Thrombocyte inhibition via vivo-morpholino knockdown of alpha IIb
Novel compounds comprising a guanidine-rich head covalently coupled to one or more oligonucleotide antisense sequences which are useful to modulate blood coagulation by affecting the expression of integrin αIIb or β3 are described herein. This invention also includes pharmaceutical compositions containing these compounds, with or without other therapeutic agents, and to methods of using these compounds as inhibitor of platelet aggregation, as thrombolytics, and/or for the treatment of other thromboembolic disorders. Vivo-MOs, which include eight guanidine groups dendrimerically arranged in the guanidine-rich head and two synthetic antisense morpholino oligonucleotides, are representative compounds of the present invention.
1. A method of identifying a gene involved in hemostasis comprising the steps of:
creating a library of knockdown agents, wherein each knockdown agent comprises an individual interfering antisense morpholino oligonucleotide that targets a specific zebrafish gene;
injecting intravenously an individual knockdown agent from the library of knockdown agents into an individual zebrafish, thereby knocking down expression of a specific zebrafish gene in the individual zebrafish;
withdrawing a blood sample from the individual zebrafish;
determining a quantitative and/or qualitative change in clotting in the blood sample relative to that of a control sample, thereby identifying the specific zebrafish gene as a gene involved in hemostasis; and
identifying a human homolog of the specific zebrafish gene.
2. The method of claim 1 , wherein the individual knockdown agent targets a splice site of αIIb.
3. The method of claim 1 , wherein each individual interfering antisense morpholino oligonucleotide of the library of knockdown agents comprises a guanidine-rich head.
4. The method of claim 3 , wherein the guanidine-rich head contains dendrimerically arranged guanidine groups.
5. The method of claim 3 , wherein the guanidine-rich head contains between 7 and 15 guanidine groups.
6. The method of claim 3 , wherein the guanidine-rich head contains 8 guanidine groups.
7. A method of identifying a gene involved in thrombocyte function comprising the steps of:
creating a library of knockdown agents, wherein each knockdown agent comprises an individual interfering antisense morpholino oligonucleotide having a guanidine-rich head of between 7 and 15 guanidine groups, wherein each individual interfering antisense morpholino oligonucleotide targets a specific zebrafish gene involved in thrombocyte function;
injecting intravenously an individual knockdown agent from the library of knockdown agents into an individual zebrafish, thereby knocking down expression of a specific zebrafish gene in the individual zebrafish;
withdrawing a blood sample from the individual zebrafish injected with the individual knockdown agent;
determining a quantitative and/or qualitative change in thrombocyte function in the blood sample relative to that of a control sample, thereby identifying the specific zebrafish gene as a gene involved in hemostasis; and
identifying a human homolog of the specific zebrafish gene.