Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1998-03-24
2001-10-16
Clark, Deborah J. R. (Department: 1633)
Chemistry: molecular biology and microbiology
Vector, per se
C435S325000, C435S368000, C536S023100, C536S024100
Reexamination Certificate
active
06303370
ABSTRACT:
BACKGROUND
1. Technical Field
The invention relates to methods and materials involved in the tissue-specific regulation of nucleic acid expression.
2. Background Information
Two components help determine cell-specific nucleic acid and polypeptide expression: transcription factors and regulatory elements. Transcription factors are polypeptides that bind nucleic acid sequences, whereas regulatory elements are specific nucleic acid sequences, usually located near a gene, that are bound by transcription factors. It is the binding of a transcription factor to a regulatory element that typically stimulates transcription of a nearby gene. Thus, the transcription factors available within a cell determine which nucleic acids and polypeptides are expressed by that cell. For example, brain cells contain a set of transcription factors that are unique to brain cells. This set of transcription factors stimulates transcription of brain-specific polypeptides since genes that encode brain-specific polypeptides have nearby regulatory elements that are recognized by transcription factors present within brain cells. Non-brain-specific genes, however, do not have nearby regulatory elements that could be recognized by transcription factors present within brain cells and thus are not expressed. Likewise, non-brain cells do not contain the same set of transcription factors and thus do not express brain-specific nucleic acid and polypeptides.
In eukaryotes, transcription factors have a limited, if not totally cell-type specific, distribution. In addition, each transcription factor typically binds a regulatory element having a very specific nucleic acid sequence. Transcription factors also can function in conjunction with other transcription factors such that the binding to a particular regulatory element is influenced. Thus, the presence of these regulatory elements provides a means for regulating gene expression. Generally, regulatory elements located near transcriptional start sites are known as promoters whereas those located at greater distances from transcriptional start sites are known as enhancers. Further, promoters are typically upstream of the transcriptional start site and only function in one orientation. For example, inverting a promoter sequence usually results in a dramatic decrease in the expression of a downstream gene. Enhancers, however, can be upstream, downstream, or within the nearby gene and can often function in both orientations. For example, an enhancer sequence can be within an intron of the gene it controls.
Many polypeptides and nucleic acids have specific and unique expression patterns. For example, an early polypeptide marker for most, but not all, stem cells in the central nervous system (CNS) is nestin, an intermediate filament polypeptide (Dahlstrand et al., 1995; Frederiksen and McKay, 1988; Hockfield S and R D G McKay,
J. Neurosci
. 5:3310-3328 (1985); Lendahl et al., 1990; Reynolds B A and S Weiss,
Science
255:1707-1710 (1992);Williams B P and J Price,
Neuron
14:1-20 (1995)). Upon neuronal differentiation, a switch in intermediate filament gene expression replaces nestin with the neurofilaments (Lendahl U et al.,
Cell
60:585-95 (1990)); reviewed in Lee M K and D W Cleveland.,
Annu. Rev. Neurosci.
19:187-217 (1996)). In the adult CNS, nestin is re-expressed in reactive astrocytes (Clarke et al., 1995; Frisén J et al.,
J. Cell Biol
. 131:453-464 (1995)) and in CNS neuroepithelial tumors, most notably gliomas and glioblastomas (Dahlstrand J et al.,
Cancer Res
. 52:5334-5341 (1992) and Tohyama T et al.,
Lab. Invest
. 66:303-313 (1992)). During embryonic development, nestin expression is not restricted to the CNS. Nestin is also found in myogenic precursors (Kachinsky A M et al.,
Dev. Biol
. 165:216-228 (1994); Lendahl U et al.,
Cell
60:585-595 (1990); Sejersen T and U Lendahl,
J. Cell Sci
. 106:1291-1300 (1993)), the peripheral nervous system (Dahlstrand J, et al.,
Dev. Brain Res
. 84:109-129 (1995); Hockfield S and RDG McKay,
J. Neurosci
. 5:3310-3328 (1985); and Stemple D L and D J Anderson,
Cell
71:973-985 (1992)), the heart (Kachinsky A M et al.,
J. Histochem. Cytochem
. 43:843-847 (1995)), the developing tooth bud (Terling C et al.,
Int. J. Dev. Biol
. 39:947-956 (1995)) and the testis (Fröjdman K et al., Differentiation 61:243-249 (1997)).
