HK1244028B - Chiral toxicity screening method - Google Patents

Description

Chiral toxicity screening method

Technical Field

The present invention relates to methods for identifying stereospecific phosphorothioate oligonucleotide variants with reduced toxicity by generating and screening libraries of stereospecific chiral phosphorothioate variants for compounds with reduced toxicity in vitro or in vivo.

Background Art

Koziolkiewicz et al. (NAR 1995, 24; 5000-5005) disclosed 15mer DNA phosphorothioate oligonucleotides in which the phosphorothioate linkages are in the [all-Rp] configuration or the [all-Sp] configuration, or a random mixture of diastereomers. They found that [all-Rp] oligonucleotides were "more susceptible" to RNase H-dependent degradation than hybrid or [all-Sp] oligonucleotides and were found to have higher duplex thermostability. They recommended that, in practical applications, [all-Rp] oligonucleotides should be protected at the 3' terminus with [Sp] phosphorothioate.

Stec et al. (J. Am. Chem. Soc. 1998, 120; 7156-7167) reported a new monomer of 5'-DMT-deoxyribonucleoside 3'-O-(2-thio-"spiro"-4,4-pentamethylene-1,2,3-oxathiaphospholane) for the stereocontrolled synthesis of PS-oligonucleotides via the oxathiaphospholane method.

Karwowski et al. (Bioorganic & Med. Chem. Letts. 2001 11; 1001-1003) used the oxathiaphospholane method to perform a stereocontrolled synthesis of LNA dinucleoside phosphorothioates. The R stereoisomer dinucleotide is susceptible to hydrolysis by snake venom phosphodiesterase.

Krieg et al. (Oligonucleotides 13; 491-499) investigated whether the immunostimulatory effects of CpG PS oligonucleotides depend on the chirality of their P chirality. CpG PS Rp oligonucleotides demonstrated greater induction of MAPK activation and IκB degradation compared to Sp oligonucleotides. There was no evidence of differential uptake of the different stereoisomers. Rp oligonucleotides had a shorter duration (less than 48 hours), likely due to rapid degradation. For immunostimulation, CpG oligonucleotides with Rp chirality are recommended for rapid, short-term effects, while Sp oligonucleotides are recommended for longer-term effects.

Levin et al., Chapter 7, Antisense Drug Technology 2008; 183–215, reviewed phosphorothioate chirality, demonstrating that the chirality of phosphorothioate DNA oligonucleotides significantly impacts their pharmacokinetics, particularly due to the exonuclease resistance of the Sp stereoisomer. It has been reported that the PK effects of phosphorothioate chirality are less pronounced in second-generation ASOs because 2′ modifications at the 3′ and 5′ termini prevent exonuclease degradation, but individual molecules with Rp terminal residues may be more susceptible to exonucleases. This suggests that molecules with terminal Sp residues may have longer half-lives.

Wave Life Sciences Poster (TIDES, May 3–6 2014, San Diego): Based on calculations of 524,288 possible stereoisomers of diandrostenone, they elucidated seven stereoisomers that differ significantly in T _m , RNAseH recruitment, lipophilicity, metabolic stability, in vivo efficacy, and specific activity.

Wan et al., Nucleic Acids Research, November 14, 2014 (Advanced Publication) disclose 31 antisense oligonucleotides in which the chirality of the gap region is controlled using a DNA-oxazaphosphopeptide approach (Oka et al., J. Am. Chem. Soc. 2008; 16031–16037.), and conclude that controlling PS chirality in the gap region of the gamper has no significant benefit for therapeutic applications relative to mixtures of stereorandom PS ASOs. Wan et al. further mention that the increased complexity and cost associated with the synthesis and characterization of chiral PS ASOs reduces their utility.

Swayze et al., 2007, NAR 35(2): 687-700 reported that LNA antisense compounds improved potency but caused significant hepatotoxicity in animals. WO 2008/049085 reported that LNA hybrid wing interspersed with 2'-O-MOE in the LNA flanking region significantly reduced the toxicity of certain LNA compounds, but significantly reduced potency.

WO2014/012081 and WO2014/010250 provide chiral reagents for synthesizing oligonucleotides.

WO2015/107425 reports the chiral design of chirally constrained oligonucleotides and reports that certain chirally constrained compounds can change the RNaseH cleavage pattern.

SUMMARY OF THE INVENTION

The present invention provides a method for reducing the toxicity of an antisense oligonucleotide sequence (parent oligonucleotide), comprising the following steps:

a. generating a library of stereospecific oligonucleotide variants (daughter oligonucleotides) that retain the core nucleobase sequence of the parent oligonucleotide,

b. screening the library generated in step a for in vitro or in vivo toxicity in cells,

c. Identifying one or more stereospecific variants present in the library that have reduced toxicity in cells compared to the parent oligonucleotide.

Wherein, the method is optionally repeated (repeated screening), for example so that one or more stereospecific variants identified by the method are used as parent oligonucleotides in the next round of screening method. The term stereospecific is interchangeable with the term stereodefined herein.

The present invention provides a method for reducing the toxicity of a phosphorothioate oligonucleotide (parent) sequence, comprising the steps of:

a. providing a stereo non-specific phosphorothioate oligonucleotide (mother) having a toxic phenotype in vivo or in vitro,

b. generating a library of stereospecific phosphorothioate oligonucleotide (progeny) libraries that retain the core nucleobase sequence of the parent gapmer oligonucleotides,

c. screening the library generated in step b in an in vivo or in vitro toxicity assay,

d. Identifying one or more stereospecific phosphorothioate oligonucleotides having reduced toxicity compared to stereononspecific phosphorothioate oligonucleotides.

In some embodiments, the methods of the present invention can be used repeatedly.

The methods of the present invention may further comprise an additional subsequent step of manufacturing one or more stereospecific phosphorothioate oligonucleotides with reduced toxicity. In some embodiments, the subsequent manufacturing scale is greater than 1 g, e.g., greater than 10 g. In some embodiments, the synthesis of the oligonucleotides used for in vivo or in vitro screening step (b) is performed on a scale of less than 1 g, e.g., less than 0.5 g, e.g., less than 0.1 g.

The daughter oligonucleotides (i.e. the members of the library of step b) or the daughter oligonucleotides with reduced toxicity identified by this method are stereospecific variants of the parent oligonucleotide, i.e. they contain at least one stereospecific phosphorothioate internucleoside linkage that is different from the parent.

In the methods of the present invention, each member of the library generated in step b) comprises at least one stereospecific phosphorothioate internucleoside linkage that is different from the parent.

In some embodiments, the method further comprises the step of determining the in vitro or in vivo potency of the library of stereospecific oligonucleotide variants or one or more stereospecific compounds present in the library identified in step c) or d).

In some embodiments, the methods of the present invention provide stereospecific phosphorothioate LNA oligonucleotides comprising at least one stereoselective phosphorothioate bond between an LNA nucleoside and a subsequent (3') nucleoside. Such LNA oligonucleotides can, for example, be LNA spacers as described herein. The term "stereospecific" is used interchangeably with stereodefined herein. Stereospecific phosphorothioate bonds can also be referred to as stereoselective or stereospecific phosphorothioate bonds.

In some embodiments, an oligonucleotide of the invention is 10-20 nucleotides in length, for example 10-16 nucleotides in length.

Oligonucleotides comprising at least one LNA nucleoside may be referred to herein as LNA oligonucleotides or LNA oligomers.

The present invention provides stereospecific phosphorothioate oligonucleotides that have reduced toxicity in vivo or in vitro compared to non-stereospecific phosphorothioate oligonucleotides having the same nucleobase sequence and chemical modifications (except for the stereospecific phosphorothioate linkage). In some embodiments, the non-stereospecific phosphorothioate oligonucleotide/stereospecific oligonucleotide can be a gapmer, such as an LNA-gapmer. For toxicity comparisons, the stereospecific phosphorothioate oligonucleotide retains the pattern of modified and unmodified nucleosides present in the parent oligonucleotide.

The present invention also provides a conjugate comprising an oligomer as described herein, comprising at least one non-nucleotide or non-polynucleotide moiety ("conjugate moiety") covalently linked to the oligomer of the invention.

The present invention provides pharmaceutical compositions comprising an oligomer or conjugate as described herein and a pharmaceutically acceptable solvent (e.g., water or saline), diluent, carrier, salt or adjuvant.

Also provided are medicaments and other compositions comprising the oligomers described herein. Also provided are methods for downregulating expression of a target nucleic acid, such as RNA, such as mRNA or microRNA, in a cell or tissue, comprising contacting the cell or tissue in vitro or in vivo with an effective amount of one or more oligomers, conjugates, or compositions described herein.

Also disclosed are methods of treating an animal (non-human animal or human) suspected of having or being susceptible to a disease or condition associated with RNA expression or overexpression by administering to the animal a therapeutically or prophylactically effective amount of one or more oligomers, conjugates, or pharmaceutical compositions described herein.

The present invention provides a method for inhibiting (e.g., by downregulating) the expression of a target nucleic acid in a cell or tissue, the method comprising the step of contacting the cell or tissue in vitro or in vivo with an effective amount of one or more oligomers, conjugates, or pharmaceutical compositions thereof to achieve downregulation of target nucleic acid expression.

In some embodiments, the LNA oligonucleotides described herein comprise at least one stereoselective phosphorothioate bond between the LNA nucleoside and the subsequent (3') nucleoside.

In some embodiments, an LNA oligonucleotide as described herein comprises at least one stereospecific phosphorothioate nucleotide pair, wherein the internucleoside linkage between the nucleosides of the stereospecific phosphorothioate nucleotide pair is in the Rp configuration or the Rs configuration, and wherein at least one nucleoside of the stereospecific phosphorothioate nucleotide pair is an LNA nucleotide. In some embodiments, the other nucleoside of the stereospecific phosphorothioate nucleotide pair is not DNA, for example, a nucleoside analog, such as another LNA nucleoside or a 2'-substituted nucleoside. The present invention also provides for the use of stereospecific phosphorothioate internucleoside linkages in oligonucleotides, wherein the oligonucleotides have reduced toxicity compared to the same oligonucleotides without stereospecific phosphorothioate internucleoside linkages.

The present invention also provides the use of stereocontrolled (also known as stereospecific) phosphorothioate monomers (e.g., phosphoramidites) for the synthesis of oligonucleotides with reduced toxicity (stereospecific phosphorothioate oligonucleotides).

The present invention also provides in vitro toxicity assays that have been found to predict the in vivo toxicity of oligonucleotides (e.g., LNA oligonucleotides). Suitable hepatotoxicity or nephrotoxicity assays are provided. It should be understood that the use of such assays is not limited to stereospecific oligonucleotides, but rather, in another aspect of the present invention, is generally applicable to oligonucleotides, such as antisense oligonucleotides including LNA oligonucleotides (e.g., β-D-oxy LNA or (S)cET) and oligonucleotides containing 2'-substituted nucleosides, such as gapmer oligonucleotides described herein.

The present invention provides the use of an in vitro primary hepatocyte assay to determine (e.g. potential) hepatotoxicity of oligonucleotides (e.g. LNA oligonucleotides).

The present invention provides a method for predicting (e.g., potential) in vivo hepatotoxicity of an oligonucleotide (e.g., an LNA oligonucleotide), comprising administering the oligonucleotide in vitro to a population of primary hepatocytes, e.g., mouse or rat primary hepatocytes (which can be obtained, for example, by liver perfusion) or human primary hepatocytes, incubating the cells in the presence of the oligonucleotide for, for example, 1-7 days, e.g., 2-4 days, e.g., 3 days, and subsequently measuring at least one biomarker of in vitro cytotoxicity, e.g., a biomarker described herein, e.g., by measuring the amount of lactate dehydrogenase (LDH) released into the culture medium and/or cellular ATP levels. Suitably, a decrease in cellular ATP levels indicates a hepatotoxic oligonucleotide, and an increase in LDH released into the culture medium indicates a hepatotoxic oligonucleotide.

The present invention provides the use of an in vitro assay to determine the (potential) hepatotoxicity of an oligonucleotide (e.g. an LNA oligonucleotide).

The present invention provides an in vitro assay for determining the (e.g., potential) nephrotoxicity of oligonucleotides (e.g., LNA oligonucleotides). Suitably, mammalian renal cortical epithelial cells, such as human renal cortical epithelial cells, which may be optionally immortalized, may be used. For example, hTERT1, such as RPTEC/TERT1 (available from ATCC, e.g., (CRL-4031 ^™ ) or RPTEC-TERT1 (Evercyte GmbH, Austria) may be used.

The present invention provides a method for predicting (e.g., potential) in vivo nephrotoxicity of an oligonucleotide (e.g., an LNA oligonucleotide), comprising administering the oligonucleotide to a population of mammalian renal cortical epithelial cells (e.g., human or rat primary proximal tubule epithelial cells, e.g., RPTEC-TERT1) in vitro, incubating the cells in the presence of the oligonucleotide for, e.g., 1-21 days, such as 2-16 days, such as 4-12 days, such as 6-10 days, such as 9 days, and then measuring at least one in vitro cytotoxicity marker, such as those described herein, for example, by measuring cellular ATP levels. Suitably, a decrease in cellular ATP levels indicates a nephrotoxic oligonucleotide.

The oligonucleotide is suitably applied to the cell culture, e.g. in PBS, to achieve a final concentration of 1-100 μM, such as 5-50 μM, for example 10 or 30 μM.

It will be appreciated that, in some embodiments, methods for predicting in vivo toxicity (e.g., nephrotoxicity or hepatotoxicity) can be used to identify stereospecific variants of a parent oligonucleotide that have reduced in vitro or in vivo toxicity.

Attached photos

Figure 1: Schematic representation of some LNA oligonucleotides of the present invention. This figure shows a 3-10-3 gapper oligonucleotide with 15 internucleoside phosphorothioate linkages. The internucleoside linkages in wings X' and Y' can be as described herein, for example, random Rp or Sp phosphorothioate linkages. The table portion of Figure 1 provides parent compounds (P) in which all internucleoside linkages in gap Y' are also randomly incorporated with Rp or Sp phosphorothioate linkages (M), and in compounds 1-10, one phosphorothioate linkage is stereospecifically an Rp phosphorothioate internucleoside linkage (R).

Figure 2: Based on Figure 1, except that in compounds 1-10, one of the phosphorothioate linkages is stereospecifically an Sp phosphorothioate internucleoside linkage (S).

Figure 3: Comparison of the hepatotoxic potential (ALT) of LNA oligonucleotides in which the three phosphorothioate internucleoside bonds are fixed in the S (Compound #10) or R (Compound #14) configuration with the ALT of the parent mixture of diastereomers (Compound #1) (a mixture of all nucleoside bonds in the R and S configuration).

Figure 4: Oligonucleotide content in liver, kidney and spleen.

Figure 5: Changes in LDH levels and intracellular ATP levels in the supernatants of cells treated with respective LNAs for 3 days. Target knockdown (Myd88) was assessed 48 hours later.

Figure 6: In vitro toxicity screening of primary hepatocytes: Changes in LDH and intracellular ATP levels in the supernatants of cells treated with the respective LNAs for 3 days. Data are mean values expressed as % of vehicle control (n = 4, triplicate experiments for #56; n = 2, triplicate experiments for all other LNAs).

Figure 7: In vitro toxicity screen in renal proximal tubule cells: Viability (cellular ATP) of PTEC-TERT1 measured after 9 days of treatment with 10 μM and 30 μM LNA Myd88 stereovariants.

Detailed Description of the Invention

The methods of the present invention provide stereospecific oligomeric compounds (also referred to herein as oligomers or oligonucleotides) for modulating, such as inhibiting, a target nucleic acid in a cell. The oligomers can be antisense gapper oligonucleotides. The oligomers identified or produced using the methods of the present invention have reduced in vitro or in vivo toxicity, such as reduced nephrotoxicity or reduced hepatotoxicity.

In the context of the present invention, the term "stereospecific" refers to an oligonucleotide wherein at least one phosphorothioate internucleoside linkage present in the oligonucleotide has a directed stereochemistry, i.e., Rp or Sp. In some embodiments, all phosphorothioate internucleoside linkages in a stereospecific oligonucleotide may be stereospecific, i.e., each phosphorothioate internucleoside linkage is independently selected from the group consisting of Rp and Sp phosphorothioate internucleoside linkages.

Typically, oligonucleotide thiophosphates are synthesized as a random mixture (also referred to as a racemic mixture) of Rp and Sp phosphorothioate bonds. In the present invention, gapmer phosphorothioate oligonucleotides are provided, wherein at least one phosphorothioate bond of the gap region oligonucleotide is stereospecific, i.e., at least 75%, such as at least 80%, or at least 85%, or at least 90% or at least 95%, or at least 97%, such as at least 98%, such as at least 99%, or (substantially) all oligonucleotide molecules present in the oligonucleotide sample are Rp or Sp. Such oligonucleotides can be referred to as stereospecific, stereoselective, or stereospecific: they contain at least one stereospecific phosphorothioate bond. The terms stereospecific and stereoselective/stereoselective can be used interchangeably herein. The terms stereospecific, stereoselective, and stereospecific can be used to describe a phosphorothioate internucleoside bond (Rp or Sp), or can be used to describe an oligonucleotide comprising such a phosphorothioate internucleoside bond. It will be appreciated that stereospecific oligonucleotides may contain a small number of alternative stereoisomers at any one position, for example, Wan et al. reported 98% stereoselectivity for the intercalator reported in NAR in November 2014.

advantage

In vivo and in vitro optimization

The present invention provides methods for optimizing oligonucleotides, such as oligonucleotides identified by gene walking, for in vivo (e.g., pharmacological) applications. In particular, the methods of the present invention can be used to reduce the in vitro or in vivo toxicity of oligonucleotides (sequences), and to synthesize and prepare oligomers having reduced toxicity and optionally other enhanced beneficial properties, such as target specificity, mismatch discrimination, serum protein binding, biodistribution, RNase H recruitment, efficacy, and cellular uptake.

Reduced toxicity

The present invention provides a method for reducing the toxicity of an antisense oligonucleotide sequence (parent oligonucleotide), comprising the following steps:

a. generating a library of stereospecific oligonucleotide variants (daughter oligonucleotides) that retain the core nucleobase sequence of the parent oligonucleotide,

b. screening the library generated in step a for in vitro or in vivo toxicity in cells,

c. Identifying one or more stereospecific variants present in the library that have reduced toxicity in cells compared to the parent oligonucleotide.

In some embodiments, toxicity (step b) is determined in vivo. In some embodiments, toxicity is determined in vitro. In some embodiments, the parent oligonucleotide is an oligonucleotide determined to be hepatotoxic in vitro or in vivo. The daughter oligonucleotides identified by the methods of the present invention have reduced toxicity, such as reduced hepatotoxicity, compared to the parent oligonucleotide.

In some embodiments, the reduced toxicity reduces hepatotoxicity. The hepatotoxicity of an oligonucleotide can be assessed in vivo, for example, in mice. In vivo hepatotoxicity assays are typically based on measuring serum markers of liver damage, such as ALT, AST, or GGT. Levels exceeding three times the upper limit of normal are considered indicative of in vivo toxicity. In vivo toxicity can be assessed in mice using, for example, a single 30 mg/kg dose of the oligonucleotide, with toxicity assessed after 7 days (7-day in vivo toxicity assay).

Suitable markers for cytotoxicity include elevated LDH or decreased cellular ATP, and these markers can be used to measure cytotoxicity in vitro, for example, using primary cells or cell cultures. To measure hepatotoxicity, mouse or rat hepatocytes, including primary hepatocytes, can be used. Suitable markers for hepatotoxicity include elevated LDH or decreased cellular ATP. If available, primary primate hepatocytes, such as human, can be used. In mammalian hepatocytes, such as mouse hepatocytes, elevated LDH indicates toxicity. Decreased cellular ATP indicates toxicity, such as hepatotoxicity. In some embodiments, the oligonucleotides of the invention have reduced in vitro hepatotoxicity, as measured in primary mouse hepatocytes using the assay provided in Example 8.

