Nucleic acid silencing sequences
The present invention features compositions and methods for introducing, into cells, nucleic acids whose expression results in chromosomal silencing. The nucleic acids are targeted to specific chromosomal regions where they subsequently reduce the expression of deleterious genes, or cause the death of deleterious cells. Where the nucleic acid sequence is a silencing sequence, it may encode an Xist RNA or other non-coding, silencing RNA. Accordingly, the present invention features, inter alia, nucleic acid constructs that include a transgene (e.g., a silencing sequence encoding an Xist RNA or other non-coding RNA that silences a segment of a chromosome); first and second sequences that direct insertion of the silencing sequence into a targeted chromosome; and, optionally, a selectable marker.
1. A composition comprising:
(i) a vector comprising a nucleic acid construct comprising:
a silencing sequence encoding an Xist RNA; and
first and second sequences that direct insertion of the silencing sequence into human chromosome 13, 18, or 21 or mouse chromosome 16; and
(ii) a nucleic acid encoding a chimeric zinc finger nuclease (ZFN), or a ZFN polypeptide, that targets the human chromosome 13, 18, or 21 or mouse chromosome 16.
2. The composition of claim 1 , wherein the silencing sequence is a full-length Xist gene sequence.
3. The composition of claim 1 , wherein the silencing sequence is an Xist gene sequence exclusive of one or more introns.
4. The composition of claim 1 , wherein the silencing sequence comprises about 6 kb to about 10 kb of exon 1 of an Xist gene sequence.
5. The composition of claim 4 , wherein the silencing sequence comprises the Xist cDNA sequence having accession number M97168 or a biologically active fragment or other variant thereof.
6. The composition of claim 1 , wherein the silencing sequence comprises a biologically active fragment or other biologically active variant of a naturally occurring Xist gene sequence.
7. The composition of claim 1 , further comprising a regulatory sequence.
8. The composition of claim 7 , wherein the regulatory sequence is a constitutively active, inducible, tissue-specific, or developmental stage-specific promoter.
9. The composition of claim 1 , wherein the first and second sequences direct insertion of the silencing sequence into a polymorphic region of the targeted chromosome.
10. An isolated trisomic cell comprising the composition of claim 1 .
11. The isolated trisomic cell of claim 10 , wherein the cell is a somatic cell or a stem cell.
12. The composition of claim 1 , wherein the vector comprising the nucleic acid construct further comprises a selectable marker.
13. A method of reducing gene expression from a trisomic human chromosome 13, 18, or 21 or mouse chromosome 16 in a cell, the method comprising:
providing a cell comprising a trisomic human chromosome 13, 18, or 21 or mouse chromosome 16; and introducing into the cell
(i) a vector comprising a nucleic acid construct comprising:
a silencing sequence encoding an Xist RNA; and first and second sequences that direct insertion of the silencing sequence into human chromosome 13, 18, or 21 or mouse chromosome 16; and
(ii) a nucleic acid encoding a chimeric zinc finger nuclease (ZFN), or a ZFN polypeptide, that targets the human chromosome 13, 18, or 21 or mouse chromosome 16,
in an amount sufficient to reduce gene expression from the trisomic chromosome.