP. The nucleic acid may be in an expression vector.
In a third, related aspect, the invention features a purified nucleic acid comprising or encoding a XIAP IRES, wherein the XIAP IRES has a nucleotide sequence substantially identical to a nucleotide sequence set forth in SEQ ID NOs: 1, 2,19-30. If nucleotides are present 5′ or 3′ to said XIAP IRES, the nucleic acid comprises at least one variant nucleotide within a 500 nucleotide region 5′ or 3′ to the XIAP IRES, the variant nucleotide being a nucleotide that is not present at the position of the variant nucleotide in a naturally occurring XIAP gene or XIAP mRNA, relative to the position of the XIAP IRES.
In a preferred embodiment of the third aspect of the invention, the nucleic acid is in an expression vector, wherein the expression vector encodes a transcription unit comprising a XIAP IRES and a coding sequence for a polypeptide. In a further embodiment, the coding sequence may encode a polypeptide that is not a XIAP polypeptide. In yet another embodiment, the expression vector may be a gene therapy vector, and the gene therapy vector may have a tissue-specific promoter. In other embodiments, the polypeptide encoded by the gene therapy vector may be selected from XIAP, NAIP, TIAP, HIAP1, HIAP2, VEGF, BCL-2, BDNF, GDNF, PDGF-B, IGF-2, NGF, CTNF, NT-3, NT-4/5, EPO, insulin, TPO, p53, VIHL, XAF, BAX, BCL-X
L1
, BAD, BCL-X
S
, and caspases 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
In a fourth aspect, the invention features a method for increasing the level of a protein in a cell, comprising introducing into a cell an expression vector comprising a promoter operably linked to a DNA sequence encoding a transcription unit. The transcription unit comprises a XIAP IRES sequence and a coding sequence for a protein, and the presence of the XIAP IRES sequence increases the level of cap-independent translation of the protein.
In various embodiments of the fourth aspect of the invention, the cell may be at risk for undergoing apoptosis, or may be undergoing apoptosis. The risk may, e.g., be due to an autoimmune disease, a degenerative disease, or an immunorejection reaction.
In other embodiments of the fourth aspect of the invention, the cell may be at risk fo
Holcik Martin
Korneluk Robert G.
Liston Peter
Apoptogen Inc.
Bierker-Brady Kristina
Clark & Elbing LLP
Elliott George C.
Epps Janet L.
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