In some embodiments, the reduced toxicity reduces nephrotoxicity. Nephrotoxicity can be assessed in vivo using markers of kidney damage, including elevated serum creatinine levels or elevated kim-1 (kidney injury marker-1) mRNA and/or protein. Suitably, mice or rodents can be used.

In vitro renal injury assays can be used to measure renal toxicity and can include an increase in kim-1 mRNA/protein or a change (decrease) in cellular ATP. In some embodiments, PTEC-TERT1 cells can be used to assay renal toxicity in vitro, for example, by measuring cellular ATP levels. In some embodiments, the oligonucleotides of the invention have reduced in vitro renal toxicity as measured in PTEC-TERT1 cells, for example, using the assay provided in Example 9.

Other in vitro toxicity assays that can be used to assess toxicity include caspase assays and cell viability assays, such as MTS assays. In some embodiments, the reduced toxicity oligonucleotides of the present invention comprise at least one stereospecific Rp internucleotide linkage, for example, at least 2, 3, or 4 stereospecific Rp internucleotide linkages. The Examples illustrate compounds comprising stereospecific Rp internucleotide linkages that exhibit reduced hepatotoxicity in vitro and in vivo. In some embodiments, at least one stereospecific Rp internucleotide linkage is present within the interstitial region of an LNA gapmer. In some embodiments, the reduced toxicity oligonucleotides of the present invention comprise at least one stereospecific Sp internucleotide linkage, for example, at least 2, 3, or 4 stereospecific Sp internucleotide linkages. The Examples illustrate compounds with reduced nephrotoxicity comprising at least one stereospecific Sp internucleotide linkage. In some embodiments, at least one stereospecific Sp internucleotide linkage is present within the interstitial region of an LNA gapmer.

The present invention provides the use of stereocontrolled (also referred to as stereospecific or stereospecific) phosphoramidite monomers for synthesizing oligonucleotides with reduced toxicity, for example, oligonucleotides with reduced hepatotoxicity or reduced nephrotoxicity. In some embodiments, the stereocontrolled phosphoramidite monomer is an LNA stereocontrolled phosphoramidite monomer. In some embodiments, the stereocontrolled phosphoramidite monomer is a DNA stereocontrolled phosphoramidite monomer. In some embodiments, the stereocontrolled phosphoramidite monomer is a 2'-modified stereocontrolled phosphoramidite monomer, such as a 2'-methoxyethyl stereocontrolled phosphoramidite RNA monomer. In certain embodiments, the stereocontrolled phosphoramidite monomer can be an oxazaphosphocholine monomer, such as a DNA-oxazaphosphocholine LNA-oxazaphosphocholine monomer.

The monomers of the present invention can be used to reduce the hepatotoxicity of LNA oligonucleotides in vitro or in vivo.

LNA hepatotoxicity can be determined using a model mouse system, see, for example, EP 1 984 381. The monomers of the present invention can be used to reduce the nephrotoxicity of LNA oligonucleotides. LNA nephrotoxicity can be determined using a model rat system, typically using the Kim-1 biomarker (see, for example, WO 2014118267). The monomers of the present invention can be used to reduce the immunogenicity of LNA oligomers in vivo. According to EP 1 984 381, LNAs with a 4'-CH ₂ -O-2' group are particularly toxic.

The oligonucleotides of the invention may have improved nuclease resistance, biostability, target affinity, RNase H activity and/or lipophilicity. Thus, the invention provides methods for enhancing the activity of oligomers in vivo and improving the pharmacological and/or toxicological profiles of oligomers.

In some embodiments, the LNA oligonucleotide has reduced toxicity compared to an equivalent non-stereoselective LNA oligonucleotide, such as reduced in vivo hepatotoxicity, for example, as measured using the assay provided in Example 6, or reduced in vitro hepatotoxicity, for example, as measured using the assay provided in Example 8, or reduced renal toxicity, for example, as measured using the assay provided in Example 9. Reduced toxicity can also be assessed using other methods known in the art, such as caspase assays and primary hepatocyte toxicity assays (e.g., Example 8).

RNase H recruitment

As illustrated in the Examples, in some embodiments, the stereospecific oligonucleotides of the present invention have enhanced RNase H recruitment activity compared to other non-stereospecific oligonucleotides (parent oligonucleotides). In fact, the inventors surprisingly discovered that, in general, the introduction of stereospecific phosphorothioate internucleoside linkages into RNase H recruiting LNA oligonucleotides, for example, LNA gapper oligonucleotides, resulted in enhanced RNase H recruitment activity of up to 30 times that of the parent (non-stereospecific) oligonucleotides. Thus, the present invention provides the use of stereocontrolled (also known as stereospecific) phosphoramidite monomers for synthesizing oligonucleotides having enhanced RNase H recruitment activity compared to otherwise identical non-stereospecific oligonucleotides.

The present invention may also include a method for enhancing the RNase H recruitment activity of an antisense oligonucleotide sequence (parent oligonucleotide) for an RNA target, comprising the steps of:

a. generating a library of stereospecific oligonucleotide variants (daughter oligonucleotides) that retain the core nucleobase sequence of the parent oligonucleotide,

b. screening the library generated in step a for in vitro RNase H recruitment activity against RNA targets,

c. Identifying one or more stereospecific variants present in the library that have enhanced RNase H recruitment activity compared to the parent oligonucleotide.

d. Optionally producing at least one stereospecific variant identified in step c.

It will be appreciated that this method may be combined with methods for reducing the toxicity of the oligonucleotides according to the invention.

The present invention provides LNA oligonucleotides having enhanced RNase H recruitment activity compared to otherwise identical non-stereospecific LNA oligonucleotides (or parent oligonucleotides). LNA oligonucleotides having enhanced RNase H recruitment activity may further have reduced in vitro or in vivo toxicity, such as reduced hepatotoxicity or reduced nephrotoxicity.

Otherwise identical non-stereospecific LNA oligonucleotides (e.g., parent oligonucleotides) are non-stereospecific phosphorothioate oligonucleotides having the same nucleobase sequence and chemical modifications, but without stereospecific phosphorothioate linkages. It will be appreciated that non-stereospecific LNA oligonucleotides may include stereospecific centers in compound moieties other than phosphorothioate internucleotide linkages, for example, within nucleosides.

The use of chirally defined phosphorothioate linkages in LNA oligonucleotides surprisingly results in increased RNase H activity. This is seen when the gap region comprises stereospecific Rp and Sp internucleoside bonds. In some embodiments, the gap region of an oligonucleotide of the invention comprises at least two Rp and at least two Sp stereospecific internucleoside bonds. In some embodiments, the ratio of Rp to Sp stereospecific internucleoside bonds within the gap region (including internucleoside bonds adjacent to the wing regions) is between about 0.25 and about 0.75. In some embodiments, the gap region of an oligonucleotide of the invention comprises at least two consecutive internucleoside bonds that are stereospecific Rp or Sp internucleoside bonds. In some embodiments, the gap region of an oligonucleotide of the invention comprises at least three consecutive internucleoside bonds that are stereospecific Rp or Sp internucleoside bonds.

In some embodiments, the LNA oligonucleotide has enhanced human RNase H recruitment activity compared to an equivalent non-stereoselective LNA oligonucleotide, for example, using the RNase H recruitment assay provided in Example 7. In some embodiments, the increase in RNase H activity is at least 2-fold, for example at least 5-fold, for example at least 10-fold, greater than the RNase H activity of an equivalent non-stereoselective LNA oligonucleotide (e.g., a parent oligonucleotide). Example 7 provides a suitable RNase H assay (also referred to as RNase H recruitment) that can be used to assess RNase H activity.

It has been found that significant improvements in RNase H activity are observed using LNA gapper compounds, wherein the gap region comprises Rp and Sp internucleoside bonds, and in some embodiments, the gap region may comprise at least two Rp internucleoside bonds and at least two Sp internucleoside bonds, for example at least three Rp internucleoside bonds and/or at least three Sp internucleoside bonds.

Significant improvements in RNase H activity have been observed with LNA gapper compounds in which the internucleoside bonds in the gap region are stereospecific. Thus, in some embodiments, at least one stereoselective phosphorothioate LNA oligonucleotide is present, comprising at least one stereoselective phosphorothioate bond between an LNA nucleoside and the subsequent (3') nucleoside. In some embodiments, at least one of the internucleoside bonds within regions X' and/or Z' is an Rp internucleoside bond. In some embodiments, the 5'-most internucleoside bond in the oligomer or region X' is an Sp internucleoside bond. In some embodiments, the flanking regions X' and Z' comprise at least one Sp internucleoside bond and at least one Rp internucleoside bond. In some embodiments, the 3' internucleoside bond in the oligomer or region Z' is an Sp internucleoside bond.

In some embodiments, the stereospecific oligonucleotides of the invention have improved potency compared to other non-stereospecific oligonucleotides or parent oligonucleotides.

Specificity and mismatch discrimination

As shown in the Examples, in some embodiments, the stereospecific oligonucleotides of the present invention can have enhanced mismatch discrimination (or enhanced target specificity) compared to otherwise non-stereospecific oligonucleotides (or parent oligonucleotides). Indeed, the present inventors surprisingly discovered that the introduction of stereospecific phosphorothioate internucleoside bonds into RNase H recruiting LNA oligonucleotides, such as LNA spacer oligonucleotides, can result in enhanced mismatch discrimination (or target specificity). Thus, the present invention provides the use of stereospecifically controlled phosphorothioate monomers for synthesizing oligonucleotides having enhanced mismatch discrimination (or target specificity) compared to otherwise identical non-stereospecific oligonucleotides.

Thus, the present invention may further include a method for enhancing mismatch discrimination (or target specificity) of an antisense oligonucleotide sequence (parent oligonucleotide) to an RNA target in a cell, comprising the steps of:

a. Generate a library of stereospecific oligonucleotide variants (daughter oligonucleotides) that retain the parent

The core nucleobase sequence of the oligonucleotide,

b. screening the library generated in step a for activity against the RNA target and for its activity against at least one other RNA present,

c. Identifying one or more stereospecific variants present in the library that have reduced activity against at least one other RNA compared to the parent oligonucleotide.

It will be appreciated that this method may be combined with methods for reducing the toxicity of the oligonucleotides according to the invention.

Reduced activity against at least one other RNA can be determined as the ratio of activity against the intended target/unintended target (at least one other RNA). This method can also be combined with methods for enhancing RNase H recruitment activity of antisense oligonucleotide sequences (parent oligonucleotides) against RNA targets to identify oligonucleotides of the invention having enhanced RNase H recruitment activity and enhanced mismatch discrimination (i.e., enhanced targeting specificity), and reduced in vitro or in vivo toxicity.

The present invention provides LNA oligonucleotides having enhanced mismatch discrimination (or enhanced target specificity) compared to otherwise identical non-stereospecific LNA oligonucleotides (or parent oligonucleotides). LNA oligonucleotides having enhanced mismatch discrimination (or enhanced target specificity) may also have reduced in vitro or in vivo toxicity, such as reduced hepatotoxicity or reduced nephrotoxicity, and optionally enhanced RNase H recruitment activity.

The present invention provides LNA oligonucleotides having reduced in vivo or in vitro toxicity and enhanced RNase H recruitment activity and enhanced mismatch discrimination (or enhanced target specificity) compared to otherwise identical non-stereospecific LNA oligonucleotides (or parent oligonucleotides).

Thus, the present invention provides the use of stereocontrolled/stereocontrolled (also referred to as stereospecific or stereospecific) phosphoramidite monomers for the synthesis of oligonucleotides having reduced toxicity, enhanced mismatch discrimination (or target specificity) and enhanced RNase H recruitment activity compared to otherwise identical non-stereospecific oligonucleotides.

In some embodiments, the stereocontrolling phosphoramidite monomer is an LNA stereocontrolling (stereospecific) phosphoramidite monomer. In some embodiments, the stereocontrolling phosphoramidite monomer is a DNA stereocontrolling phosphoramidite monomer. In some embodiments, the stereocontrolling (stereospecific) phosphoramidite monomer is a 2'-modified stereocontrolling (stereospecific) phosphoramidite monomer, such as a 2'-methoxyethyl stereocontrolling (stereospecific) phosphoramidite RNA monomer. In certain embodiments, the stereocontrolling (stereospecific) phosphoramidite monomer can be an oxazaphosphorylcholine monomer, such as a DNA-oxazaphosphorylcholine LNA-oxazaphosphorylcholine monomer. In some embodiments, the stereocontrolling (stereospecific) phosphoramidite monomer can comprise a nucleobase selected from A, T, U, C, 5-methyl-C, or G nucleobases.

Biodistribution

The present invention provides a method for altering the biodistribution of an antisense oligonucleotide sequence (parent oligonucleotide), comprising the following steps:

a. generating a library of stereospecific oligonucleotide variants (daughter oligonucleotides) that retain the core nucleobase sequence of the parent oligonucleotide,

b. screening the libraries generated in step a for their biodistribution,

c. identifying one or more stereospecific variants present in the library that have an altered (eg, preferred) biodistribution compared to the parent oligonucleotide,

d. Optionally producing one or more stereospecific variants identified in step c.

The above methods can be used in conjunction with the methods for reducing toxicity disclosed herein.

Recognizing that in some embodiments, a parent oligonucleotide may be a mixture of different stereoisomeric forms, the methods of the present invention may include methods for identifying a single stereospecific oligonucleotide or single stereoisomer (daughter oligonucleotide) having a reduced toxicity, and optionally one or more other improved properties, such as enhanced specificity, altered biodistribution, enhanced potency, compared to the parent oligonucleotide.

In some embodiments, compounds of the invention or identified by the methods of the invention have enhanced biodistribution to the liver.

In some embodiments, compounds of the invention or identified by the methods of the invention have an enhanced liver/kidney biodistribution ratio.

In some embodiments, compounds of the invention or identified by the methods of the invention have an enhanced kidney/liver biodistribution ratio.

In some embodiments, compounds of the invention or identified by the methods of the invention have enhanced biodistribution to the kidney.

In some embodiments, compounds of the invention or identified by the methods of the invention have enhanced cellular uptake in hepatocytes.

In some embodiments, compounds of the invention or identified by the methods of the invention have enhanced cellular uptake in renal cells.

When referring to compounds having enhanced functional characteristics, the enhancement may be made with respect to a parent oligonucleotide, e.g., an otherwise identical non-stereospecific oligonucleotide.

Although biodistribution studies are typically performed in vivo, they can also be performed in in vitro systems, for example by comparing cellular uptake in different cell types, such as in vitro hepatocytes (e.g. primary hepatocytes) or renal cells (e.g. renal epithelial cells, such as PTEC-TERT1 cells).

Oligonucleotides

The term "oligomer" or "oligomeric compound" in the context of the present invention refers to a molecule (i.e., an oligonucleotide) formed by the covalent linkage of two or more nucleotides. Herein, a single nucleotide (unit) may also be referred to as a monomer or unit. In some embodiments, the terms "nucleoside," "nucleotide," "unit," and "monomer" are used interchangeably. It should be understood that when referring to a sequence of nucleotides or monomers, this refers to a base sequence such as A, T, G, C, or U. The term oligonucleotide is used interchangeably with the terms oligomeric compound and oligomer herein.

In some embodiments, the oligonucleotide is 10-20 nucleotides in length, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In some embodiments, the parent and daughter oligonucleotides are LNA oligomers. In some embodiments, the LNA oligomer comprises at least one stereoselective phosphorothioate bond between an LNA nucleoside and a subsequent (3') nucleoside. In some embodiments, the LNA oligomer comprises at least one stereospecific phosphorothioate nucleotide pair, wherein the internucleoside bond between the nucleosides of the stereospecific phosphorothioate nucleotide pair is in the Rp configuration or the Rs configuration, and wherein at least one nucleoside of the nucleotide pair is an LNA nucleotide. In some embodiments, the other nucleotide of the nucleotide pair is not DNA, e.g., a nucleoside analog, e.g., another LNA nucleoside or a 2'-substituted nucleoside.

In some embodiments, the oligomer is a stereospecific (stereoselective) phosphorothioate LNA oligonucleotide comprising at least one stereoselective phosphorothioate bond between an LNA nucleoside and a subsequent (3') nucleoside. Such an LNA oligonucleotide can, for example, be an LNA gapmer as described herein.

In some embodiments, the oligonucleotide comprises a central region (Y') of at least 5 or more contiguous nucleosides, a 5' wing region (X') comprising 1-6 LNA nucleosides, and a 3' wing region (Z') comprising 1-6 LNA nucleosides, wherein at least one of the internucleoside linkages of the central region is stereospecific, and wherein the central region comprises Rp and Sp internucleoside linkages.

parent oligonucleotides

In some embodiments, the parent oligonucleotide can be a non-stereospecific phosphorothioate oligonucleotide, i.e., an oligonucleotide comprising a mixture of individual molecules having unrestricted chirality of the phosphorothioate linkage, e.g., a racemic mixture. In other words, in some embodiments, the parent oligonucleotide can have only stereounrestricted phosphorothioate internucleoside linkages (i.e., a stereounrestricted oligonucleotide).

In some embodiments, the parent oligonucleotide comprises non-stereospecific phosphorothioate internucleoside linkages. In some embodiments, all internucleoside linkages present in the parent oligonucleotide are non-stereospecific internucleoside linkages, such as non-stereospecific internucleoside phosphorothioate linkages.

In some embodiments, the parent oligonucleotide may comprise one or more stereospecific phosphorothioate internucleoside linkages. In some embodiments, all phosphorothioate internucleoside linkages of the parent oligonucleotide are stereospecific phosphorothioate internucleoside linkages. In some embodiments, the parent oligonucleotide is identified by an earlier iteration of the method of the present invention.

Variant Library

The method of the present invention comprises the step of producing a library of parent oligonucleotide variants, wherein the variants have at least one stereospecific phosphorothioate internucleoside bond that is different from the parent oligonucleotide. Suitably, each member of the variant library has a different pattern of stereospecific phosphorothioate internucleoside bonds that are different from the parent.

In some embodiments, each member of the daughter oligonucleotide library (stereospecifically defined oligonucleotide variants) comprises at least 2, such as at least 3, such as at least 4 stereospecific phosphorothioate bonds, wherein the remaining bonds may optionally be non-stereospecific defined phosphorothioate bonds. Suitably, the at least 2, at least 3, or at least 4 stereospecific phosphorothioate bonds present in the daughter oligonucleotide are different from those of the parent (e.g., the parent does not comprise stereospecific phosphorothioate internucleoside bonds, or the parent comprises different stereospecific phosphorothioate bonds, or a different pattern or stereospecific phosphorothioate bonds).

In some embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the bonds in the (e.g., daughter) oligomer are stereospecific phosphorothioate bonds. In some embodiments, all phosphorothioate bonds in the (e.g., daughter) oligomer are stereospecific phosphorothioate bonds. In some embodiments, all internucleoside bonds in the (e.g., daughter) oligomer are stereospecific phosphorothioate bonds. It should be understood that stereospecificity (stereospecificity) refers to the incorporation of a high proportion, i.e., at least 75%, of Rp or Sp internucleoside bonds at defined internucleoside linkages.

In some embodiments, in step b) of the method of the present invention, each member of the library of stereospecific oligonucleotide variants can be generated by inserting at least one stereospecific phosphorothioate internucleoside linkage into the interstitial region of a parent interstitial. Suitably, the inserted stereospecific phosphorothioate internucleoside linkage is different from the equivalent internucleoside linkage of the parent, or the parent does not contain a stereospecific internucleoside linkage at the equivalent position.

In some embodiments, each member of the library of stereospecific oligonucleotide variants is generated by inserting at least one stereospecific phosphorothioate internucleoside linkage into the gap region of a gapmer.

