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Publication Number
US-11203755-B2
Publication Date
2021-12-21
Expiration Date
Abstract
An objective of the present invention is to provide a chemically-modified siRNA that has polydeoxyadenylic acid added to the 5′ end of the sense strand, and, when complexed with schizophyllan, is high in resistance against RNase and moreover effectively exhibits RNAi activity. To achieve the objective, in the chemically-modified siRNA with polydeoxyadenylic acid added to the 5′ end of the sense strand, specific chemical modification is performed for dinucleotide sequences of CA, UA, and UG in the base sequence of the sense strand and dinucleotide sequences of CA, UA, and UG in the base sequence at and after the eighth base from the 5′ end of the antisense strand.
Core Innovation
The invention provides a chemically-modified siRNA with polydeoxyadenylic acid added to a 5′ end of a sense strand. The sense strand base sequence includes at least one dinucleotide sequence selected from CA, UA, and UG, and a base sequence beginning at or after the eighth base from a 5′ end of an antisense strand includes at least one dinucleotide sequence selected from CA, UA, and UG, with specified positional constraints on which antisense residues are chemically modified or not chemically modified.
For each dinucleotide motif present in the sense strand and the antisense strand, the invention defines 2′-position substitutions on cytidylic, uridylic, and guanylic residues and a methoxy substitution on the corresponding adenylic or guanylic residue within the dinucleotide. The first to seventh ribonucleotide residues from the 5′ end of the antisense strand remain unmodified, together with additional provisos that control when sense-end and antisense-end residues remain unmodified.
The invention further specifies a chemically-modified siRNA in which the sense strand has a polydeoxyadenylic acid sequence at the 5′ end, and the siRNA includes defined fluoro- and methoxy-substituted 2′ positions. The chemically-modified siRNA is complexed with schizophyllan (SPG) to form an SPG/siRNA complex.
Claims Coverage
The partial content provides six independent claims. Across these claims, the inventive subject matter centers on a chemically-modified siRNA with a 5′ sense-strand polydeoxyadenylic acid and specific 2′ fluoro/methoxy substitution rules or specified modified nucleotide sequences, and optionally a schizophyllan (SPG) complex.
Dinucleotide-driven 2′ fluoro/methoxy modification with antisense positional unmodified residues
A chemically-modified siRNA with polydeoxyadenylic acid added to a 5′ end of a sense strand, wherein a base sequence of the sense strand includes at least one kind of dinucleotide sequence selected from CA, UA, and UG, and a base sequence beginning at or after the eighth base from a 5′ end of an antisense strand includes at least one kind of dinucleotide sequence selected from CA, UA, and UG, with specified 2′ hydroxy substitutions within the dinucleotide motifs, unmodified antisense first-to-seventh ribonucleotide residues, and additional provisos controlling modification at the sense first ribonucleotide and antisense nineteenth ribonucleotide positions.
Specified modified nucleotide sequence with 5′ sense polydeoxyadenylic acid
A chemically-modified siRNA comprising a sense strand and an antisense strand having the sequences defined using dt, U(F), G(M), C(F), and A(M), wherein the sense strand has a polydeoxyadenylic acid sequence at the 5′ end.
Schizophyllan siRNA complex
A chemically-modified siRNA complexed with schizophyllan (SPG), forming an SPG/siRNA complex.
Overall, the independent claims cover chemically-modified siRNA defined either by dinucleotide-based 2′ fluoro/methoxy modification rules with antisense positional unmodified constraints, or by specific modified sense/antisense sequences using dt, U(F), G(M), C(F), and A(M). The claims also require a 5′ sense-strand polydeoxyadenylic acid sequence, and the partial content further narrows by specifying polydeoxyadenylic acid length, phosphorothioated phosphodiester bonds, and forming a schizophyllan (SPG) complex.
Stated Advantages
Improved RNase stability.
Improved RNA interference (RNAi).
RNase resistance.
Effective RNAi.
Documented Applications
Gene knockdown of CD40.
Serum stability testing (mouse/human serum/FBS context).
Ex vivo MLR in RAW264 cells.
PBMC assays.
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