Chiral control
Inventors
Butler, David Charles Donnell • Iwamoto, Naoki • MEENA,, null • Svrzikapa, Nenad • Verdine, Gregory L. • ZLATEV, Ivan
Assignees
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Publication Number
US-11643657-B2
Publication Date
2023-05-09
Expiration Date
Abstract
The present invention relates to chirally controlled oligonucleotides, chirally controlled oligonucleotide compositions, and the method of making and using the same. The invention specifically encompasses the identification of the source of certain problems with prior methodologies for preparing chiral oligonucleotides, including problems that prohibit preparation of fully chirally controlled compositions, particularly compositions comprising a plurality of oligonucleotide types. In some embodiments, the present invention provides chirally controlled oligonucleotide compositions. In some embodiments, the present invention provides methods of making chirally controlled oligonucleotides and chirally controlled oligonucleotide compositions.
Core Innovation
The invention describes a method for making a chirally controlled double-stranded RNAi agent capable of inhibiting the expression of PCSK-9. The method includes repeating steps of coupling, capping, modifying, and deblocking until a desired length is achieved, and at least one coupling step uses a phosphoramidite comprising a chiral auxiliary from a chiral agent having the structure of Formula 3-AB.
The resulting chirally controlled double-stranded RNAi agent comprises a sense strand and an antisense strand, each having 21 nucleotides. The sense strand includes a terminal chirally controlled internucleotidic linkage with the linkage phosphorus in the Rp or Sp configuration, and the antisense strand includes terminal chirally controlled internucleotidic linkages at the 5′ end and the 3′ end, each being a phosphorothioate internucleotidic linkage with terminal chiral modifications at the first internucleotidic linkage at the 3′ end in the Sp configuration and at the first internucleotidic linkage at the 5′ end in the Rp configuration.
The sense strand is 5′-uucuAGAccuGuuuuGcuudTsdT-3′ (SEQ ID NO: 121) and the antisense strand is 5′-asAGcAAAAcAGGUCuAGAAdTsdT-3′ (SEQ ID NO: 122), where upper case nucleotides are RNA nucleotides, lower case nucleotides are 2′-OMe nucleotides, dT represents thymidine, and s represents a phosphorothioate internucleotidic linkage. The Formula 3-AB chiral auxiliary is defined by substituent options for G1, G2, G3, G4, and G5, including formation of an optionally substituted saturated or partially unsaturated heteroatom-containing ring.
Claims Coverage
One independent claim is provided. It covers a method for making a chirally controlled double-stranded RNAi agent capable of inhibiting PCSK-9, using a repeating coupling/capping/modifying/deblocking cycle and a chiral-auxiliary phosphoramidite coupling partner defined by Formula 3-AB, together with terminal phosphorothioate linkage stereochemistry in 21-nucleotide sense and antisense strands.
Chirally controlled double-stranded RNAi agent for PCSK-9 inhibition
A method for making a chirally controlled double-stranded RNAi agent capable of inhibiting the expression of PCSK-9.
Repeating coupling, capping, modifying, and deblocking
A method comprising coupling, capping, modifying, deblocking, and repeating the steps until a desired length is achieved.
Phosphoramidite coupling partner with Formula 3-AB chiral auxiliary
At least one coupling step uses a phosphoramidite comprising a chiral auxiliary from a chiral agent having the structure of Formula 3-AB, with defined G1, G2, G3, G4, and G5 group relationships and an optionally substituted saturated or partially unsaturated heteroatom-containing ring.
Defined 21-nucleotide sense and antisense strands with terminal phosphorothioate stereochemistry
The chirally controlled double-stranded RNAi agent comprises a sense strand and an antisense strand, each strand having 21 nucleotides, where the sense strand has a terminal chirally controlled internucleotidic linkage with linkage phosphorus in the Rp or Sp configuration, and the antisense strand has terminal chirally controlled internucleotidic linkages at the 5′ and 3′ ends, each being a phosphorothioate internucleotidic linkage with specified Sp and Rp terminal chiral modifications.
Specified sense and antisense sequences
The sense strand is 5′-uucuAGAccuGuuuuGcuudTsdT-3′ (SEQ ID NO: 121) and the antisense strand is 5′-asAGcAAAAcAGGUCuAGAAdTsdT-3′ (SEQ ID NO: 122), with RNA nucleotides, 2′-OMe nucleotides, thymidine, and phosphorothioate linkage notation.
The claim coverage centers on producing a 21-nucleotide-per-strand chirally controlled double-stranded RNAi agent for PCSK-9 inhibition by repeating coupling/capping/modifying/deblocking steps, using a Formula 3-AB chiral auxiliary phosphoramidite, and specifying terminal phosphorothioate internucleotidic linkage stereochemistry on the sense and antisense strands.
Stated Advantages
Stronger and longer-lasting ApoB knockdown in mice versus a stated control.
Inhibition of PCSK9 mRNA with stereochemistry-dependent potency.
Improved potency over stereorandom controls for specific stereochemical arrangements.
Nuclease resistance, delivery, and duration of action are linked to the stereochemistry of the chiral phosphorothioate backbone.
Overcoming stereorandom diastereomer loss during purification.
Enabling predetermined diastereomer/purity profiles.
Stereochemistry-dependent biological effects on PCSK9 mRNA knockdown.
Stereochemistry-dependent in vivo reduction of ApoB protein in huApoB transgenic mice, with differences versus a stereorandom control.
Capable of inhibiting the expression of PCSK-9.
Improved full-length purity.
Unexpected stability of certain sulfurization products, including phosphorothioate precursor species along a beta-elimination pathway.
Avoids batch inconsistency associated with HPLC purification of stereorandom oligonucleotide mixtures by using chirally controlled synthesis with predetermined diastereomeric ratios.
Documented Applications
In vivo ApoB knockdown in mice using chirally controlled oligonucleotides/siRNA agents.
In vitro inhibition of PCSK9 mRNA in Hep3B and HeLa cells using chirally controlled siRNA duplexes.
A chirally controlled double-stranded RNAi agent capable of inhibiting the expression of PCSK-9.
RNA interference context including siRNA, miRNA-related agents, antagomirs, and RISC mediated gene silencing.
PCSK9-targeting chirally controlled double-stranded siRNA used for cellular evaluation of PCSK9 mRNA knockdown and for in vivo ApoB protein reduction in huApoB transgenic mice.
Inhibiting the expression of PCSK-9 using a chirally controlled double-stranded RNAi agent.
Making a chirally controlled double-stranded RNAi agent capable of inhibiting the expression of PCSK-9.
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