In some embodiments, each member of the stereospecific oligonucleotide variant library is generated by inserting at least one stereospecific phosphorothioate internucleoside linkage into one or both wings of a gapmer.

In some embodiments, each member of the stereospecific oligonucleotide variant library is generated by inserting at least one stereospecific phosphorothioate internucleoside linkage into one or both wing regions of a gapmer and at least one stereospecific phosphorothioate internucleoside linkage into the gap region of a gapmer.

It will be appreciated that in some embodiments, the remaining internucleoside linkages of a daughter oligonucleotide, e.g., a gap region or a gapmer compound, can be the same as the parent, or in some embodiments can be different from the parent.

In some embodiments, the daughter oligonucleotide produced in step b) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 independent stereospecific phosphorothioate internucleoside linkages. In some embodiments, all phosphorothioate internucleoside linkages in the daughter oligonucleotide produced in step b) are stereospecific phosphorothioate internucleoside linkages. In some embodiments, all internucleoside linkages in the daughter oligonucleotide produced in step b) are stereospecific phosphorothioate internucleoside linkages.

In some embodiments, each member of the stereospecific oligonucleotide variant library comprises at least 2, such as at least 3, such as at least 4 stereospecific phosphorothioate linkages, wherein the remaining linkages may optionally be non-stereospecific defined phosphorothioate linkages.

In some embodiments, all phosphorothioate bonds present in each member of the stereospecific oligonucleotide variant library are stereospecifically defined phosphorothioate bonds.

In some embodiments, all internucleoside linkages present in each member of the library of the daughter oligonucleotides or gapmer regions thereof are stereospecific phosphorothioate linkages.

In some embodiments, each member of the oligonucleotide variant library of stereospecific definition retains the modification and unmodified nucleoside pattern present in the parent oligonucleotide, such as antisense gapmer oligonucleotide. In some embodiments, parent and daughter oligonucleotide share the same nucleoside modification pattern Formula, such as, the (such as) gapmer design of parent oligonucleotide is retained in daughter oligonucleotide or at least In a portion of daughter oligonucleotide. However, it is recognized that the variant library can include daughter oligonucleotides, although the overall gapmer design of the parent oligonucleotide is retained, a minority, such as 1 or 2 or 3 or 4 nucleosides can be included, wherein the sugar chemical property of the parent has been changed, such as by using in the wing Alternative nucleosides, such as using alternative high-affinity nucleosides in the wing region, or in the gap region Increase or decrease or offset.

In some embodiments, the mother and daughter oligonucleotides are LNA gapmer oligonucleotides.

Preserve core nucleobase sequence

The daughter oligonucleotide library comprises two or more stereospecific phosphorothioate oligonucleotides that retain the core nucleobase sequence of the parent compound.

In some embodiments, the daughter oligonucleotides can have the same length as the parent oligonucleotide and retain the same nucleobase sequence. However, it is contemplated that in some embodiments, the daughter oligonucleotides can be truncated, for example, by removing the 5' and/or 3' terminal nucleotides, or in some embodiments, can have additional nucleotides at the 5' and/or 3' termini. Since inserting one or more LNA nucleosides into the interstitial region increases affinity for the RNA target, removing one or more terminal high-affinity nucleosides, such as LNA nucleosides, allows the oligonucleotide's affinity for the RNA target to be maintained. It is contemplated that in some embodiments, the daughter oligonucleotide library can include variants with different flanking regions, some truncated, some with additional nucleosides, some with one or two offset nucleosides (e.g., measured for RNA targets), and some with additional high-affinity nucleosides in the flanking regions, thereby creating a complex library of stereospecific phosphorothioate oligonucleotides with heterogeneous phosphorothioate internucleoside linkages, thereby allowing for the simultaneous selection of daughter oligonucleotides with reduced toxicity compared to the parent.

The parent and daughter oligonucleotides share a core nucleobase sequence. Typical core nucleobase sequences are typically at least 10 nucleobases long, e.g., at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 nucleobases long, and in some embodiments may be the same nucleobase sequence as the parent oligonucleotide. In some embodiments, the parent and (at least a portion of) the daughter oligonucleotide have the same nucleobase sequence over the entire length of the oligonucleotide. However, it is contemplated that, in some embodiments, a portion of the daughter oligonucleotide may include additional 5' or 3' nucleotides, e.g., and an additional 1, 2, or 3 5' or 3' nucleotides. Additionally or alternatively, in some embodiments, a portion of the daughter oligonucleotide may be truncated relative to the parent, e.g., may include a 1, 2, or 3 nt truncation at the 5' or 3' end. In some embodiments, the additional nucleobases or truncation of the nucleobase sequence of (a portion of) the daughter oligonucleotide is a single nucleobase addition or truncation. In some embodiments, when aligned with the target sequence, the daughter oligonucleotide or a portion thereof may be offset by a single nucleobase or by 2 or 3 nucleobases compared to the parent oligonucleotide (effectively truncated at one end and added at the other end). The additional nucleotides retain complementarity to the target nucleic acid sequence.

Gap oligonucleotides

In some embodiments, parent oligonucleotide and daughter oligonucleotide are gap body oligonucleotide.Gap body oligonucleotide is widely used in suppressing target RNA such as mRNA in cell by antisense mechanism Or viral RNA (and therefore also can be referred to as antisense gap body oligonucleotide).Gap body oligonucleotide Acid comprises the region of at least 5 continuous nucleotides that can or raise RNase H (gap region), Such as the region of DNA nucleotide, for example, 6-14 DNA nucleotide, its flank is 5 ' and 3 ' Region, the region comprises the nucleoside of the modification of enhancing affinity, such as LNA or 2 ' substituted nucleotide Acid.In some embodiments, flanking region can be 1-8 nucleotides in length.

In some embodiments, the parent and daughter oligonucleotides are gapmer oligonucleotides comprising a central region (Y') of at least 5 or more consecutive nucleosides (e.g., at least 5 consecutive DNA nucleosides), a 5' wing region (Z') comprising 1-6 high affinity nucleoside analogs, e.g., LNA nucleosides, and a 3' wing region (Z') comprising 1-6 high affinity nucleoside analogs (e.g., LNA 1-6 nucleosides). LNA gapmer oligonucleotides are oligonucleotides comprising at least one LNA nucleotide in a wing region and, for example, may comprise at least one LNA in both the 5' and 3' wing regions.

In some embodiments, the daughter oligonucleotide is a gapmer, wherein at least one of the internucleoside linkages of the central region is stereospecific, and wherein the central region comprises Rp and Sp internucleoside linkages.

A gapmer oligonucleotide may comprise a central region (Y') of at least 5 or more consecutive nucleosides capable of recruiting RNase H, a 5' wing region (X') comprising 1-6 LNA nucleosides, and a 3' wing region (Z') comprising 1-6 LNA nucleosides. Suitable region Y' may have 6, 7, 8, 9, 10, 11, 12, 13, or 14 (e.g., 6-12) consecutive nucleotides, such as DNA nucleotides, and the nucleotides of regions X' and Z' adjacent to region Y' are LNA nucleotides. In some embodiments, regions X' and Z' have 1-6 nucleotides, at least one of which is an LNA in each wing (X' and Z'). In some embodiments, all nucleotides in regions X' and Z' are LNA nucleotides. In some embodiments, the oligonucleotides of the present invention comprise both LNA and DNA nucleosides. In some embodiments, the oligonucleotides of the present invention may be mixed-wing LNA gapmers, in which at least one LNA nucleoside in one wing region (or at least one LNA in each wing) is replaced with a 2'-substituted nucleoside, such as a 2'MOE nucleoside. In some embodiments, the LNA gapmer does not contain 2'-substituted nucleosides in the wing region. The internucleoside bonds between nucleotides in the contiguous nucleotide sequence of nucleotides in the region X'-Y'-Z' may all be phosphorothioate internucleoside bonds.

In some embodiments, in the daughter oligonucleotide (and optionally the parent), at least one of the internucleoside linkages of the central region is stereospecific, and wherein the central region comprises Rp and Sp internucleoside linkages.

In some embodiments, in the daughter oligonucleotide (and optional parent), the internucleoside bonds within region Y' are all stereospecific phosphorothioate internucleoside bonds. In some embodiments, in the daughter oligonucleotide (and optional parent), the internucleoside bonds within regions X' and Y' are stereospecific phosphorothioate internucleoside bonds. In some embodiments, in the daughter oligonucleotide (and optional parent), the internucleoside bonds between regions X' and Y' and between regions Y' and Z' are stereospecific phosphorothioate internucleoside bonds. In some embodiments, in the daughter oligonucleotide (and optional parent), all internucleoside bonds within consecutive nucleosides of region X'-Y'-Z' are stereospecific phosphorothioate internucleoside bonds.

Introducing at least one stereospecific phosphorothioate bond in the gap region of an oligonucleotide can be used to regulate the biological characteristics of the oligonucleotide, for example, it can regulate toxicity characteristics. In some embodiments, 2, 3, 4 or 5 phosphorothioates in the gap region in a sub-oligonucleotide (and optional parent) Acid ester bonds are stereospecific. In some embodiments, the remaining internucleoside bonds in the gap region are not stereospecific: they exist as a racemic mixture of Rp and Sp in an oligonucleotide species population. In some embodiments, in a sub-oligonucleotide (and optional parent), the remaining internucleoside bonds in an oligonucleotide are not stereospecific. In some embodiments, in a sub-oligonucleotide (and Optional parent), all internucleoside bonds in the gap region are stereospecific. Herein, the gap region (referred to as Y ') is a nucleotide region that can recruit RNase H, and can, for example, be a region of at least 5 continuous DNA nucleosides.

In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the bonds in the interstitial region of the oligomer are stereoselective phosphorothioate bonds.

In some embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the linkages in an oligomer (e.g., a gapmer) are stereoselective phosphorothioate linkages. In some embodiments, all phosphorothioate linkages in an oligomer are stereoselective phosphorothioate linkages. In some embodiments, all internucleoside linkages in an oligomer are stereospecific phosphorothioate linkages.

effect

The sub-oligonucleotides identified by the method of the present invention can be carried out as a part of the inventive method Row test, to determine that they are effective antisense oligonucleotides, such as they can inhibit their target nucleic acid. Therefore, the method of the present invention can include screening the sub-oligonucleotide library (such as before step b, During or after) the additional step, to obtain its effect in regulating, such as inhibiting their target. Alternatively, the method of the present invention can include testing the stereo-directed variant of the selected toxicity with reduction Towards the additional step of determining its effect as antisense oligonucleotide. In some embodiments In, the ability of oligonucleotide to raise RNase H is determined, or in some embodiments party It can be the ability of regulating target expression in cells in vitro or in some embodiments in vivo Power.

In some embodiments, the present invention provides the oligonucleotide of the present invention.It should be appreciated that the selected sub-oligonucleotide of toxicity reduction need not maintain the external or internal effectiveness of the parent oligonucleotide, but preferably they are effective antisense oligonucleotides with reduced toxicity.Suitably, when evaluated in vivo, the therapeutic index of oligonucleotide can be improved.The therapeutic index is usually calculated as maximum tolerated dose (MTD) (for example, three times the upper limit of normal for liver toxicity) divided by ED _50.For experimental purposes, assuming that the target sequence is present in mice, MTD and ED ₅₀ can be measured in mice in the mouse study of 7 days.If the sequence conservation in mice is disadvantageous, other model species can be used, for example, rat, monkey, dog or pig.

In some embodiments, the selected daughter oligonucleotide identified in step c retains at least 25%, such as at least 50%, such as at least 75%, such as at least 90% of the in vitro (e.g., IC ₅₀ ) or in vivo (e.g., ED ₅₀ or EC ₅₀ ) potency of the parent. In some embodiments, the selected daughter oligonucleotide identified in step c has similar in vitro (e.g., IC ₅₀ ) or in vivo (e.g., ED ₅₀ or EC ₅₀ ) potency as the parent (i.e., +/- 10%) or has enhanced in vitro (e.g., IC ₅₀ ) or in vivo (e.g., ED ₅₀ or EC ₅₀ ) potency compared to the parent. IC ₅₀ or ED ₅₀ should be evaluated in target cells expressing the intended target.

In some embodiments, the daughter oligonucleotide identified in step c has an improved EC ₅₀ value of the parent compound. In some embodiments, the daughter oligonucleotide identified in step c retains a similar EC ₅₀ value (i.e., +/- 10%) to the parent compound. In some embodiments, the daughter oligonucleotide identified in step c has an improved EC ₅₀ value of the parent compound.

In some embodiments, the daughter oligonucleotide identified in step c has an _EC50 value no greater than two times (2x), or in some embodiments three times (3x) that of the parent compound (i.e., +/- 10%). In some embodiments, the daughter oligonucleotide identified in step c retains an _EC50 value no greater than that of the parent compound.

LNA

In some embodiments, the parent and daughter oligonucleotides are LNA oligonucleotides, i.e., they comprise at least one LNA nucleoside.

LNA monomers (also known as bicyclic nucleic acids, BNAs) are ribose sugars with a diradical between the 2' and 4' positions of the ribose ring. The 2'-4' diradical is also known as a bridge. When incorporated into oligonucleotides, LNA monomers are known to enhance the binding affinity of the oligonucleotide to a complementary DNA or RNA sequence, typically measured or calculated as an increase in the temperature ( _Tm ) required to melt the oligonucleotide/target duplex.

LNA oligomers contain at least one "locked nucleic acid" (LNA) nucleoside, e.g., a nucleoside containing a covalent bridge (also known as a free radical) between the 2' and 4' positions (2'-4' bridge). LNA nucleosides are also known as "bicyclic nucleosides." LNA oligomers are typically single-stranded antisense oligonucleotides.

In some embodiments, the LNA oligomer comprises or is a gapmer. In some embodiments, all nucleoside analogs present in the oligomer are LNA.

In various embodiments, the compounds of the present invention do not include RNA (units). In some embodiments, the oligomer has a single continuous sequence that is a linear molecule or is synthesized as a linear molecule. Therefore, the oligomer can be a single-stranded molecule. In some embodiments, the oligomer does not include a short region of at least 3,4, or 5 consecutive nucleotides that are complementary to the equivalent region within the same oligomer (i.e., duplex). In some embodiments, the oligomer may not be (substantially) double-stranded. In some embodiments, the oligomer is not substantially double-stranded, for example, not siRNA. In some embodiments, the oligomeric compound is not in the duplex form with (substantially) complementary oligonucleotides--for example, not siRNA.

Target

The target of an oligonucleotide is typically a selected nucleic acid with which the oligonucleotide is capable of hybridizing under physiological conditions. For antisense therapy, the target nucleic acid is indicated by a medical condition. The target nucleic acid can be, for example, an mRNA or microRNA (also encompassed by the term target gene). Such oligonucleotides are referred to as antisense oligonucleotides.

Suitable oligonucleotides (oligomers) used in the methods or prepared by the methods of the present invention are capable of downregulating (e.g., reducing or eliminating) the expression of a target nucleic acid (also referred to as a target gene). In this regard, they can affect the inhibition of the target gene, typically in mammalian, such as human, cells. In some embodiments, the oligomer binds to the target nucleic acid and achieves at least 10% or 20% inhibition of expression compared to normal expression levels, more preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% inhibition compared to normal expression levels (e.g., expression levels in the absence of the oligomer or conjugate). In some embodiments, such modulation is observed when using a compound of the invention at a concentration of 0.04 to 25 nM, e.g., 0.8 to 20 nM. In the same or different embodiments, the inhibition of expression is less than 100%, e.g., less than 98% inhibition, less than 95% inhibition, less than 90% inhibition, less than 80% inhibition, e.g., less than 70% inhibition. Modulation of expression levels can be determined by measuring protein levels, for example, by methods such as SDS-PAGE followed by Western blotting using an appropriate antibody raised against the target protein. Alternatively, modulation of expression levels can be determined by measuring mRNA levels, for example, by Northern blotting or quantitative RT-PCR. When measured by mRNA levels, when an appropriate dose (e.g., 0.04 to 25 nM, e.g., 0.8 to 20 nM concentration) is used, the level of downregulation, in some embodiments, is typically 10-20% of the normal level in the absence of the compound, conjugate, or composition of the invention.

Thus, oligonucleotides can be used to downregulate or inhibit the expression of a target protein and/or target RNA in a cell expressing the target protein and/or target RNA, the method comprising administering an oligomer or conjugate according to the invention to the cell to downregulate or inhibit the expression of the target protein or RNA in the cell. Suitably, the cell is a mammalian cell, such as a human cell. In some embodiments, administration may occur in vitro. In some embodiments, administration may occur in vivo.

The oligomer may comprise or consist of a contiguous nucleotide sequence that corresponds to the reverse complement of a nucleotide sequence present in the target nucleic acid.

When determining the degree of "complementarity" between an oligomer of the invention (or a region thereof) and a target region of a nucleic acid, the degree of "complementarity" (also referred to as "homology" or "identity") is expressed as the percent identity (or percent homology) between the sequence of the oligomer (or a region thereof) and the sequence of the target region (or the reverse complement of the target region) with which it is optimally aligned. The percentage is calculated by dividing the number of aligned bases that are identical between the two sequences by the total number of consecutive monomers in the oligomer and multiplying by 100. In such a comparison, if gaps are present, it is preferred that such gaps are merely mismatches, rather than regions in which the number of monomers in the gap differs between the oligomer of the invention and the target region.

As used herein, the terms "homologous" and "homology" are interchangeable with the terms "identity" and "identical."

The terms "corresponding to" and "corresponding to" refer to a comparison between the nucleotide sequence of an oligomer (i.e., a nucleobase or base sequence) or contiguous nucleotide sequence (first region) and an equivalent contiguous nucleotide sequence of another sequence selected from i) a subsequence of the reverse complement of a nucleic acid target, e.g., an mRNA encoding a target protein. WO2014/118267 provides a number of target mRNAs of therapeutic relevance and oligomer sequences that can be optimized using the present invention (see, e.g., Table 3, NCBI Genbank references such as those disclosed in WO2014/118257).

Table 3

The terms "corresponding nucleotide analogue" and "corresponding nucleotide" are intended to indicate that the nucleotide in the nucleotide analogue is identical to the naturally occurring nucleotide. For example, when the 2-deoxyribose unit of the nucleotide is linked to adenine, the "corresponding nucleotide analogue" contains a pentose unit (other than 2-deoxyribose) linked to adenine.

As used herein, the terms "reverse complement," "reverse complementary," and "reverse complementarity" are interchangeable with the terms "complement," "complementary," and "complementarity."

length

The oligomer may consist of or comprise a contiguous nucleotide sequence of 7-30, for example 7-26 or 8-25, such as 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides in length, for example 10-20 nucleotides in length. In some embodiments, the LNA oligomer is 10-16 nucleotides in length, for example 12, 13 or 14 nucleotides.

In some embodiments, the oligomer comprises or consists of a contiguous nucleotide sequence having a total length of 10-22, e.g., 12-18, e.g., 13-17 or 12-16, e.g., 13, 14, 15, 16 contiguous nucleotides.

In some embodiments, the oligomer comprises or consists of a contiguous nucleotide sequence that is a total of 10, 11, 12, 13, or 14 contiguous nucleotides in length.

In some embodiments, the oligomer according to the present invention is composed of no more than 22 nucleotides, for example no more than 20 nucleotides, for example no more than 18 nucleotides, for example 15, 16 or 17 nucleotides. In some embodiments, the oligomer of the present invention comprises less than 20 nucleotides. It should be understood that when a range of oligomer or continuous nucleotide sequence length is given, it includes the upper and lower length limits provided within the range, for example from 10-30 (or between 10-30) including 10 and 30.

In some embodiments, the length of the oligomer is less than 20, such as less than 18, for example 16 nts or less or 15 or 14 nts or less. LNA oligomers are typically less than 20 in length.

In some embodiments, the oligomer comprises or consists of a contiguous nucleotide sequence that is a total of 10, 11, 12, 13, or 14 contiguous nucleotides in length.

