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, the method comprising:
identifying a subject with a trisomic human chromosome 13, 18, or 21 or mouse chromosome 16;
harvesting hematopoietic stem cells from the subject;
transfecting the hematopoietic stem cells with a vector comprising a nucleic acid construct comprising:
a silencing sequence encoding an XIST/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; and
administering to the subject a sufficient number of the transfected hematopoietic stem cells to reduce gene expression from the trisomic chromosome.
2. The method of claim 1 , wherein the silencing sequence is a full-length XIST/Xist gene sequence.
3. The method of claim 1 , wherein the silencing sequence is an XIST/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/Xist gene sequence.
5. The method of claim 4 , wherein the silencing sequence comprises the XIST/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/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 subject is a human.