Recently, the regulation of nestin gene expression during development was studied using transgenic mice (Zimmerman L B et al.,
Neuron
12:11-24 (1994)). Interestingly, the upstream promoter region of the gene does not possess any enhancer elements specific for neural stem cells. Instead, two separate enhancer regulatory elements were identified within the rat nestin gene: a myogenic precursor cell-specific enhancer within the first intron and a CNS cell-specific enhancer within the second intron. These results demonstrate the presence of multiple regulatory elements within a single gene that each control its expression in distinct tissues.
Tissue-specific regulatory elements are powerful tools that can direct heterologous nucleic acid and polypeptide expression. Indeed, the second intron from the nestin gene directs heterologous gene expression to the developing nervous system (Lardelli M et al.,
Mech. Dev
. 59:177-190 (1996); Ringstedt T et al.,
Developnzent
124:2603-2613 (1997); and Lothian C and U Lendahl,
Eur. J. Neurosci
. 9:452-462 (1997)). Most, if not all, gene therapy approaches benefit from tissue-specific regulatory elements since these elements direct expression of the nucleic acid and/or polypeptide of interest to specific target tissues.
SUMMARY
The present invention relates to tissue-specific nucleic acid expression. Specifically, this invention relates to the discovery of regulatory elements that direct the expression of nucleic acid in a tissue-specific manner. The invention provides midbrain-specific and nervous system (NS)-specific regulatory elements as well as elements that potentiate nucleic acid expression and cooperate with other regulatory elements. In addition, the invention provides regulatory elements that direct the expression of nucleic acid in a temporally regulated manner. These elements are particularly useful for gene therapy approaches that require expression in only a specific cell population or at only particular times. The invention also provides nucleic acid constructs that contain these regulatory elements in combination with selected nucleic acid sequences. In addition, the invention provides cells and animals that contain these regulatory elements and constructs as well as methods of providing an animal with a selected nucleic acid sequence that is expressed in a tissue-specific or temporal manner.
One aspect of the invention provides an isolated midbrain-specific regulatory element. An isolated midbrain-specific regulatory element can be isolated from a nestin gene and can contain the nucleic acid sequence of SEQ ID NO:1.
Another aspect of the invention provides an isolated potentiating regulatory element. Isolated potentiating regulatory elements can contain the nucleic acid sequence of SEQ ID NO:2.
Another aspect of the invention provides an isolated nervous system-specific regulatory element that is less than about 400 bases. In addition, an isolated nervous system-specific regulatory element can be less than about 200 bases or less than about 100 bases. Isolated nervous system-specific regulatory elements can contain the nucleic acid sequence of SEQ ID NO:3.
Another aspect of the invention provides an isolated cooperating regulatory element. Isolated cooperating regulatory elements can contain the nucleic acid sequence of SEQ ID NO:4.
Another aspect of the invention provides an isolated temporal midbrain-specific regulatory element. Isolated temporal midbrain-specific regulatory elements can contain the nucleic acid sequence of SEQ ID NO:5.
Another aspect of the invention provides a nucleic acid construct containing an isolated midbrain-specific regulatory element and a selected nucleic acid sequence. A selected nucleic acid sequence can encode a polypep
Kappen Claudia
Yaworsky Paul J.
Clark Deborah J. R.
Fish & Richardson P.C. P.A.
Kaushal Sumesh
Mayo Foundation for Medical Education and Research
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