In some embodiments, the oligomer according to the present invention is composed of no more than 22 nucleotides, for example no more than 20 nucleotides, for example no more than 18 nucleotides, for example 15, 16 or 17 nucleotides. In some embodiments, the oligomer of the present invention comprises less than 20 nucleotides. It should be understood that when a range of oligomer or continuous nucleotide sequence length is given, it includes the upper and lower length limits provided within the range, for example from 10-30 (or between 10-30) including 10 and 30.

Screening methods

The present invention provides a method for reducing the toxicity of stereo-nonspecific phosphorothioate oligonucleotide sequences, comprising the steps of:

a. providing a stereo-nonspecific phosphorothioate oligonucleotide (parent) having a toxic phenotype in vivo or in vitro,

b. generating a library of stereospecific phosphorothioate oligonucleotides (children) that retain the core nucleobase sequence of the parent gapmer oligonucleotides,

c. Screening the library generated in step b in an in vivo or in vitro toxicity assay to

d. Identifying one or more stereospecific phosphorothioate oligonucleotides having reduced toxicity compared to stereononspecific phosphorothioate oligonucleotides.

The stereospecific phosphorothioate oligonucleotide can be an oligonucleotide of the invention as disclosed herein. In some embodiments, the parent oligonucleotide is a gapmer oligonucleotide, such as an LNA gapmer oligonucleotide disclosed herein. In some embodiments, the stereospecific phosphorothioate oligonucleotide library comprises at least 2, for example, at least 5, or at least 10, or at least 15, or at least 20 stereospecific phosphorothioate oligonucleotides.

The screening method may further comprise the step of screening the sub-oligonucleotides for at least one other functional parameter, such as one or more of RNase H recruitment activity, RNase H cleavage specificity, target specificity, target binding affinity and/or in vivo or in vitro potency.

Thus, the methods of the present invention can be used to reduce the toxicity associated with the parent oligonucleotide. The toxicity of the oligonucleotide can be evaluated in vitro or in vivo. In vitro assays include caspase assays (see, for example, the caspase assay disclosed in WO2005/023995) or hepatotoxicity assays (see, for example, Soldatow et al., Toxicol Res (Camb). 2013 Jan 1; 2(1): 23–39.). In vivo toxicity is typically identified in preclinical screening, for example in mice or rats. In vivo toxicity can be hepatotoxicity, which is typically measured by analyzing serum levels of liver transaminases, such as ALT and/or AST, or can be, for example, nephrotoxicity, which can be measured by measuring molecular markers of nephrotoxicity, such as serum creatinine levels or levels of the kidney injury marker mRNA, kim-1.

Therefore, the selected daughter oligonucleotides identified by the screening method are therefore safer and effective antisense oligonucleotides.

Interstitial region with stereospecific phosphorothioate linkages

As reported by Wan et al., there is little benefit in introducing an all-Rp or all-Sp interstitial region into an interstitial body compared to a random racemic mixture of phosphorothioate bonds. The present invention is based on the surprising benefit that the introduction of at least one stereospecific phosphorothioate bond can substantially improve the biological properties of oligonucleotides, for example, reduced toxicity. This can be achieved by introducing one or more, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 stereospecific phosphorothioate bonds, or by having all phosphorothioate bonds in a stereo-defined interstitial region.

In some embodiments, only 1, 2, 3, 4, or 5 of the internucleoside linkages in the central region (Y') are stereoselective phosphorothioate linkages, while the remaining internucleoside linkages are random Rp or Sp.

In some embodiments, all internucleoside linkages of the central region (Y') are stereoselective phosphorothioate linkages.

In some embodiments, the central region (Y') comprises at least 5 consecutive phosphorothioate-linked DNA nucleosides. In some embodiments, the central region is at least 8 or 9 DNA nucleosides in length. In some embodiments, the central region is at least 10 or 11 DNA nucleosides in length. In some embodiments, the central region is at least 12 or 13 DNA nucleosides in length. In some embodiments, the central region is at least 14 or 15 DNA nucleosides in length.

Stereoselective DNA motifs

We have previously identified certain DNA dinucleotides that may contribute to the toxicity profile of antisense oligonucleotides (Hagedorn et al., Nucleic Acid Therapeutics 2013, 23; 302–310). In some embodiments of the present invention, the toxicity of DNA dinucleotides in antisense oligonucleotides (e.g., LNA gapmer oligonucleotides described herein) can be modulated by introducing stereoselective phosphorothioate internucleoside bonds between the DNA nucleosides of the DNA dinucleotide, particularly dinucleotides known to promote toxicity, such as hepatotoxicity. In some embodiments, the oligonucleotides identified by the methods of the present invention comprise a DNA dinucleotide motif selected from cc, tg, tc, ac, tt, gt, ca, and gc, wherein the internucleoside bond between the DNA nucleosides of the dinucleotide is a stereospecific phosphorothioate bond, such as an Sp or Rp phosphorothioate internucleoside bond. Typically, such dinucleotides can be located within the gap region of a gapmer oligonucleotide, such as an LNA gapmer oligonucleotide. In some embodiments, the oligonucleotide identified by this method comprises at least two, such as at least three, dinucleotides, which are dependently or independently selected from the above-mentioned list of DNA dinucleotide motifs.

RNase recruitment

It will be appreciated that oligomeric compounds may act by non-RNase-mediated degradation of target mRNA, such as by steric hindrance of translation or other methods. In some embodiments, the oligomers of the invention are capable of recruiting endonuclease (RNase), such as RNase H.

Preferably, such an oligomer comprises a contiguous nucleotide sequence (region Y') comprising at least 6, For example, at least 7 contiguous nucleotide units, For example, at least 8 or at least 9 contiguous nucleotide units (residues), including 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 contiguous nucleotides, which, when forming a duplex with a complementary target RNA, can recruit an RNase. The continuous sequence capable of recruiting an RNase can be the region Y' referred to in the context of a gapmer as described herein. In some embodiments, the size of the contiguous sequence capable of recruiting an RNase, For example, region Y', can be higher, For example 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotide units.

EP 1 222 309 provides an in vitro method for determining RNase H activity, which can be used to determine the ability to recruit RNase H. Using the method provided in Examples 91-95 of EP 1 222 309, an oligomer is considered capable of recruiting RNase H if, when provided with a complementary RNA target, the oligomer has an initial rate measured in pmol/l/min of at least 1%, such as at least 5%, such as at least 10% or 20% or more of the initial rate determined using a DNA-only oligonucleotide having the same base sequence but containing only DNA monomers, no 2' substitutions, and having phosphorothioate linkages between all monomers in the oligonucleotide.

In some embodiments, using the methods provided in Examples 91-95 of EP 1 222 309, an oligomer is considered to be substantially incapable of recruiting RNase H if the oligonucleotide, when provided with a complementary RNA target and RNase H, has an initial rate of RNase H measured in pmol/l/min that is less than 1%, such as less than 5%, such as less than 10% or less than 20% of the initial rate determined using an equivalent DNA-only oligonucleotide having no 2' substitutions and having phosphorothioate linkages between all nucleotides in the oligonucleotide.

In other embodiments, using the methods provided in Examples 91-95 of EP 1 222 309, an oligomer is considered capable of recruiting RNase H when provided with a complementary RNA target and RNase H if the initial rate of RNase H, measured in pmol/l/min, is at least 20%, such as at least 40%, such as at least 60%, such as at least 80% of the initial rate determined using an equivalent DNA-only oligonucleotide having no 2' substitutions and having phosphorothioate linkages between all nucleotides in the oligonucleotide.

Typically, a region of an oligomer that forms contiguous nucleotide units, when duplexed with a complementary target RNA, is capable of recruiting an RNase composed of nucleotide units that form a DNA/RNA-like duplex with the RNA target. An oligomer of the invention, such as the first region, may comprise a nucleotide sequence comprising nucleotides and nucleotide analogs, and may be in the form of, for example, a gapmer, a headmer, or a tailmer.

A "header" is defined as an oligomer comprising a region X' and an adjacent region Y', wherein the 5'-most monomer of region Y' is linked to the 3'-most monomer of region X'. Region X' comprises a continuous stretch of non-RNase-recruiting nucleoside analogs, and region Y' comprises a continuous stretch of DNA monomers (e.g., at least 7 consecutive monomers) or nucleoside analog monomers that are recognized and cleaved by RNases.

A "tail" is defined as an oligomer comprising a region X' and an adjacent region Y', wherein the 5'-most monomer of region Y is linked to the 3'-most monomer of region X'. Region X' comprises a continuous stretch (e.g., at least 7 continuous monomers) of DNA monomers or nucleoside analog monomers that are recognizable and cleavable by RNases, and region X' comprises a continuous stretch of non-RNase recruiting nucleoside analogs.

In some embodiments, in addition to enhancing the affinity of the oligomer for the target region, some nucleoside analogs also mediate the binding and cleavage of RNases (e.g., RNase H). Since α-L-LNA (BNA) monomers recruit RNase H activity to some extent, in some embodiments, the interstitial region of the oligomer containing α-L-LNA monomers (e.g., region Y' described herein) is composed of fewer monomers that can be recognized and cleaved by RNase H, thereby introducing greater flexibility in hybrid construction.

Gap body design

In some embodiments, the oligomers of the present invention comprise or are LNA gapmers. A gapmer oligomer is an oligomer comprising a continuous nucleotide stretch capable of recruiting an RNase, such as RNase H, for example, a region of at least 5, 6, or 7 DNA nucleotides, referred to herein as region Y' (Y'), wherein region Y' is flanked 5' and 3' by affinity-enhancing nucleotide analogs, for example, 1-6 affinity-enhancing nucleotide analogs 5' and 3' of the continuous nucleotide stretch capable of recruiting an RNase - these regions are referred to as regions X' (X') and Z' (Z'), respectively. Examples of gapmers are disclosed in WO2004/046160, WO2008/113832, and WO2007/146511. The LNA gapmer oligomers of the present invention comprise at least one LNA nucleoside in region X' or Z', for example, at least one LNA nucleoside in region X' and at least one LNA nucleotide in region Z'.

In some embodiments, the monomer capable of recruiting RNase is selected from a DNA monomer, an α-L-LNA monomer, a C4'alkylated DNA monomer (see PCT/EP2009/050349 and Vester et al., Bioorg. Med. Chem. Lett. 18 (2008) 2296-2300, which are incorporated herein by reference) and a UNA (unlinked nucleic acid) nucleotide (see Fluiter et al., Mol. Biosyst., 2009, 10, 1039, incorporated herein by reference). UNA is an unlocked nucleic acid, typically wherein the C2-C3C-C bond of the ribose sugar is removed to form an unlocked "sugar" residue. Preferably, the gapmer comprises a (poly)nucleotide sequence of the formula X'-Y'-Z' (5' to 3'), wherein: region X' (X') (5' region) consists of or comprises at least one high-affinity nucleotide analog, such as at least one LNA unit, for example 1-6 affinity-enhancing nucleotide analogs, such as LNA units, and region Y' (Y') consists of or comprises at least five consecutive nucleotides capable of recruiting an RNase (when forming a duplex with a complementary RNA molecule, such as an mRNA target), such as DNA nucleotides, and region Z' (Z') (3' region) consists of or comprises at least one high-affinity nucleotide analog, such as at least one LNA unit, for example 1-6 affinity-enhancing nucleotide analogs, such as LNA units.

In some embodiments, region X' comprises or consists of 1, 2, 3, 4, 5 or 6 LNA units, for example 2-5 LNA units, for example 3 or 4 LNA units; and/or region Z' consists of or comprises 1, 2, 3, 4, 5 or 6 LNA units, for example 2-5 LNA units, for example 3 or 4 LNA units.

In some embodiments, region X' may comprise 1, 2, 3, 4, 5 or 6 2' substituted nucleotide analogues, such as 2'MOE; and/or region Z' comprises 1, 2, 3, 4, 5 or 6 2' substituted nucleotide analogues, such as 2'MOE units.

In some embodiments, the substituent at the 2' position is selected from F; _CF3 , CN, _N3 , NO, _NO2 , O-, S-, or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, O-alkyl-N-alkyl or N-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl groups may be substituted or unsubstituted _C1 - _C10 alkyl or _C2 - _C10 alkenyl and alkynyl groups. Examples of 2' substituents include, but are not limited to, O( _CH2 ) _OCH3 , and O( _CH2 ) _NH2 , wherein n is 1 to about 10, such as MOE, DMAOE, DMAEOE.

In some embodiments, Y' consists of or comprises 5, 6, 7, 8, 9, 10, 11 or 12 consecutive nucleotides that are capable of recruiting RNases, or 6-10, or 7-9, such as 8 consecutive nucleotides that are capable of recruiting RNases. In some embodiments, region Y' consists of or comprises at least one DNA nucleotide unit, such as 1-12 DNA units, preferably 4-12 DNA units, more preferably 6-10 DNA units, such as 7-10 DNA units, for example 8, 9 or 10 DNA units.

In some embodiments, region X' consists of 3 or 4 nucleotide analogs, such as LNA, region X' consists of 7, 8, 9 or 10 DNA units, and region Z' consists of 3 or 4 nucleotide analogs, such as LNA. Such designs include (X'-Y'-Z')3-10-3, 3-10-4, 4-10-3, 3-9-3, 3-9-4, 4-9-3, 3-8-3, 3-8-4, 4-8-3, 3-7-3, 3-7-4, and 4-7-3.

Other gapmer designs are disclosed in WO2004/046160, which is incorporated herein by reference. WO2008/113832 (which requires priority to U.S. Provisional Application 60/977,409, which is incorporated herein by reference) relates to "shortmer" gapmer oligomers. In some embodiments, the oligomer presented here can be such a shortmer gapmer.

In some embodiments, the oligomer, for example, region X', consists of a contiguous nucleotide sequence of a total of 10, 11, 12, 13 or 14 nucleotide units, wherein the contiguous nucleotide sequence comprises or is of the formula (5'-3'), X'-Y'-Z' wherein; X' consists of 1, 2 or 3 affinity-enhancing nucleotide analog units, such as LNA units; Y' consists of 7, 8 or 9 contiguous nucleotide units, which are capable of recruiting RNases when forming a duplex with a complementary RNA molecule (e.g., an mRNA target); and Z' consists of 1, 2 or 3 affinity-enhancing nucleotide analog units, such as LNA units.

In some embodiments, the oligomer comprises a contiguous nucleotide sequence of a total of 10, 11, 12, 13, 14, 15, or 16 nucleotide units, wherein the contiguous nucleotide sequence comprises or is of the formula (5'-3'), X'-Y'-Z', wherein: X' comprises 1, 2, 3, or 4 LNA units; Y' consists of 7, 8, 9, or 10 contiguous nucleotide units, which, when duplexed with a complementary RNA molecule (e.g., an mRNA target), such as a DNA nucleotide, is capable of recruiting an RNase; and Z' comprises 1, 2, 3, or 4 LNA units.

In some embodiments, X' consists of 1 LNA unit. In some embodiments, X' consists of 2 LNA units. In some embodiments, X' consists of 3 LNA units. In some embodiments, Z' consists of 1 LNA unit. In some embodiments, Z' consists of 2 LNA units. In some embodiments, Z' consists of 3 LNA units. In some embodiments, Y' consists of 7 nucleotide units. In some embodiments, Y' consists of 8 nucleotide units. In some embodiments, Y' consists of 9 nucleotide units. In certain embodiments, region Y' consists of 10 nucleoside monomers. In certain embodiments, region Y' consists of or comprises 1-10 DNA monomers. In some embodiments, Y' comprises 1-9 DNA units, for example, 2, 3, 4, 5, 6, 7, 8, or 9 DNA units. In some embodiments, Y' consists of DNA units. In some embodiments, Y' comprises at least one LNA unit in the α-L configuration, for example 2, 3, 4, 5, 6, 7, 8 or 9 LNA units in the α-L configuration. In some embodiments, Y' comprises at least one α-L-oxy LNA unit, or all LNA units in the α-L configuration are α-L-oxy LNA units. In some embodiments, the number of nucleotides present in X'-Y'-Z' is selected from (nucleotide analogue unit-region Y'-nucleotide analogue unit): 1-8-1, 1-8-2, 2-8-1, 2-8-2, 3-8-3, 2-8-3, 3-8-2, 4-8-1, 4-8-2, 1-8-4, 2-8-4, or; 1-9-1, 1-9-2, 2-9-1, 2-9-2, 2-9-3, 3-9-2, 1-9-3, 3-9-1, 4-9-1, 1-9-4, or; 1-10-1, 1-10-2, 2-10-1, 2-10-2, 1-10-3, 3-10-1. In some embodiments, the number of nucleotides in X'-Y'-Z' is selected from the group consisting of: 2-7-1, 1-7-2, 2-7-2, 3-7-3, 2-7-3, 3-7-2, 3-7-4, and 4-7-3. In certain embodiments, regions X' and Y' each consist of three LNA monomers, and region Y' consists of 8, 9, or 10 nucleoside monomers, preferably DNA monomers. In some embodiments, X' and Z' each consist of two LNA units, and Y' consists of 8 or 9 nucleotide units, preferably DNA units. In various embodiments, other gapmer designs include those in which regions X' and/or Z' consist of 3, 4, 5, or 6 nucleoside analogs, such as monomers containing 2'-O-methoxyethyl-ribose (2'-MOE) or monomers containing 2'-fluorodeoxyribose, and Y' consists of 8, 9, 10, 11, or 12 nucleosides, such as DNA monomers, wherein the region X'-Y'-Z' has 3-9-3, 3-10-3, 5-10-5, or 4-12-4 monomers. WO 2007/146511A2, incorporated herein by reference, discloses other gapmer designs.

In the gapmer designs reported herein, the gap region (Y') may comprise one or more stereospecific phosphorothioate linkages, and the remaining internucleoside linkages of the gap region may, for example, be non-stereospecific internucleoside linkages, or may also be stereospecific phosphorothioate linkages.

internucleotide bonds

The oligomer identified by the method of the present invention comprises at least one stereospecific phosphorothioate bond. Although most compounds for therapeutic use use phosphorothioate internucleotide bonds, it is possible to use other internucleoside bonds. However, in some embodiments, all internucleoside bonds of the oligomer of the present invention are phosphorothioate internucleoside bonds. In some embodiments, the bonds in the gap region are all phosphorothioate, and the internucleoside bonds in the wing region can be phosphorothioate or phosphodiester bonds.

The nucleoside monomers of the oligomers described herein are coupled together by [internucleoside] linking groups. Suitably, each monomer is linked to the 3' adjacent monomer by a linking group.

One of ordinary skill in the art will understand that, in the context of the present invention, the 5' monomer at the end of the oligomer does not contain a 5' linking group, although it may or may not contain a 5' terminal group.

The term "linking group" or "internucleotide bond" is intended to mean a group capable of covalently coupling two nucleotides. Specific and preferred examples include phosphate and phosphorothioate groups.

The nucleotide of oligomer of the present invention or its continuous nucleotide sequence is coupled together through linking group.Each nucleotide is suitably connected to 3 ' adjacent nucleotide through linking group.

Suitable internucleotide linkages include those listed in WO2007/031091, for example the internucleotide linkages listed in the first paragraph on page 34 of WO2007/031091 (incorporated herein by reference).

In some embodiments, it is desirable to modify the internucleotide linkage from its normal phosphodiester to a nucleotide linkage that is more resistant to nuclease attack, such as phosphorothioate or boranophosphate - both of which can be cleaved by RNase H and also allow for antisense inhibition pathways that reduce expression of the target gene.

Suitable sulfur (S) containing internucleotide linkages as provided herein may be preferred, such as phosphorothioates or phosphorodithioates.

For gapmers, the internucleotide linkages in the oligomer can be, for example, phosphorothioate or borophosphate to allow RNA-targeted RNase H cleavage. Phosphorothioates are generally preferred for improved nuclease resistance and other reasons, such as ease of manufacturing.

