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 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 a vector comprising a nucleic acid construct comprising:
a silencing sequence encoding an Xist RNA; and
first and second sequences homologous to a site of desired integration in human chromosome 13, 18, or 21 or mouse chromosome 16 that specifically direct insertion of the silencing sequence into human chromosome 13, 18, or 21 or mouse chromosome 16 by homologous recombination.
2. The method of claim 1 , wherein the silencing sequence is a full-length Xist gene sequence.
3. The method of claim 1 , wherein the silencing sequence is an Xist gene sequence exclusive of one or more introns.
4. The method 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 method 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 method 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 method of claim 1 , further comprising a regulatory sequence.
8. The method of claim 7 , wherein the regulatory sequence is a constitutively active, inducible, tissue-specific, or developmental stage-specific promoter.
9. The method of claim 1 , wherein the first and second sequences direct insertion of the silencing sequence into a polymorphic region of the targeted chromosome.
10. The method of claim 1 , wherein the first and second sequences direct insertion of the silencing sequence into an APP gene.
11. The method of claim 1 , wherein the vector comprising the nucleic acid construct further comprises a selectable marker.
12. The method of claim 1 , wherein the cell is a somatic cell, a germ cell, a stem cell, or a precursor cell.
13. The method of claim 12 , wherein the stem cell is an embryonic stem cell, an adult stem cell, or an induced pluripotent stem cell.
14. The method of claim 13 , wherein the adult stem cell is a hematopoietic stem cell or a neural stem cell.
15. The method of claim 1 , wherein the cell is a differentiated cell.
16. The method of claim 1 , wherein the differentiated cell is a fibroblast or neuron.