Modified siRNA molecules incorporating 5-fluoro-2′-deoxyuridine residues to enhance cytotoxicity
Inventors
SCHMITZ, SR., John C. • Chu, Edward • Gmeiner, William H.
Assignees
WAKE FOREST UNIVERSITY HEALTH SCIENCES AN EDUCATIONAL INSTITUTION • University of Pittsburgh • US Department of Veterans Affairs • Wake Forest University Health Sciences
Publication Number
US-9096853-B2
Publication Date
2015-08-04
Expiration Date
2033-09-18
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Abstract
A synthesized siRNA molecule having the sense strand with one or more uridine bases replaced by one or more respective nucleoside analogs, such as 5-fluoro-2′-deoxyuridine (FdU).
Core Innovation
The invention is directed to modified small interfering RNA (siRNA) molecules that incorporate nucleoside analogs, specifically 5-fluoro-2′-deoxyuridine (FdU), into the siRNA backbone to enhance cytotoxicity, particularly for cancer treatment. The modified siRNA retains gene-silencing activity while releasing cytotoxic nucleoside analogs upon intracellular degradation, resulting in enhanced therapeutic effects.
The problem addressed is that traditional chemically modified siRNAs, while improving stability and reducing off-target effects, release nucleosides such as deoxythymidine (dT) upon degradation, which can rescue cancer cells from cytotoxic effects of siRNA targeting thymidylate synthase (TS). There was a need to design siRNAs that not only silence target genes but also release cytotoxic agents to improve cancer cell killing.
Claims Coverage
The patent includes four independent claims directed to nucleic acid sequences comprising 5-fluoro-2′-deoxyuridine (FdU) residues incorporated at specific positions in siRNA sequences.
Incorporation of FdU at specific position in nucleotide sequence
A nucleic acid comprising the nucleotide sequence of SEQ ID NO: 5 with position 4 replaced by a 5-fluoro-2′-deoxyuridine (FdU) residue.
Incorporation of FdU at specific position in nucleotide sequence
A nucleic acid comprising the nucleotide sequence of SEQ ID NO: 7 with position 21 replaced by a 5-fluoro-2′-deoxyuridine (FdU) residue.
Incorporation of multiple FdU residues at 3′-end overhang
A nucleic acid comprising the nucleotide sequence of SEQ ID NO: 9 with positions 21 to 25 replaced by 5-fluoro-2′-deoxyuridine (FdU) residues.
Incorporation of multiple FdU residues in control sequence
A nucleic acid comprising the nucleotide sequence of SEQ ID NO: 21 with positions 21 to 25 replaced by 5-fluoro-2′-deoxyuridine (FdU) residues.
The claims cover siRNA molecules modified to contain one or more FdU nucleoside analogs at defined positions, enabling a dual function of gene silencing and enhanced cytotoxicity through intracellular release of FdU.
Stated Advantages
The modified siRNAs have enhanced cytotoxic capability compared to unmodified siRNAs.
The incorporation of FdU leads to formation of an inhibitory ternary complex with thymidylate synthase, resulting in increased apoptosis and DNA damage in cancer cells.
Modification enhances therapeutic potential by combining target mRNA silencing with intracellular release of cytotoxic nucleoside analogs.
The design approach allows rational synthesis of dual-acting siRNA molecules with potential to treat human cancers effectively.
Documented Applications
Use of modified siRNA molecules incorporating 5-fluoro-2′-deoxyuridine residues as therapeutic agents for the treatment of human cancers, particularly colon cancer.
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