WO09124238 refers to oligomeric compounds having at least one bicyclic nucleoside (LNA) linked to the 3' or 5' terminus via a neutral internucleoside bond. Thus, the oligomers of the present invention may have at least one bicyclic nucleoside linked to the 3' or 5' terminus via a neutral internucleoside bond, such as one or more phosphotriesters, methylphosphonates, MMIs, amide-3's, methylacetals, or thiomethylacetals. The remaining bonds may be phosphorothioates.

Stereo-controlled monomers

Stereo controlled monomers are monomers used in oligonucleotide synthesis that confer stereospecific phosphorothioate internucleoside bonds, i.e., Sp or Rp, in oligonucleotides. In some embodiments, monomers can be amidites, such as phosphoramidites. Therefore, in some embodiments, monomers can be stereo controlled/controlled amidites, such as stereo controlled/controlled phosphoramidites. Suitable monomers are provided in the Examples or in Oka et al., J.AM.CHEM.SOC.2008, 130, 16031 16037 9 16031. See also WO10064146, WO11005761, WO13012758, WO14010250, WO14010718, WO14012081 and WO15107425. The terms stereo controlled/stereo control are used interchangeably herein and may also be referred to as stereospecific/stereospecific monomers.

Because the stereocontrolled monomers can therefore be referred to as stereocontrolled "phosphorothioate" monomers. The terms stereocontrolled and stereocontrolling are used interchangeably herein. In some embodiments, the stereocontrolled monomers, when used with a sulfarizing agent during oligonucleotide synthesis, produce stereospecific internucleoside linkages at the 3' side of a newly introduced nucleoside (or the 5' side of a growing oligonucleotide chain).

Nucleosides and nucleoside analogs

In some embodiments, the terms "nucleoside analog" and "nucleotide analog" are used interchangeably.

As used herein, the term "nucleotide" refers to a glycoside comprising a sugar moiety, a base moiety, and a covalent linking group (linker), such as a phosphate or phosphorothioate internucleotide linker, and encompasses naturally occurring nucleotides, such as DNA or RNA, as well as non-naturally occurring nucleotides comprising modified sugar and/or base moieties, which are also referred to herein as "nucleotide analogs." Herein, a single nucleotide (unit) may also be referred to as a monomer or nucleic acid unit.

In the field of biochemistry, the term "nucleoside" is generally used to refer to a glycoside comprising a sugar moiety and a base moiety, and thus may be used when referring to a nucleotide unit covalently linked by an internucleotide bond between the nucleotides of an oligomer. In the field of biotechnology, the term "nucleotide" is generally used to refer to a nucleic acid monomer or unit, and thus in the context of an oligonucleotide, may refer to a base—e.g., "nucleotide sequence"—generally refers to the nucleobase sequence (i.e., the presence of a sugar backbone and internucleoside bonds is implicit). Similarly, particularly in the context of an oligonucleotide in which one or more internucleoside linking groups are modified, the term "nucleotide" may refer to a "nucleoside," and for example, the term "nucleotide" may be used even when specifying the presence or nature of the linkage between the nucleosides.

As will be appreciated by one of ordinary skill in the art, the 5' terminal nucleotide of an oligonucleotide does not contain a 5' internucleotide linking group, although a 5' terminal group may or may not be included.

Non-naturally occurring nucleotides include nucleotides with modified sugar moieties, such as bicyclic nucleotides or 2' modified nucleotides, such as 2' substituted nucleotides.

"Nucleotide analogs" are variants of natural nucleotides, such as DNA or RNA nucleotides, due to modifications of the sugar and/or base moieties. In principle, an analog may simply be "silent" or "equivalent" to a natural nucleotide in the context of an oligonucleotide, i.e., have no functional effect on the way the oligonucleotide works to inhibit target gene expression. However, such "equivalent" analogs may still be useful if, for example, they are easier or cheaper to manufacture, or more stable to storage or manufacturing conditions, or represent a tag or label. Preferably, however, the analog will have a functional effect on the way the oligomer acts to inhibit expression; for example, by producing increased binding affinity for the target and/or increased resistance to intracellular nucleases and/or increased ease of transport into cells. Specific examples of nucleoside analogs are described by, for example, Freier &Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, and Scheme 1:

Solution 1

Thus, the oligomer may comprise or consist of a simple sequence of naturally occurring nucleotides, preferably 2'-deoxynucleotides (generally referred to herein as "DNA"), or ribonucleotides (generally referred to herein as "RNA"), or a combination of such naturally occurring nucleotides and one or more non-naturally occurring nucleotides, i.e., nucleotide analogs. Such nucleotide analogs may appropriately enhance the affinity of the oligomer for the target sequence.

Examples of suitable and preferred nucleotide analogues are provided in WO 2007/031091 or referenced herein.

Incorporation of affinity-enhancing nucleotide analogs into oligomers (e.g., LNA or 2'-substituted sugars) can reduce the size of specifically binding oligomers and also lower the upper limit of oligomer size before nonspecific or aberrant binding occurs. The oligomers of the present methods refer to both parent and daughter oligonucleotides.

In some embodiments, the parent and daughter oligonucleotides comprise at least one nucleotide analog. In some embodiments, the parent and daughter oligonucleotides comprise at least two nucleotide analogs. In some embodiments, the parent and daughter oligonucleotides comprise 3-8 nucleotide analogs, e.g., 6 or 7 nucleotide analogs. In some embodiments, at least one of the nucleotide analogs is a locked nucleic acid (LNA); e.g., at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or 8 nucleotide analogs may be LNA. In some embodiments, all nucleotide analogs may be LNA.

It will be appreciated that when reference is made to a preferred nucleotide sequence motif or nucleotide sequence consisting solely of nucleotides, an oligomer defined by that sequence may comprise corresponding nucleotide analogues in place of one or more nucleotides present in the sequence, such as LNA units or other nucleotide analogues, which increase the duplex stability/ _Tm of the oligomer/target duplex (i.e. affinity-enhancing nucleotide analogues).

Examples of such modifications of nucleotides include modification of the sugar moiety to provide 2'-substituents or to create bridged (locked nucleic acid) structures that enhance binding affinity and may also provide increased nuclease resistance.

Preferred nucleotide analogs are LNA, such as oxy-LNA (e.g., β-D-oxy-LNA and α-L-oxy-LNA) and/or amino-LNA (e.g., β-D-amino-LNA and α-L-amino-LNA) and/or thio-LNA (e.g., β-D-thio-LNA and α-L-thio-LNA) and/or ENA (e.g., β-D-ENA and α-L-ENA). β-D-oxy-LNA is most preferred.

In some embodiments, the nucleotide analogs present in the oligomers of the present invention (e.g., regions X' and Y' as referred to herein) are independently selected from, for example, 2'-O-alkyl-RNA units, 2'-amino-DNA units, 2'-fluoro-DNA units, LNA units, arabinoic acid (ANA) units, 2'-fluoro-ANA units, HNA units, INA (Intercalating Nucleic Acids - Christensen, 2002. Nucl. Acids. Res 2002 30: 4918-4925, incorporated herein by reference) units, and 2'MOE units. In some embodiments, only one of the above-mentioned nucleotide analogs is present in the oligomers of the present invention or in their contiguous nucleotide sequences.

In some embodiments, the nucleotide analogs are 2'-O-methoxyethyl-RNA (2'MOE), 2'-fluoro-DNA monomers, or LNA nucleotide analogs. Thus, the oligonucleotides of the present invention may comprise nucleotide analogs independently selected from these three types of analogs, or may include only one type of analog selected from the three types. In some embodiments, at least one of the nucleotide analogs is 2'-MOE-RNA, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-MOE-RNA nucleotide units. In some embodiments, at least one of the nucleotide analogs is 2'-fluoro-DNA, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-fluoro-DNA nucleotide units.

In some embodiments, oligomers according to the method inventions comprise at least one locked nucleic acid (LNA) unit, for example, 1, 2, 3, 4, 5, 6, 7, or 8 LNA units, for example, 3-7 or 4 to 8 LNA units, or 3, 4, 5, 6, or 7 LNA units. In some embodiments, all nucleotide analogs are LNA. In some embodiments, the oligomer may comprise β-D-oxy-LNA and one or more of the following LNA units: thio-LNA, amino-LNA, oxy-LNA, and/or ENA, in the β-D or α-L configuration, or a combination thereof. In some embodiments, all LNA cytosine units are 5'-methyl-cytosine. In some embodiments of the present invention, the oligomer may comprise both LNA and DNA units. Preferably, the total number of LNA and DNA units combined is 10-25, for example, 10-24, preferably 10-20, such as 10-18, and even more preferably 12-16. In some embodiments of the present invention, the nucleotide sequence of the oligonucleotide, for example, a contiguous nucleotide sequence, consists of at least one LNA, with the remaining nucleotide units being DNA units. In some embodiments, the oligomer comprises only LNA nucleotide analogs and naturally occurring nucleotides (e.g., RNA or DNA, most preferably DNA nucleotides), optionally with modified internucleotide linkages such as phosphorothioate.

The term "nucleobase" refers to the base portion of a nucleotide and covers naturally occurring as well as non-naturally occurring variants. Thus, "nucleobase" encompasses not only the known purine and pyrimidine heterocycles, but also heterocyclic analogs and tautomers thereof.

Examples of nucleobases include, but are not limited to, adenine, guanine, cytosine, thymidine, uracil, xanthine, hypoxanthine, 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine.

In some embodiments, at least one of the nucleobases present in the oligomer is a modified nucleobase selected from 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine.

LNA

The term "LNA" refers to a class of bicyclic nucleoside analogs known as "locked nucleic acids." It can refer to an LNA monomer or, when used in the context of an "LNA oligonucleotide," an oligonucleotide containing one or more of these bicyclic nucleotide analogs. LNA nucleotides are characterized by the presence of a linker (e.g., a bridge) between the C2' and C4' ends of the ribose sugar ring, such as the diradical R4 ^* -R2 ^* described below.

The LNA used in the oligonucleotide compound of the present invention preferably has the structure of formula I

where for all chiral centers, the asymmetric groups can be found in either R or S orientation;

wherein X is selected from -O-, -S-, -N(RN ^* )-, -C( ^{R6R6 *} )-, such as in certain embodiments -O-;

B is selected from hydrogen, optionally substituted C _1-4 -alkoxy, optionally substituted C _1-4 -alkyl, optionally substituted C _1-4 -acyloxy, nucleobases, including naturally occurring and nucleobase analogs, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups and ligands; preferably, B is a nucleobase or a nucleobase analog;

P represents an internucleotide bond to an adjacent monomer or a 5'-terminal group, such internucleotide bond or 5'-terminal group optionally including a substituent ^R5 or a similarly applicable substituent R5 ^* ;

P* represents an internucleotide bond to an adjacent monomer or 3′-terminal group;

R ^4* and R ^2* together represent ^a divalent linker group consisting of 1 to 4 groups/atoms selected from -C( ^RaRb )-, -C( ^Ra )=C( ^Rb )-, -C( ^Ra )=N-, -O-, -Si(Ra ⁾ _2- , _-S- , -SO2-, -N( ^Ra )-, and >C=Z, wherein Z is selected from -O-, -S-, and -N( ^Ra )-, ^Ra and ^Rb are each independently selected from hydrogen, optionally substituted _C1-12 -alkyl, optionally substituted _C2-12 -alkenyl, optionally substituted _C2-12 -alkynyl, hydroxy, optionally substituted _C1-12 -alkoxy, _C2-12 -alkoxyalkyl, _C2-12 -alkenyloxy, carboxyl, _C1-12 -alkoxycarbonyl, _C1-12 -alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(Ci _-6 -alkyl)amino, carbamoyl, mono- and di(Ci-6-alkyl)-amino-carbonyl, amino-Ci _-6 -alkyl-aminocarbonyl, mono- and di(Ci- ₆ -alkyl)amino-Ci- ₆ -alkyl-aminocarbonyl, Ci- ₆ -alkyl-carbonylamino, urea, Ci- ₆ -alkanoyloxy, sulfonyl, Ci _{-6 -} alkylsulfonyloxy, nitro, azido, sulfanyl, Ci _-6 -alkylthio, halogen, DNA intercalator, photochemically active group, thermochemically active group, chelating group, reporter group and ligand, wherein aryl and heteroaryl can be optionally substituted, and wherein the two geminal substituents ^R and R ^b together may represent an optionally substituted methylene (=CH ₂ ), wherein for all chiral centers, the asymmetric groups may be found in R or S orientation;

The substituents R ^1* , R ² , R ³ , R ⁵ , R ^5* , R ⁶ and R ^6* present are each independently selected from hydrogen, optionally substituted C _1-12 -alkyl, optionally substituted C _2-12 -alkenyl, optionally substituted C _2-12 -alkynyl, hydroxy, C 1-12 -alkoxy, C _2-12 -alkoxyalkyl, C _2-12 -alkenyloxy, carboxyl, _{C 1-12} -alkoxycarbonyl, C _1-12 -alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di-(C _1-6 -alkyl)amino, carbamoyl, mono- and di-(C _1-6 -alkyl)-amino-carbonyl, amino-C _1-6 -alkyl-aminocarbonyl, mono- and di-(C [0014] The present invention also includes alkyl groups selected from the group consisting of alkyl ( _Ci-6 -alkyl)amino-Ci _{- 6} -alkyl-aminocarbonyl, Ci _-6 -alkyl-carbonylamino, urea, Ci _-6 -alkanoyloxy, sulfonyl, Ci _-6 -alkylsulfonyloxy, nitro, azido, sulfanyl, Ci-6-alkylthio, halogen, DNA intercalator, photochemically active group, thermochemically active group, chelating group, reporter group and ligand, wherein aryl and heteroaryl groups may be optionally substituted, and wherein two geminal substituents together may represent oxo, thioxo, imino or optionally substituted methylene; wherein ^RN is selected from hydrogen and Ci _-4 -alkyl, and wherein two adjacent (non-geminal) substituents may represent an additional bond, resulting in a double bond; and when present and not participating in a diradical, RN ^* is selected from hydrogen and Ci _-4 -alkyl; and basic salts and acid addition salts thereof. For all chiral centers, asymmetric groups may be found in either R or S orientation.

In some embodiments, R ^4* and R ^2* together represent ^a diradical consisting of a group selected from C( ^RaRb )-C( ^RaRb )-, C( ^RaRb )-O- ^, C( ^{RaRb )} -NRa-, C( ^{RaRb )} -S-, and C( ^{RaRb ) -C(RaRb )} -O-, ^wherein each ^Ra and ^Rb can be optionally independently selected. In some embodiments, ^Ra and ^Rb can be optionally independently selected from hydrogen and _C1-6 alkyl, ^such as methyl, such as hydrogen.

In some embodiments, R ^4* and R ^2* together represent the diradical O—CH(CH ₂ OCH ₃ )-(2O-methoxyethyl bicyclic nucleic acid—Seth at al., 2010, J. Org. Chem) in the R- or S-configuration.

In some embodiments, R ^4* and R ^2* together represent a diradical -O-CH(CH ₂ CH ₃ )-(2'O-ethylbicyclic nucleic acid-Seth at al., 2010, J. Org. Chem) in the R- or S-configuration.

In some embodiments, R ^4* and R ^2* together represent a diradical —O—CH(CH ₃ )— in the R- or S-configuration. In some embodiments, R ^4* and R ^2* together represent a diradical O—NR—CH ₃ — (Seth at al., 2010, J. Org. Chem).

In some embodiments, R ^4* and R ^2* together represent a diradical —O—NR—CH ₃ — (Seth at al., 2010, J. Org. Chem).

In some embodiments, the LNA unit has a structure selected from the group consisting of:

In some embodiments, R ^1* , R ² , R ³ , R ⁵ , R ^5* are independently selected from hydrogen, halogen, C _1-6 alkyl, substituted C _1-6 alkyl, C _2-6 alkenyl, substituted C _2-6 alkenyl, C _2-6 -alkynyl or substituted C _2-6 alkynyl, C _1-6 alkoxy, substituted C _1-6 alkoxy, acyl, substituted acyl, C _1-6 aminoalkyl or substituted C _1-6 aminoalkyl. For all chiral centers, asymmetric groups can be found in either R or S orientation.

In some embodiments, R ^1* , R ² , R ³ , R ⁵ , R ^5* are hydrogen.

In some embodiments, R ^1* , R ² , and R ³ are independently selected from hydrogen, halogen, C _1-6 alkyl, substituted C _1-6 alkyl, C _2-6 alkenyl, substituted C _2-6 alkenyl, C _2-6 alkynyl or substituted C _2-6 alkynyl, C _1-6 alkoxy, substituted C _1-6 alkoxy, acyl, substituted acyl, C _1-6 aminoalkyl or substituted C _1-6 aminoalkyl. For all chiral centers, asymmetric groups can be found in either R or S orientation.

In some embodiments, R ^1* , R ² , and R ³ are hydrogen.

In some embodiments, R ⁵ and R ^5* are each independently selected from H, —CH ₃ , —CH ₂ —CH ₃ , —CH ₂ —O—CH ₃ , and —CH═CH ₂ . Suitably, in some embodiments, R ⁵ or R ^5* is hydrogen, wherein the other group (R ⁵ or R ^5* , respectively) is selected from C _1-5 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, substituted C _1-6 alkyl, substituted C _2-6 alkenyl, substituted C _2-6 alkynyl or substituted acyl (-C(=O)-); wherein each substituent is mono- or poly-substituted with a substituent independently selected from halogen, C _1-6 alkyl, substituted C _1-6 alkyl, C _2-6 alkenyl, substituted C 2-6 alkenyl, C _2-6 alkynyl, substituted C _2-6 alkynyl, OJ ₁ , SJ ₁ , NJ ₁ J ₂ , N ₃ , COOJ ₁ , CN, OC(=O ₎ NJ ₁ J ₂ , N(H)C(=NH)NJ, J ₂ or N(H)C(=X)N(H)J ₂ wherein X is O or S; and each J ₁ and J ₂ are independently H, C _1-6 alkyl, substituted C _1-6 alkyl, C _2-6 alkenyl, substituted C _2-6 alkenyl, C _2-6 alkynyl, substituted C _2-6 alkynyl, C _1-6 aminoalkyl, substituted C _1-6 aminoalkyl or a protecting group. In some embodiments, R ⁵ or R ^5* is substituted C _1-6 alkyl. In some embodiments, R ⁵ or R ^5* is substituted methylene, wherein preferred substituents include one or more groups independently selected from F, NJ ₁ J ₂ , N ₃ , CN, OJ ₁ , SJ ₁ , OC(=O)NJ ₁ J ₂ , N(H)C(=NH)NJ, J ₂ or N(H)C(O)N(H)J ₂ . In some embodiments, each J ₁ and J ₂ is independently H or C _1-6 alkyl. In some embodiments, R ⁵ or R ^5* is methyl, ethyl, or methoxymethyl. In some embodiments, R ⁵ or R ^5* is methyl. In another embodiment, R ⁵ or R ^5* is ethylene. In some embodiments, R ⁵ or R ^5* is substituted acyl. In some embodiments, R ⁵ or R ^5* is C(═O)NJ ₁ J ₂ . For all chiral centers, asymmetric groups can be found in either R or S orientation. Such 5'-modified bicyclic nucleotides are disclosed in WO 2007/134181, the entire contents of which are incorporated herein by reference.

In some embodiments, B is a nucleobase, including nucleobase analogs and naturally occurring nucleobases, such as a purine or pyrimidine or a substituted purine or substituted pyrimidine, such as a nucleobase described herein, such as a nucleobase selected from the group consisting of adenine, cytosine, thymine, adenine, uracil and/or a modified or substituted nucleobase such as 5-thiazolo-uracil, 2-thio-uracil, 5-propynyl-uracil, 2'-thiothymine, 5-methylcytosine, 5-thiazolo-cytosine, 5-propynyl-cytosine and 2,6-diaminopurine.

