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:
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 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. The composition of claim 1 , wherein the first and second sequences direct insertion of the silencing sequence into an APP gene.
11. The composition of claim 1 , wherein the vector comprising the nucleic acid construct further comprises a selectable marker.
12. An isolated trisomic cell comprising the composition of claim 1 .
13. The isolated trisomic cell of claim 12 , wherein the cell is a somatic cell or a stem cell.
14. An isolated cell comprising the composition of claim 1 .
15. The isolated cell of claim 14 , wherein the cell is a germ cell, a stem cell, or a precursor cell.
16. The isolated cell of claim 15 , wherein the stem cell is an embryonic stem cell, an adult stem cell, or an induced pluripotent stem cell.
17. The isolated cell of claim 16 , wherein the adult stem cell is a hematopoietic stem cell or a neural stem cell.
18. The isolated cell of claim 14 , wherein the cell is a differentiated cell.
19. The isolated cell of claim 18 , wherein the differentiated cell is a fibroblast or neuron.
20. 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 neural cells from the subject;
transfecting the neural 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 neural cells to reduce gene expression from the trisomic chromosome.
21. The method of claim 20 , wherein the neural cells are neural stem cells.
22. The method of claim 20 , wherein the silencing sequence is a full-length XIST/Xist gene sequence.
23. The method of claim 20 , wherein the silencing sequence is an XIST/Xist gene sequence exclusive of one or more introns.
24. The method of claim 20 , wherein the silencing sequence comprises about 6 kb to about 10 kb of exon 1 of an XIST/Xist gene sequence.
25. The method of claim 24 , wherein the silencing sequence comprises the XIST/Xist cDNA sequence having accession number M97168 or a biologically active fragment or other variant thereof.
26. The method of claim 20 , wherein the silencing sequence comprises a biologically active fragment or other biologically active variant of a naturally occurring XIST/Xist gene sequence.
27. The method of claim 20 , further comprising a regulatory sequence.
28. The method of claim 27 , wherein the regulatory sequence is a constitutively active, inducible, tissue-specific, or developmental stage-specific promoter.
29. The method of claim 20 , wherein the first and second sequences direct insertion of the silencing sequence into a polymorphic region of the targeted chromosome.
30. The method of claim 20 , wherein the first and second sequences direct insertion of the silencing sequence into an APP gene.
31. The method of claim 20 , wherein the vector comprising the nucleic acid construct further comprises a selectable marker.
32. The method of claim 20 , wherein the subject is a human.