In some embodiments, R ^4* and R ^2* together represent a group selected from -C(R ^a R ^b )-O-, -C(R ^a R ^b )-C(R ^c R ^d )-O-, -C(R ^a R ^b )-C(R ^c R ^d ) -C(R ^e R ^f ) -O -, -C(R ^a R ^b )-OC(R ^c R ^d )-, -C(R ^a R ^b )-OC(R ^c R ^d )-O-,-C(R ^a R ^b )-C(R ^c R ^d )-,-C(R ^a R ^b )-C(R ^c R ^d )-C(R ^e R ^f )-, -C(R ^a )＝C(R ^b )-C(R ^c R ^d )-, -C(R ^a R ^b )-N(R ^c )-, -C(R ^a R ^b )-C(R ^c R ^d )-N(R ^e )-, -C( ^{RaRb )} -N( ^Rc )-O-, and -C( ^{RaRb )} -S-, -C( ^RaRb )-C( ^{RcRd )} -S-, wherein ^Ra , ^Rb , ^Rc , ^Rd , ^{Re ,} and ^Rf are each independently selected from hydrogen, optionally substituted _C1-12 -alkyl, optionally substituted _C2-12 -alkenyl, optionally substituted _C2-12 -alkynyl, hydroxy, C1-12-alkoxy, _C2-12 -alkoxyalkyl, _C2-12 -alkenyloxy, carboxyl, _C1-12 -alkoxycarbonyl, _C1-12 -alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- _and di-(C [ _{0014 ] The present} invention also includes _, but is _not limited _{to ,} ^alkylamino _, ^thioalkyl _...

In another embodiment, R ^4* and R ^2* together represent a group selected from -CH ₂ -O-, -CH ₂ -S-, -CH ₂ -NH-, -CH ₂ -N(CH ₃ )-, -CH ₂ -CH ₂ -O-, -CH ₂ -CH(CH ₃ )-, -CH ₂ -CH ₂ -S-, -CH ₂ -CH ₂ -NH-, -CH ₂ -CH ₂ -CH ₂ -,-CH ₂ -CH ₂ -CH ₂ -O-,-CH ₂ -CH ₂ -CH(CH ₃ )-, -CH＝CH-CH ₂ -,-CH ₂ -O-CH ₂ -O-,-CH ₂ -NH-O-,-CH ₂ -N(CH ₃ )-O-,-CH ₂ -O-CH ₂ -,-CH(CH ₃ )-O-, and –CH(CH ₂ -O-CH ₃ )-O-, and/or, -CH ₂ -CH ₂ -, and -CH=CH- diradicals (divalent radicals). For all chiral centers, asymmetric groups are found in either R or S orientation.

In some embodiments, R ^4* and R ^2* together represent a diradical C( ^RaRb )-N( ^Rc )-O-, wherein ^Ra and ^Rb are independently selected from hydrogen, halogen, ^Ci_6 alkyl, substituted _{Ci_6} alkyl, _{C2_6} alkenyl, substituted _{C2_6} alkenyl, _{C2_6} alkynyl or substituted _{C2_6} alkynyl, _{Ci_6} alkoxy, substituted _{Ci_6} alkoxy, acyl, substituted _acyl , _{Ci_6} aminoalkyl or substituted _{Ci_6} aminoalkyl, such as hydrogen; wherein R ^c is selected from hydrogen, halogen, Ci_6 alkyl, substituted Ci_6 alkyl, _{C2_6} alkenyl, substituted _{C2_6} alkenyl, _{C2_6} alkynyl or substituted _{C2_6} alkynyl, _{Ci_6} alkoxy, substituted _{Ci_6} alkoxy, acyl, substituted acyl, _{Ci_6 aminoalkyl} or substituted Ci_6 aminoalkyl, such as hydrogen; _{C 1-6} aminoalkyl or substituted C _1-6 aminoalkyl such as hydrogen.

In some embodiments, R ^4* and ^{R 2*} together represent a diradical C( ^RaRb )-OC( ^RcRd )-O-, wherein ^Ra , ^Rb , ^Rc , and ^Rd are independently selected from hydrogen, halogen, _C1-6 alkyl, substituted _C1-6 alkyl, _C2-6 alkenyl, substituted _C2-6 alkenyl, _C2-6 alkynyl or substituted _C2-6 alkynyl, ^C1-6 alkoxy, substituted _C1-6 alkoxy, acyl, substituted acyl, _C1-6 aminoalkyl or substituted _C1-6 aminoalkyl _, for example hydrogen.

In some embodiments, R ^4* and R ^2* form a diradical -CH(Z)-O-, wherein Z is selected from C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, substituted C _1-6 alkyl, substituted C _{2-6 alkenyl, substituted C 2-6 alkynyl} , acyl, substituted acyl, substituted amide, thiol or substituted thio; and wherein each substituent is independently mono- or polysubstituted with optionally protected substituents independently selected from halogen, oxo, hydroxy, OJ ₁ , NJ ₁ J ₂ , SJ 1 , N ₃ , OC(＝X)J ₁ , OC(＝X)NJ _{1 J 2} , NJ ³ C(＝X)NJ ₁ J ₂ and CN, wherein each J ₁ , J ₂ and J ₃ is independently H or C _1-6 alkyl, and X is O, S or NJ ₁ . In some embodiments, Z is C _1-6 alkyl or substituted C _1-6 alkyl. In some embodiments, Z is methyl. In some embodiments, Z is substituted C _1-6 alkyl. In some embodiments, the substituent is C _1-6 alkoxy. In some embodiments, Z is CH ₃ OCH ₂ -. For all chiral centers, asymmetric groups can be found in either R or S orientation. This bicyclic nucleotide is disclosed in US 7,399,845, the entire contents of which are incorporated herein by reference. In some embodiments, R ^1* , R ² , R ³ , R ⁵ , and R ^5* are hydrogen. In certain embodiments, R ^1* , R ² , and R ^3* are hydrogen, and one or both of R ⁵ and R ^5* may be different from the hydrogen described above and in WO 2007/134181.

In some embodiments, R ^4* and R ^2* together represent a diradical comprising a substituted amino group in the bridge, for example consisting of or comprising the diradical —CH ₂ —N(R ^c )—, wherein R ^c is C _1-12 alkoxy. In some embodiments, R ^4* and R ^2* together represent a diradical -C _{q 3} q ₄ -NOR-, wherein q ₃ and q ₄ are independently selected from hydrogen, halogen, C _1-6 alkyl, substituted C _1-6 alkyl, C _2-6 alkenyl, substituted C _2-6 alkenyl, C _2-6 alkynyl or substituted C _2-6 alkynyl, C _{1-6 alkoxy, substituted C 1-6 alkoxy} , acyl, substituted acyl, C _1-6 aminoalkyl or substituted C _1-6 aminoalkyl; wherein each substituent is independently mono- or polysubstituted with substituents independently selected from halogen, OJ ₁ , SJ ₁ , NJ ₁ J ₂ , COOJ ₁ , CN, OC(═O)NJ ₁ J ₂ , N(H)C(═NH)NJ ₁ J ₂ or N(H)C(═X═N(H)J ₂ , wherein X is O or S; J ₁ and J ₂ are each independently H, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 aminoalkyl or a protecting group. For all chiral centers, asymmetric groups can be found in the R or S orientation. Such bicyclic nucleotides are disclosed in WO2008/150729, which is incorporated herein by reference. In some embodiments, R ^1* , R ² , R ³ , R ⁵ , R ^5* are independently selected from hydrogen, halogen, C _1-6 alkyl, substituted C _1-6 alkyl, C _2-6 alkenyl, substituted C _2-6 alkenyl, C _2-6 alkynyl or substituted C _2-6 alkynyl, C _1-6 alkoxy, substituted C _1-6 alkoxy, acyl, substituted acyl, C _1-6 aminoalkyl or substituted C _1-6 aminoalkyl. In some embodiments, R ^1* , R ² , R ³ , R ⁵ , R ^5* are hydrogen. In some embodiments, R ^1* , R ² , R ³ are hydrogen and one or both of R ⁵ , R ^5* may be different from the hydrogen described above and in WO 2007/134181. In some embodiments, R ^4* and R ^2* together represent a diradical ( ^divalent group) C( ^RaRb )-O-, wherein ^Ra and ^Rb are each independently halogen, _C1 - _C12 alkyl, substituted _C1 - _C12 alkyl, _C2 - _C12 alkenyl, substituted _C2 - _C12 alkenyl, _C2 - _C12 alkynyl, substituted _C2 - _C12 alkynyl, _C1 - _C12 alkoxy, substituted _C1 - _C12 alkoxy, OJ ₁ SJ ₁ , SOJ ₁ , SO ₂ J ₁ , NJ ₁ J ₂ , N ₃ , CN, C(=O) _OJ1 , C(=O) _{NJ1 J2} , C(=O) _J1 , OC(=O) _{NJ1 J2} , N(H)C(=NH) _{NJ1 J2} , N(H)C(=O) _{NJ1 J2} or N(H)C(=S) _{NJ1 J2} ; or ^Ra and ^Rb together are =C(q3)(q4); _q3 and _q4 are each independently H, halogen, _C1 - _C12 alkyl or substituted _C1 - _C12 alkyl; each substituent is independently mono- or poly-substituted with a substituent independently selected from halogen, _C1 - _C6 alkyl, substituted _C1 - _C6 alkyl, _C2 - _C6 alkenyl, substituted _C2 - _C6 alkenyl, _C2 - _C6 alkynyl, substituted _C2 -C ₆ alkynyl, OJ ₁ , SJ ₁ , NJ ₁ J ₂ , N ₃ , CN, C(═O)OJ ₁ , C(═O)NJ ₁ J ₂ , C(═O)J ₁ , OC(═O)NJ ₁ J ₂ , N(H)C(═O)NJ ₁ J ₂ or N(H)C(═S)NJ ₁ J ₂ ; each J ₁ and J ₂ is independently H, C ₁ -C ₆ alkyl, substituted C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, substituted C ₂ -C ₆ alkynyl, C ₁ -C ₆ aminoalkyl, substituted C 1 -C 6 ₆ -aminoalkyl or a protecting group. Such compounds are disclosed in WO2009006478A, the entire contents of which are incorporated herein by reference.

In some embodiments, R ^4* and R ^2* form a diradical -Q-, wherein Q is C(q ₁ )(q ₂ )C(q ₃ )(q ₄ ), C(q ₁ )＝C(q ₃ ), C[＝C(q ₁ )(q ₂ )]-C(q ₃ )(q ₄ ) or C(q ₁ )(q ₂ )-C[＝C(q ₃ )(q ₄ )]; q ₁ , q ₂ , q ₃ , q ₄ are each independently H, halogen, C _1-12 alkyl, substituted C _1-12 alkyl, C _2-12 alkenyl, substituted C _1-12 alkoxy, OJ ₁ , SJ ₁ , SOJ ₁ , SO ₂ J ₁ , NJ ₁ J ₂ , N ₃ , CN, C(＝O)OJ ₁ , C(＝O)-NJ ₁ J ₂ , C(＝O)J ₁ , -C(＝O)NJ ₁ J ₂ , N(H)C(＝NH)NJ ₁ J ₂ , N(H)C(＝O)NJ ₁ J ₂ or N(H)C(＝S)NJ ₁ J ₂ ; each J ₁ and J ₂ are independently H, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 aminoalkyl or a protecting group; and optionally, wherein when Q is C(q ₁ )(q ₂ )(q ₃ )(q ₄ ) and one of q ₃ or q ₄ is CH ₃ , at least one of q ₃ or q ₄ or one of q ₁ and q ₂ is not H. In some embodiments, R ^1* , R ² , R ³ , R ⁵ , R ^5* are hydrogen. For all chiral centers, asymmetric groups can be found in either R or S orientation. Such bicyclic nucleotides are disclosed in WO2008/154401, which is incorporated herein by reference in its entirety. In some embodiments, R ^1* , R ² , R ³ , R ⁵ , R ^5* are independently selected from hydrogen, halogen, C _1-6 alkyl, substituted C _1-6 alkyl, C _2-6 alkenyl, substituted C _2-6 alkenyl, C _2-6 alkynyl or substituted C _2-6 alkynyl, C _1-6 alkoxy, substituted C _1-6 alkoxy, acyl, substituted acyl, C _1-6 aminoalkyl or substituted C _1-6 aminoalkyl. In some embodiments, R ^1* , R ² , R ³ , R ⁵ , R ^5* are hydrogen. In some embodiments, R ^1* , R ² , and R ³ are hydrogen, and one or both of R ⁵ and R ^5* may be different from hydrogen as described above and in WO 2007/134181 or WO 2009/067647 (α-L-bicyclic nucleic acid analogs).

Other bicyclic nucleoside analogs and their use in antisense oligonucleotides are disclosed in WO2011 115818, WO2011/085102, WO2011/017521, WO09100320, WO10036698, WO09124295 & WO09006478. In certain aspects, these nucleoside analogs can be used in the compounds of the present invention.

In some embodiments, the LNA used in the oligonucleotide compounds of the present invention preferably has the structure of formula II:

wherein Y is selected from -O-, -CH ₂ O-, -S-, -NH-, N( ^Re ) and/or -CH ₂ -; Z and Z* are independently selected from an internucleotide bond, ^RH , a terminal group or a protecting group; B constitutes a natural or non-natural nucleotide base moiety (nucleobase), and ^RH is selected from hydrogen and C _{1-4 -alkyl} ; ^Ra , ^Rb , ^Rc , ^Rd and ^Re are optionally independently selected from hydrogen, optionally substituted C _1-12 -alkyl, optionally substituted C _2-12 -alkenyl, optionally substituted C _2-12 -alkynyl, hydroxy, C _1-12 -alkoxy, C 2-12 -alkoxyalkyl, C _2-12 -alkenyloxy, carboxyl, C _1-12 -alkoxycarbonyl, C _1-12 -alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy, 6 -alkyl)amino, carbamoyl, mono- and di-(C _1-6 -alkyl)-amino-carbonyl, amino-C 1-6 -alkyl-aminocarbonyl, mono- and di-(C 1-6 -alkyl)amino-C _1-6 -alkyl-aminocarbonyl, C _1-6 -alkyl-carbonylamino, urea, C _1-6 -alkanoyloxy, sulfonyl, C _1-6 -alkylsulfonyloxy, nitro, azido, sulfanyl, _{C 1-6 -alkylthio} , halogen, DNA intercalator, photochemically active group, thermochemically active group, chelating group, reporter group and ligand, wherein aryl and heteroaryl may be optionally substituted and wherein _the two geminal substituents ^Ra and ^Rb together may represent an optionally substituted methylene (=CH ₂ ) _; and R ^H is selected from hydrogen and C _1-4 -alkyl. In some embodiments, ^Ra , ^{Rb, Rc} , ^Rd and ^Re are optionally independently selected from hydrogen and _C1-6 alkyl, such as methyl. For all chiral centers, asymmetric groups can be found in either R or S orientation, for example, two exemplary stereochemical isomers include β-D and α-L isoforms, which can be shown as follows:

A specific exemplary LNA unit is shown below:

The term "thio-LNA" encompasses locked nucleotides wherein Y in the above formula is selected from S or _-CH2 -S-. Thio-LNA can be in both the β-D and α-L configurations.

The term "amino-LNA" encompasses locked nucleotides wherein Y in the above formula is selected from -N(H)-, N(R)-, _CH2 -N(H)-, and -CH2- _N (R)-, wherein R is selected from hydrogen and _C1-4 -alkyl. Amino-LNA can be in the β-D and α-L configurations.

The term "oxy-LNA" includes locked nucleotides wherein Y in the general formula represents -O-. Oxy-LNA can be in both the β-D and α-L configurations.

The term "ENA" encompasses locked nucleotides wherein Y in the above formula is -CH2 _- O- (wherein the oxygen atom of -CH2 _- O- is attached at the 2'-position relative to base B). ^Re is hydrogen or methyl.

In some exemplary embodiments, the LNA is selected from β-D-oxy-LNA, α-L-oxy-LNA, β-D-amino-LNA and β-D-thio-LNA, in particular β-D-oxy-LNA.

Conjugate

In this context, the term "conjugate" is intended to mean a heterologous molecule formed by covalently linking ("conjugating") an oligomer as described herein to one or more non-nucleotide or non-polynucleotide moieties. Examples of non-nucleotide or non-polynucleotide moieties include macromolecular agents such as proteins, fatty acid chains, sugar residues, glycoproteins, polymers, or combinations thereof. Typically, the protein may be an antibody to the target protein. A typical polymer may be polyethylene glycol.

Thus, in various embodiments, the oligomers of the invention may comprise a polynucleotide region generally consisting of a continuous nucleotide sequence and an additional non-nucleotide region. When referring to an oligomer of the invention consisting of a continuous nucleotide sequence, the compound may comprise a non-nucleotide component, such as a conjugate component.

In various embodiments of the present invention, the oligomeric compound is linked to a ligand/conjugate that can be used to increase the cellular uptake of the oligomeric compound. Suitable ligands and conjugates are provided in WO2007/031091, which is incorporated herein by reference.

The present invention also provides conjugates comprising a compound of the invention as described herein and at least one non-nucleotide or non-polynucleotide moiety covalently attached to the compound. Thus, in various embodiments in which a compound of the invention consists of a specific nucleic acid or nucleotide sequence disclosed herein, the compound may further comprise at least one non-nucleotide or non-polynucleotide moiety covalently attached to the compound (e.g., not comprising one or more nucleotides or nucleotide analogs).

Conjugation (to a conjugate moiety) can enhance the activity, cellular distribution, or cellular uptake of the oligomers of the invention. These moieties include, but are not limited to, antibodies, polypeptides, lipid moieties, such as cholesterol moieties, bile acid, thioethers, such as hexyl-s-tritylthiol, thiocholesterol, aliphatic chains, such as dodecandiol or undecyl residues, phospholipids, such as di-hexadecyl-rac-glycerol or 1,2-di-O-hexadecyl-rac-glycero-3-h-phosphonic acid triethylammonium, polyamines or polyethylene glycol chains, adamantaneacetic acid, palmityl moieties, octadecylamine, or hexylamino-carbonyl-hydroxycholesterol moieties.

The oligomers of the present invention may also be conjugated to active drug substances such as aspirin, ibuprofen, sulfonamides, antidiabetics, antibacterials or antibiotics.

In certain embodiments, the conjugated moiety is a sterol, such as cholesterol.

In various embodiments, the conjugated moiety comprises or consists of a positively charged polymer, such as a positively charged peptide, e.g., 1-50, such as 2-20, such as 3-10 amino acid residues long, and/or a polyalkylene oxide such as polyethylene glycol (PEG) or polypropylene glycol - see WO 2008/034123, which is incorporated herein by reference. Suitably, the positively charged polymer, e.g., polyalkylene oxide, may be attached to the oligomer of the invention via a linker, such as a releasable linker as described in WO 2008/034123.

For example, the following GalNAc conjugate moieties can be used in the conjugates of the present invention:

The present invention also provides conjugates comprising an oligomer of the present invention, comprising at least one non-nucleotide or non-polynucleotide moiety covalently linked to an oligomer of the present invention ("conjugated moiety"). In some embodiments, the conjugate of the present invention is covalently linked to the oligomer via a biocleavable linker, which can be, for example, a region of phosphodiester-linked nucleotides, such as 1-5 phosphodiester-linked DNA nucleosides ( WO 2014/076195 , incorporated herein by reference). Preferred conjugated groups include carbohydrate conjugates, such as GalNAc conjugates, such as trivalent GalNAc conjugates (see, for example, WO 2014/118267 , incorporated herein by reference), or lipophilic conjugates, such as sterols, e.g., cholesterol ( WO 2014/076195 , incorporated herein by reference).

Activated oligomers

As used herein, the term " activated oligomer " refers to an oligomer of the present invention, which is covalently linked (i.e., functionalized) to at least one functional moiety that allows the oligomer to be covalently linked to one or more conjugated moieties (i.e., itself not a part of a nucleic acid or monomer) to form a conjugate as described herein. Typically, the functional moiety will comprise a chemical group that can be covalently bonded to the oligomer by, for example, the 3'-hydroxyl group of an adenine base or an outer ring NH ₂ group, preferably a hydrophilic spacer and an end group (e.g., amino, sulfhydryl or hydroxyl) that can be bound to the conjugated moiety. In some embodiments, the end group is not protected, for example, is an NH ₂ group. In other embodiments, the end group is, for example, protected by any suitable protecting group, for example, by those protecting groups described in " Protective Groups in Organic Synthesis " in Theodora W Greene and Peter GM Wuts, 3rd edition (John Wiley & Sons, 1999). Examples of suitable hydroxy protecting groups include esters such as acetate, aralkyl groups such as benzyl, diphenylmethyl or trityl, and tetrahydropyranyl. Examples of suitable amino protecting groups include benzyl, α-methylbenzyl, diphenylmethyl, triphenylmethyl, benzyloxycarbonyl, tert-butoxycarbonyl, and acyl groups such as trichloroacetyl or trifluoroacetyl. In some embodiments, the functional moiety is self-cleavable. In other embodiments, the functional moiety is biodegradable. See, for example, U.S. Patent No. 7,087,229, the entire contents of which are incorporated herein by reference.

In some embodiments, the oligomers of the present invention are functionalized at the 5' terminus to covalently attach the conjugate moiety to the 5' terminus of the oligomer. In other embodiments, the oligomers of the present invention may be functionalized at the 3' terminus. In other embodiments, the oligomers of the present invention may be functionalized along the backbone or heterocyclic base moiety. In other embodiments, the oligomers of the present invention may be functionalized at more than one position independently selected from the group consisting of the 5' terminus, the 3' terminus, the backbone, and the base.

In some embodiments, the activated oligomers of the present invention are synthesized by incorporating one or more monomers covalently linked to a functional moiety during the synthesis process. In other embodiments, the activated oligomers of the present invention are synthesized using monomers that are not functionalized, and the oligomers are functionalized upon completion of the synthesis. In some embodiments, the oligomers are functionalized with hindered esters containing aminoalkyl linkers, wherein the alkyl moiety has the formula (CH ₂ ) _w , wherein w is an integer from 1 to 10, preferably about 6, wherein the alkyl portion of the alkylamino group can be linear or branched, and wherein the functional group is linked to the oligomer via an ester group (-OC(O)-(CH ₂ ) ₂ NH).

In other embodiments, the oligomer is functionalized with a hindered ester containing a (CH ₂ ) ₂ -thiol (SH) linker, wherein w is an integer from 1 to 10, preferably about 6, wherein the alkyl portion of the alkylamino group can be linear or branched, and wherein the functional group is attached to the oligomer through an ester group (-OC(O)-(CH ₂ ) ₂ SH).

In some embodiments, the thiol-activated oligonucleotide is conjugated to a polymeric moiety, such as polyethylene glycol or a peptide (via disulfide bond formation).

Activated oligomers containing the aforementioned hindered esters can be synthesized by any method known in the art, in particular by the methods disclosed in PCT Publication No. WO 2008/034122 and the Examples therein, the entire contents of which are incorporated herein by reference.

In other embodiments, the oligomers of the present invention are functionalized by introducing a sulfhydryl, amino, or hydroxyl group into the oligomer using a functionalizing agent substantially as described in U.S. Patent Nos. 4,962,029 and 4,914,210, i.e., a substantially linear reagent having a phosphoramidite at one end connected to an opposite end via a hydrophilic spacer chain, the opposite end containing a protected or unprotected sulfhydryl, amino, or hydroxyl group. These reagents react primarily with the hydroxyl group of the oligomer. In some embodiments, the activated oligomer has a functionalizing agent coupled to the 5'-hydroxyl group of the oligomer. In other embodiments, the activated oligomer has a functionalizing agent coupled to the 3'-hydroxyl group. In other embodiments, the activated oligomer of the present invention has a functionalizing agent coupled to the hydroxyl group on the oligomer backbone. In other embodiments, the oligomers of the present invention are functionalized with more than one functionalizing agent, as described in U.S. Patent Nos. 4,962,029 and 4,914,210, the entire contents of which are incorporated herein by reference. Methods for synthesizing these functionalizing agents and incorporating them into monomers or oligomers are disclosed in U.S. Patent Nos. 4,962,029 and 4,914,210.

In some embodiments, the 5'-terminus of a solid-phase-bound oligomer is functionalized with a dienyl phosphoramidite derivative and the deprotected oligomer is then conjugated to, for example, an amino acid or peptide via a Diels-Alder cycloaddition reaction.

In various embodiments, incorporation of monomers containing 2'-sugar modifications (e.g., 2'-carbamate-substituted sugars or 2'-(O-pentyl-N-phthalimido)-deoxyribose) into oligomers facilitates covalent attachment of the conjugated moiety to the oligomer's sugar. In other embodiments, oligomers having an amino-containing linker at the 2'-position of one or more monomers are prepared using reagents such as 5'-dimethoxytrityl-2'-O-(ε-phthalimidoaminopentyl)-2'-deoxyadenosine-3'-N,N-diisopropyl-cyanoethoxyphosphoramidite. See, for example, Manoharan, et al., Tetrahedron Letters, 1991, 34, 7171.

In additional embodiments, the oligomers of the invention may have amine-containing functional moieties at the nucleobases, including at the N6 purine amino group, at the exocyclic N2 of guanine, or at the N4 or 5 position of cytosine. In various embodiments, such functionalization can be achieved by using commercial reagents that have already been functionalized during oligomer synthesis.

Some functional moieties are commercially available, for example, heterobifunctional and homobifunctional linking moieties are available from Pierce Co. (Rockford, 111.). Other commercially available linking groups are 5'-amino-modifier C6 and 3'-amino-modifier reagents, both available from Glen Research Corporation (Sterling, Va.). 5'-amino-modifier C6 is also available as Aminolink-2 from ABI (Applied Biosystems Inc., Foster City, Calif.), and 3'-amino-modifiers are also available from Clontech Laboratories Inc. (Palo Alto, Calif.).

Composition

The oligomers of the present invention can be used in pharmaceutical formulations and compositions. Suitably, such compositions comprise a pharmaceutically acceptable diluent, carrier, salt, or adjuvant. Suitable and preferred pharmaceutically acceptable diluents, carriers, and adjuvants are provided in PCT/DK2006/000512, which is incorporated herein by reference. PCT/DK2006/000512 (which is also incorporated herein by reference) also provides suitable dosages, formulations, routes of administration, compositions, dosage forms, combinations with other therapeutic agents, and prodrug formulations.

application

The oligomers of the present invention are useful as research reagents, for example, for diagnosis, treatment and prevention.

In research, such oligomers can be used to specifically inhibit the synthesis of a target protein in cells and experimental animals (usually by degrading or inhibiting the mRNA, thereby preventing protein formation), thereby facilitating functional analysis of the target or evaluation of its usefulness as a target for therapeutic intervention.

In diagnostics, oligomers can be used to detect and quantify target expression in cells and tissues by Northern blotting, in situ hybridization, or similar techniques.

For therapeutic purposes, an animal or human suspected of having a disease or condition can be treated by administering an oligomeric compound of the invention to treat the animal or human by modulating target expression. Also provided are methods for treating a mammal, such as a human, suspected of having or susceptible to a disease or condition associated with target expression by administering a therapeutically or prophylactically effective amount of one or more oligomers or compositions of the invention. The oligomers, conjugates, or pharmaceutical compositions of the invention are typically administered in an effective amount.

The present invention also provides use of a compound of the invention or a conjugate as described herein in the preparation of a medicament for treating a condition as described herein or in a method of treating a condition as described herein.

The present invention also provides a method of treating the conditions described herein, which method comprises administering a compound of the invention and/or a conjugate according to the invention and/or a pharmaceutical composition according to the invention as described herein to a patient in need thereof.

Medical indications

Oligomers and other compositions according to the invention can be used to treat disorders associated with overexpression or expression of mutant forms of a target.

The present invention also provides the use of a compound of the present invention in the preparation of a medicament for treating a disease, disorder or condition described herein.

Generally speaking, one aspect of the present invention relates to a method for treating a mammal suffering from or susceptible to a condition associated with abnormal levels of a target, comprising administering to the mammal a therapeutically effective amount of a target-targeting oligomer comprising one or more LNA units. The oligomer, conjugate, or pharmaceutical composition of the present invention is typically administered in an effective amount.

Example

sequence

The compound used herein has the following nucleobase sequence:

actgctttccactctg SEQ ID NO 1 tcatggctgcagct SEQ ID NO 2 gcattggtattca SEQ ID NO 3 cacattccttgctctg SEQ ID NO 4 gcaagcatcctgt SEQ ID NO 5

Example 1

DNA 3'-O-oxazaphospholidine monomers were synthesized as previously described (Oka et al., J. Am. Chem. Soc. 2008 130:16031–16037, and Wan et al., NAR 2014, November, published online).

Synthesis of LNA 3'-O-oxazaphosphacyclopentane monomer

Synthesis scheme

α-Phenyl-2-pyrrolidinemethanol (P5-L and P5-D) were synthesized as described in the literature (Oka et al., JACS, 2008, 16031-16037.).

3-OAP-LNA T

Synthesis of L-3-OAP-LNA T:

_PCl3 (735 μL, 6.30 mmol) was dissolved in toluene (7 mL) and cooled to 0°C (ice bath). A solution of P5-L (1.12 g, 6.30 mmol) and NMM (1.38 mL, 12.6 mmol) in toluene (7 mL) was added dropwise. The reaction mixture was stirred at room temperature for 1 hour and then cooled to -72°C. The precipitate was filtered under argon, washed with toluene (4 mL), and the filtrate was concentrated at 40°C under reduced pressure (Schlenk technique). The residue was dissolved in THF (8 mL) and used in the next step.

To a solution of 5'-ODMT-LNA-T (2.40 g, 4.20 mmol) in THF (16 mL) was added _NEt (4.10 mL, 29.4 mmol). The reaction mixture was cooled to -74°C and a solution of 2-chloro-1,3,2-oxazaphospholidine in THF was added dropwise. The reaction mixture was stirred at room temperature for 4 hours. EtOAc was added, and the reaction mixture was washed _twice with saturated NaHCO, extracted with brine, dried over _NaSO , and evaporated. The residue was purified by column chromatography (eluent: hexane/EtOAc _30/70 + NEt ₆ %). The product was isolated as a white foam (1.00 g, 30% yield). ¹ H-NMR spectrum (400MHz): (DMSO-d ₆ )δ: 11.53(1H,s),7.64(1H,m),7.46-7.41(2H,m), 7.40-7.19(12H,m),6.92-6.83(4H,m),5.51(1H,d,J＝6.3Hz),5.49(1H, s),4.78(1H,d,J=7.4Hz),4.37(1H,s),3.91(1H,m),3.76-3.67(2H,m), 3.72(3H,s),3.71(3H,s),3.50(1H,m),3.41(2H,s),2.90(1H,m),1.60-1.46 (2H,m),1.51(3H,s),1.15(1H,m),0.82(1H,m). ³¹ P-NMR spectrum(160 MHz):(DMSO-d ₆ )δ:151.3.LCMS ESI(m/z):776.2[MH] ^- .

Synthesis of D-3-OAP-LNA T

_PCl3 (1.05 mL, 9.0 mmol) was dissolved in toluene (12 mL) and cooled to 0°C (ice bath). A solution of P5-D (1.13 g, 12 mmol) and NMM (2.06 mL, 24 mmol) in toluene (12 mL) was added dropwise. The reaction mixture was stirred at room temperature for 1 hour and then cooled to -72°C. The precipitate was filtered under argon, washed with toluene, and the filtrate was concentrated at 40°C under reduced pressure (Schlenk technique). The residue was dissolved in THF (18 mL) and used in the next step.

To a solution of 5'-ODMT-LNA-T (3.44 g, 6.0 mmol) in THF (30 mL) was added _NEt₃ (5.82 mL, 42 mmol). The reaction mixture was cooled to -74°C and a solution of 2-chloro-1,3,2-oxazaphosphacyclopentane in THF was added dropwise. The reaction mixture was stirred at room temperature for 4 hours. EtOAc was added, and the reaction mixture was extracted with saturated _NaHCO₃ (twice), brine, dried _{over Na₂SO₄} , and evaporated. The residue was purified by column chromatography (eluent: hexane/EtOAc 30/70 + _NEt₃ 6%). The product was isolated as a white foam (1.86 g, yield 36%). ¹ H-NMR spectrum (400 MHz): (DMSO-d ₆ ) δ: 11.55 (1H, s), 7.60 (1H, m), 7.46-7.41 (2H, m), 7.39-7.22 (12H, m), 6.91-6.84 (4H, m), 5.66 (1H, d, J = 6.3 Hz), 5.51 (1H, s), 4.60 (1H, d, J = 7.4 Hz), 4.41 (1H, s), 3.80-3.70 (3H, m), 3.72 (3H, s), 3.71 (3H, s), 3.48-3.37 (3H, m), 2.96 (1H, m), 1.61-1.43 (2H m),1.51(3H,s)1.10(1H,m),0.80(1H,m). ³¹ P-NMR spectrum (160MHz): (DMSO-d ₆ )δ: 152.5. LCMS ESI (m/z): 776.2 [MH] ^- .

3-OAP-LNA MeC

Synthesis of L-3-OAP-LNA MeC

_PCl3 (110 μL, 1.25 mmol) was dissolved in toluene (3 mL) and cooled to 0°C (ice bath). A solution of P5-L (222 mg, 1.25 mmol) and NMM (275 μL, 2.5 mmol) in toluene (3 mL) was added dropwise. The reaction mixture was stirred at room temperature for 45 minutes and then cooled to -72°C. The precipitate was filtered under argon, washed with toluene, and the filtrate was concentrated under reduced pressure at 40°C (Schlenk technique). The residue was dissolved in THF (5 mL) and used in the next step.

To a solution of 5'-ODMT-LNA-C (338 mg, 0.50 mmol) in THF (2.5 mL) was added _NEt₃ (485 μL, 3.6 mmol). The reaction mixture was cooled to -70°C and a solution of the phosphor _2- chloro-1,3,2-oxazaphosphacyclopentane was added dropwise. The reaction mixture was stirred at room temperature for 1.45 hours. EtOAc (30 mL) was added, and the reaction mixture was extracted with saturated _NaHCO₃ (2 × 20 mL) and brine (20 _mL ), dried over Na₂SO₄, and evaporated. The residue was purified by column chromatography (eluent: EtOAc in hexane from 20% to 30% + toluene 10% + _NEt₃ 7%). The product was isolated as a white foam (228 mg, 47% yield). ¹ H-NMR spectrum (400MHz): (CD ₃ CN) δ: 13.3(1H,br s), 8.41-8.25(2H,m),7.88(1H,m),7.59(1H,m),7.54-7.47(4H,m),7.41-7.19 (12H,m),6.90-6.79(4H,m),5.62(1H,m),5.58(1H,s),4.79(1H,d,J＝7.5 Hz),4.47(1H,s),3.93(1H,m),3.86(1H,m),3.75(1H,m),3.76(3H,s),3.75 (3H,s),3.60-3.47(3H,m),2.99(1H,m),1.83(3H,d,J＝1.2Hz),1.65-1.51 (2H,m),1.17(1H,m),0.89(1H,m). ³¹ P-NMR spectrum (160MHz): (CD ₃ CN)δ: 153.4.LCMS ESI(m/z):881.2[M+H] ⁺ .

Synthesis of D-3-OAP-LNA MeC

_PCl3 (1.10 mL, 12.3 mmol) was dissolved in toluene (10 mL) and cooled to 0°C (ice bath). A solution of P5-D (2.17 g, 12.3 mmol) and NMM (2.70 mL, 2.5 mmol) in toluene (10 mL) was added dropwise. The reaction mixture was stirred at room temperature for 45 minutes and then cooled to -72°C. The precipitate was filtered under argon, washed with toluene, and the filtrate was concentrated under reduced pressure at 40°C (Schlenk technique). The residue was dissolved in THF (10 mL) and used in the next step.

To a solution of 5-ODMT-LNA-C (3.38 g, 5 mmol) in THF (20 mL) was added _NEt₃ (4.85 mL, 35 mmol). The reaction mixture was cooled to -70°C and a solution of the phosphor 2-chloro-1,3,2-oxazaphosphacyclopentane was added dropwise. The reaction mixture was stirred at room temperature for 1.45 hours. EtOAc was added, and the reaction mixture was washed twice with saturated _NaHCO₃ , extracted with brine, dried over _Na₂SO₄ , and evaporated. The residue was purified by column chromatography ₍ eluent: EtOAc in hexane from 20% to 30% + toluene 10% + NEt₃ ₇ %). The product was isolated as a white foam (1.09 g, 23% yield). ¹ H-NMR spectrum (400MHz): (CD ₃ CN) δ: 12.8 (1H, br s), 8.34-8.24 (2H, m), 7.85 (1H, d, J = 1.2 Hz), 7.57 (1H, m), 7.53-7.45 (4H, m), 7.41-7.22 (12H, m), 6.89-6.84(4H,m),5.72(1H,d,J＝6.5Hz),5.59(1H,s),4.62(1H,d,J＝8.0 Hz),4.52(1H,s),3.82(2H,dd,J＝24.4 8.2Hz),3.77(1H,m),3.76(3H,s), 3.75(3H,s),3.51(2H,s),3.46(1H,m),3.05(1H,m),1.81(3H,s),1.65-1.47 (2H,m),1.12(1H,m),0.85(1H,m). ³¹ P-NMR spectrum (160MHz): (CD ₃ CN)δ: 153.5.LCMS ESI(m/z):881.2[M+H] ⁺ .

3-OAP-LNA A

Synthesis of L-3-OAP-LNA A

_PCl₃ (184 μL, 2.1 mmol) was dissolved in toluene (5 mL) and cooled to 0°C (ice bath). A solution of P5-L (373 mg, 2.10 mmol) and NMM (463 μL, 4.20 mmol) in toluene (5 mL) was added dropwise. The reaction mixture was stirred at room temperature for 45 minutes and then cooled to -72°C. The precipitate was filtered under argon, washed with toluene (4 mL), and the filtrate was concentrated under reduced pressure at 40°C (Schlenk technique). The residue was dissolved in THF (5 mL) and used in the next step.

To a solution of 5'-ODMT-LNA-A (960 mg, 1.40 mmol) in THF (7 mL) was added _NEt₃ (1.36 mL, 9.80 mmol). The reaction mixture was cooled to -70°C and a solution of the phosphor ₂ -chloro-1,3,2-oxazaphosphacyclopentane was added dropwise. The reaction mixture was stirred at room temperature for 4 hours. EtOAc (50 mL) was added, and the reaction mixture was extracted with saturated _NaHCO₃ (2 x 30 mL), brine (30 mL ₎ , dried over Na₂SO₄, and evaporated. The residue was purified by column chromatography (eluent: hexane/EtOAc 30/70 + _NEt₃ 6-7%). The product was isolated as a white foam (455 mg, 35% yield). ¹ H-NMR spectrum (400MHz): (DMSO-d ₆ )δ: 11.33(1H,s),8.76(1H,s),8.53(1H,s), 8.11-8.02(2H,m),7.66(1H,m),7.60-7.53(2H,m),7.44-7.38(2H,m), 7.35-7.18(10H,m),7.05-6.99(2H,m),6.89-6.82(4H,m),6.21(1H,s),5.27 (1H,d,J＝6.6Hz),5.19(1H,d,J＝7.9Hz),4.81(1H,s),3.93(2H,dd,J＝29.0 8.2Hz),3.77(1H,m),3.71(6H,s),3.51-3.35(3H,m),2.70(1H,m),1.56-1.34(2H,m),1.10(1H,m),0.73(1H,m). ³¹ P-NMR spectrum (160MHz): (DMSO-d ₆ )δ:149.9.LCMSESI(m/z):891.1[M+H] ⁺ .

Synthesis of D-3-OAP-LNA A

PCl3 ₍ 0.84 mL, 9.63 mmol) was dissolved in toluene (12 mL) and cooled to 0°C (ice bath). A toluene solution (12 mL) of P5-D (1.70 g, 9.63 mmol) and NMM (2.12 mL, 19.3 mmol) was added. The reaction mixture was stirred at room temperature for 45 minutes and then cooled to -72°C. The precipitate was filtered under argon, washed with toluene, and the filtrate was concentrated under reduced pressure at 40°C (Schlenk technique). The residue was dissolved in THF (12 mL) and used in the next step.

To a solution of 5'-ODMT-LNA-A (3.7 g, 5.50 mmol) in THF (20 mL) was added _NEt₃ (5.30 mL, 38.5 mmol). The reaction mixture was cooled to -70°C and a solution of the phosphor 2-chloro-1,3,2-oxazaphosphacyclopentane was added dropwise. The reaction mixture was stirred at room temperature for 4 hours. EtOAc was added, and the reaction mixture was extracted with saturated _NaHCO₃ and brine, dried over _Na₂SO₄ , and evaporated. The residue was purified by column chromatography (eluent: hexane/EtOAc _30/70 + _NEt₃ 6-7%). The product was isolated as a white foam (1.86 g, 36% yield). ¹ H-NMR spectrum (400MHz): (DMSO-d ₆ )δ: 11.28(1H,s),8.78(1H,s),8.54(1H,s),8.09-8.04(2H,m),7.67(1H,m), 7.60-7.54(2H,m),7.42-7.15(14H,m),6.89-6.82(4H,m),6.21(1H,s),5.58 (1H,d,J＝6.7Hz),5.02(1H,d,J＝8.1Hz),4.89(1H,s),3.96(2H,dd,J＝35.4 8.2Hz),3.71(3H,s),3.70(3H,s),3.53-3.33(4H,m),2.90(1H,m), 1.54-1.37(2H,m),0.98(1H,m),0.71(1H,m). ³¹ P-NMR spectrum (160MHz): (DMSO-d ₆ )δ: 150.6, 150.5 (2%), 150.4. LCMS ESI (m/z): 891.1[M+H] ⁺ .

3-OAP-LNA G

Synthesis of D-3-OAP-LNA G

_PCl3 (1.09 mL, 12.4 mmol) was dissolved in toluene (12.5 mL) and cooled to 0°C (ice bath). A toluene solution (12.5 mL) of P5-D (2.20 g, 12.4 mmol) and NMM (2.73 mL, 27.8 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 45 minutes and then cooled to -72°C. The precipitate was filtered under argon, washed with toluene, and the filtrate was concentrated under reduced pressure at 40°C (Schlenk technique). The residue was dissolved in THF (19 mL) and used in the next step.

Prior to synthesis, 5'-ODMT-LNA-G was co-evaporated with toluene and then with pyridine (the order was necessary). NEt ₃ (4.85 mL, 35.0 mmol) was added to a solution of 5'-ODMT-LNA-G (3.26 g, 5.0 mmol) in THF (15 mL) and pyridine (8 mL). The reaction mixture was cooled to -70 ° C and a solution of the phosphor 2-chloro-1,3,2-oxazaphosphacyclopentane was added dropwise. The reaction mixture was stirred at room temperature for 2.5 hours. EtOAc was added, and the reaction mixture was extracted with saturated NaHCO ₃ and brine, dried over Na ₂ SO ₄ and evaporated. The residue was purified by column chromatography (eluent THF in EtOAc from 10% to 20% + NEt ₃ 6%). The product was isolated as a white foam 1.49 g (yield 33%). ¹ H-NMR spectrum (400MHz): (DMSO-d ₆ )δ: 11.42(1H,s),8.56(1H,s), 7.95(1H,s),7.49-7.38(2H,m),7.36-7.16(12H,m),6.90-6.83(4H,m),5.96 (1H,s),5.58(1H,d,J＝6.7Hz),4.99(1H,d,J＝8.2Hz),4.76(1H,s), 3.96-3.85(2H,m),3.72(6H,s),3.62-3.54(1H,m),3.45(2H,s),3.40-3.33 (1H,m),3.08(3H,s),2.99(3H,s),2.93-2.84(1H,m),1.53-1.39(2H,m), 1.06-0.97(1H,m),0.79-0.63(1H,m). ³¹ P-NMR spectrum (160MHz): (DMSO-d ₆ )δ: 151.6.LCMS ESI(m/z):858.2[M+H] ⁺ .

Synthesis of L-3-OAP-LNA G

_PCl3 (1.00 mL, 11.4 mmol) was dissolved in toluene (10 mL) and cooled to 0°C (ice bath). A solution of P5-L (2.02 g, 11.4 mmol) and NMM (2.50 mL, 22.7 mmol) in toluene (10 mL) was added dropwise. The reaction mixture was stirred at room temperature for 45 minutes and then cooled to -72°C. The precipitate was filtered under argon, washed with toluene, and the filtrate was concentrated under reduced pressure at 40°C (Schlenk technique). The residue was dissolved in THF (7 mL) and used in the next step.

Prior to synthesis, 5'-ODMT-LNA-G was co-evaporated with toluene and then with pyridine (sequence is necessary). NEt ₃ (4.40 mL, 31.8 mmol) was added to a solution of 5-ODMT-LNA-G (2.86 g, 4.54 mmol) in THF (20 mL) and pyridine (12 mL). The reaction mixture was cooled to -70 ° C and a solution of phosphor 2-chloro-1,3,2-oxazaphosphacyclopentane was added dropwise. The reaction mixture was stirred at room temperature for 2.5 hours. EtOAc was added, and the reaction mixture was extracted with saturated NaHCO ₃ and brine, dried over Na ₂ SO ₄ and evaporated. The residue was purified by column chromatography (eluent THF in EtOAc from 10% to 20% + NEt ₃ 6%). The product was isolated as a white foam 1.44 g (yield 34%). ¹ H-NMR (400mHz, DMSO-d ₆ ): δ: 11.44(1H,s),8.42(1H,s),7.94 (1H,s),7.44-7.38(2H,m),7.34-7.23(10H,m),7.03-6.98(2H,m),5.94(1H, s),5.17(1H,d,J＝6.5Hz),5.07(1H,d,J＝7.8Hz),4.68(1H,s),3.88(1H,d, J＝8.2Hz),3.84(1H,d,J＝8.2Hz),3.73(3H,s),3.72(3H,s),3.68(1H,m), 3.46-3.36(3H,m),3.05(3H,s),2.95(3H,s),2.77(1H,m),1.55-1.38(2H,m), 1.07(1H,m),0.75(1H,m). ³¹ P-NMR(160MHz,DMSO-d ₆ ): δ: 148.4.LCMS ESI (m/z): 858.5[M+H] ⁺ ; 856.5[MH] ^- .

General synthesis description

Synthesis of the phosphor 2-chloro-1,3,2-oxazaphosphorolane: _PCl₃ (1 equivalent) was dissolved in toluene and cooled to 0°C (ice bath). A toluene solution of P5-L (1 equivalent) and NMM (2.1 equivalents) was added dropwise. The reaction mixture was stirred at room temperature and then cooled to -72°C. The precipitate was filtered under argon, washed with toluene, and the filtrate was concentrated under reduced pressure at 40°C (Schlenk technique). The residue was dissolved in THF and used in the next step.

To a solution of 5'-ODMT-LNA nucleoside (1 equivalent) in THF (pyridine in the case of the G nucleoside) was added _NEt₃ (7 eq). The reaction mixture was cooled to -70°C and a solution of the phosphor 2-chloro-1,3,2-oxazaphosphacyclopentane (2.5 eq) was added dropwise. The reaction mixture was stirred at room temperature. EtOAc was added, _and the reaction mixture was extracted with saturated _NaHCO₃ , brine, dried over _Na₂SO₄ , and evaporated. The residue was purified by column chromatography.

Structure of LNA monomer

The following LNA-oxazaphosphorylcholine LNA monomer was synthesized using the method disclosed by Oka et al., J. Am. Chem. Soc. 2008; 16031-16037.

The above LNA monomers were used for oligonucleotide synthesis and stereo controlled phosphoramidite LNA oligonucleotides were shown as determined by HPLC.

Example 2

The following Myd88-targeting LNA oligonucleotides were synthesized.

A _x ^m C _x T _x g _x c _x t _x t _x t _x c _x c _x a _x c _x t _x ^m C _x T _x G(Parent#1)(SEQ ID NO 1)

A _x ^m C _x T _x g _x c _x t _x t _x t _x c _x c _x a _x c _x t _x ^m C _x T _x G(Parent#1)

A _x ^m C _x T _x g _x c _s t _x t _x t _x c _x c _x a _x c _x t _x ^m C _x T _x G(Comp#2)

A _x ^m C _x T _x g _x c _x t _x t _x t _x c _s c _x a _x c _x t _x ^m C _x T _x G(Comp#3)

A _x ^m C _x T _x g _x c _x t _x t _x t _x c _x c _x a _x c _s t _x ^m C _x T _x G(Comp#4)

A _x ^m C _x T _x g _x c _r t _x t _x t _x c _x c _x a _x c _x t _x ^m C _x T _x G(Comp#5)

A _x ^m C _x T _x g _x c _x t _x t _x t _x c _r c _x a _x c _x t _x ^m C _x T _x G(Comp#6)

A _x ^m C _x T _x g _x c _x t _x t _x t _x c _x c _x a _x c _r t _x ^m C _x T _x G(Comp#7)

A _x ^m C _x T _x g _x c _s t _x t _x t _x c _s c _x a _x c _x t _x ^m C _x T _x G(Comp#8)

A _x ^m C _x T _x g _x c _s t _x t _x t _x c _x c _x a _x c _s t _x ^m C _x T _x G(Comp#9)

A _x ^m C _x T _x g _x c _s t _x t _x t _x c _s c _x a _x c _s t _x ^m C _x T _x G(Comp#10)

A _x ^m C _x T _x g _x c _x t _x t _x t _x c _s c _x a _x c _s t _x ^m C _x T _x G(Comp#11)

A _x ^m C _x T _x g _{x c r} t _x t _x t _x c _r c _x a _x c _x t _x ^m C _x T _x G(Comp#12)

A _x ^m C _x T _x g _x c _r t _x t _x t _x c _x c _x a _x c _r t _x ^m C _x T _x G(Comp#13)

A _x ^m C _x T _x g _{x c} r t _x t _x t _x c _r c _x a _x c _r t _x ^m C _x T _x G(Comp#14)

A _x ^m C _x T _x g _x c _x t _x t _x t _x c _r c _x a _x c _r t _x ^m C _x T _x G(Comp#15)

A _x ^m C _x T _x g _x c _x t _x t _x t _x c _r c _x a _x c _s t _x ^m C _x T _x G(Comp#16)

A _x ^m C _x T _x g _x c _x t _x t _x t _x c _s c _x a _x c _r t _x ^m C _x T _x G(Comp#17)

A _x ^m C _x T _x g _x c _s t _x t _x t _x c _r c _x a _x c _x t _x ^m C _x T _x G(Comp#18)

A _x ^m C _x T _x g _x c _s t _x t _x t _x c _x c _x a _x c _r t _x ^m C _x T _x G(Comp#19)

A _x ^m C _x T _x g _x c _s t _x t _x t _x c _r c _x a _x c _r t _x ^m C _x T _x G(Comp#20)

A _x ^m C _x T _x g _x c _s t _x t _x t _x c _r c _x a _x c _s t _x ^m C _x T _x G(Comp#21)

A _x ^m C _x T _x g _x c _s t _x t _x t _x c _s c _x a _x c _r t _x ^m C _x T _x G(Comp#22)

A _x ^m C _x T _x g _x c _r t _x t _x t _x c _s c _x a _x c _x t _x ^m C _x T _x G(Comp#23)

A _x ^m C _x T _x g _x c _r t _x t _x t _x c _x c _x a _x c _s t _x ^m C _x T _x G(Comp#24)

A _x ^m C _x T _x g _x c _r t _x t _x t _x c _s c _x a _x c _s t _x ^m C _x T _x G(Comp#25)

A _x ^m C _x T _x g _x c _r t _x t _x t _x c _s c _x a _x c _r t _x ^m C _x T _x G(Comp#26)

A _x ^m C _x T _x g _{x c r} t _x t _x t _x c _r c _x a _x c _s t _x ^m C _x T _x G(Comp#27)

Capital letters are β-D-oxy LNA nucleosides, and lowercase letters are DNA nucleosides.

Subscript x = randomly incorporated phosphorothioate linkages from the racemic mixture of Rp and Sp monomers.

Subscript s = stereocontrolled phosphoramidite bond from the Sp monomer.

Subscript r = stereocontrolled phosphoramidite bond from the Rp monomer.

The superscript m before the capital letter C indicates 5-methylcytosine LNA nucleoside.

Example 3

Parent compound #1 has been determined to be hepatotoxic in mice. Compounds #1-27# were evaluated for hepatotoxicity in an in vivo assay: 5 NMRI female mice per group were administered 15 mg/kg of compound on days 0, 3, 7, 10, and 14, and sacrificed on day 16. Serum ALT was measured. Hepatotoxicity can also be measured as described in EP 1 984 381, Example 41, but using NMRI mice, or using an in vitro hepatotoxicity assay.

Example 4

The following LNA oligonucleotides were synthesized as identified as toxic in Seth et al J. Med. Chem 2009, 52, 10-13.

T _x ^m C _x a _x t _x g _x g _x c _x t _x g _x c _x a _x g _x ^m C _x T(Parent#28)(SEQ ID NO 2)

T _x ^m C _{x a x} t _s g _x g _x c _x t _x g _x c _x a _x g _x ^m C _x T(Comp#29)

T _x ^m C _{x a x t x} g _x g _s c _x t _x g _x c _x a _x g _x ^m C _x T(Comp#31)

T _x ^m C _{x a x t x} g _x g _x c _x t _x g _x c _s a _x g _x ^m C _x T(Comp#32)

T _x ^m C _{x a} x _{t s} g _x g _s c _x t _x g _x c _x a _x g _x ^m C _x T(Comp#33)

T _x ^m C _{x a x t s} g _x g _x c _x t _x g _x c _s a _x g _x ^m C _x T(Comp#34)

T _x ^m C _{x a} x _{t s} g _x g _s c _x t _x g _x c _s a _x g _x ^m C _x T(Comp#35)

T _x ^m C _{x a x t x} g _x g _s c _x t _x g _x c _s a _x g _x ^m C _x T(Comp#36)

T _x ^m C _{x a} x t _s g _x g _r c _x t _x g _x c _x a _x g _x ^m C _x T(Comp#37)

T _x ^m C _{x a x} t _s g _x g _x c _x t _x g _x c _r a _x g _x ^m C _x T(Comp#38)

T _x ^m C _{x a} x t _s g _x g _r c _x t _x g _x c _r a _x g _x ^m C _x T(Comp#39)

T _x ^m C _{x a x} t _x g _x g _s c _x t _x g _x c _r a _x g _x ^m C _x T(Comp#40)

T _x ^m C _{x a} x _{t s} g _x g _r c _x t _x g _x c _s a _x g _x ^m C _x T(Comp#41)

T _x ^m C _{x a} x t _s g _x g _s c _x t _x g _x c _r a _x g _x ^m C _x T(Comp#42)

T _x ^m C _x a _x t _r g _x g _x c _x t _x g _x c _x a _x g _x ^m C _x T(Comp#43)

T _x ^m C _{x a x} t _x g _x g _r c _x t _x g _x c _x a _x g _x ^m C _x T(Comp#44)

T _x ^m C _{x a x} t _x g _x g _x c _x t _x g _x c _r a _x g _x ^m C _x T(Comp#45)

T _x ^m C _{x a x} t _r g _x g _r c _x t _x g _x c _x a _x g _x ^m C _x T(Comp#46)

T _x ^m C _{x a x} t _r g _x g _x c _x t _x g _x c _r a _x g _x ^m C _x T(Comp#47)

T _x ^m C _{x a x} t _r g _x g _r c _x t _x g _x c _r a _x g _x ^m C _x T(Comp#48)

T _x ^m C _{x a x} t _x g _x g _r c _x t _x g _x c _r a _x g _x ^m C _x T(Comp#49)

T _x ^m C _{x a x} t _r g _x g _s c _x t _x g _x c _x a _x g _x ^m C _x T(Comp#50)

T _x ^m C _x a _x t _r g _x g _x c _x t _x g _x c _s a _x g _x ^m C _x T(Comp#51)

T _x ^m C _{x a x} t _r g _x g _s c _x t _x g _x c _s a _x g _x ^m C _x T(Comp#52)

T _x ^m C _{x a x} t _x g _x g _r c _x t _x g _x c _s a _x g _x ^m C _x T(Comp#53)

T _x ^m C _{x a x} t _r g _x g _s c _x t _x g _x c _r a _x g _x ^m C _x T(Comp#54)

T _x ^m C _{x a x} t _r g _x g _r c _x t _x g _x c _s a _x g _x ^m C _x T(Comp#55)

Capital letters are β-D-oxy LNA nucleosides, and lowercase letters are DNA nucleosides.

Subscript x = randomly incorporated phosphorothioate linkages from the racemic mixture of Rp and Sp monomers.

Subscript s = stereocontrolled phosphoramidite bond from the Sp monomer.

Subscript r = stereocontrolled phosphoramidite bond from the Rp monomer.

The superscript m before the capital letter C indicates 5-methylcytosine LNA nucleoside.

Example 5

The parent compound #28 has been determined to be hepatotoxic in mice. Compounds #28–27# were evaluated for hepatotoxicity in an in vivo assay: 5 NMRI female mice per group were administered 15 mg/kg of compound on days 0, 3, 7, 10, and 14, and sacrificed on day 16. Serum ALT was measured. Hepatotoxicity can also be measured as described in EP 1 984 381, Example 41, but using NMRI mice, or using an in vitro hepatotoxicity assay.

Example 6. In vivo tolerance and tissue content of a compound library with three fixed PS internucleoside linkages

C57BL6/J mice (5 animals/gr) were injected intravenously on day 0 with a single dose of saline or 30 mg/kg LNA antisense oligonucleotide in saline (seq ID#1, 10 or 14) and sacrificed on day 8.

Serum was collected and ALT was measured in all groups. Oligonucleotide levels were measured in the LNA dose groups using ELISA.

in conclusion:

The hepatotoxic potential (ALT) of LNA oligonucleotide subsets (in which the three phosphorothioate internucleoside bonds were fixed in the S (Compound #10) or R (Compound #14) configuration) was compared to the ALT of the parent diastereomeric mixture (Compound #1), a mixture of all internucleoside bonds in the R and S configuration. As can be seen (Figure 3), for one subset (Compound #14), the ALT reading was significantly lower than that of the parent mixture (Compound #1), while the other subset compound (Compound #10) had an ALT reading similar to that of the parent. Furthermore, the liver uptake curve (Figure 4a) showed that the LNA oligonucleotide subset with a low ALT reading (Compound #14) was absorbed into the liver to the same extent as the parent LNA mixture (Compound #1), while the other subset (Compound #10), with an ALT comparable to that of the parent mixture (Compound #1), was less absorbed into the liver. Renal uptake (Figure 4b) was similar for the parent LNA (Compound #1) and one subgroup (Compound #10), while higher for another LNA oligonucleotide subgroup (Compound #14). Splenic uptake was similar for all three groups of compounds (Figure 4c). It can be seen that fixing the stereochemistry at certain positions and thus generating subgroups of LNA oligonucleotides induces differences in properties such as uptake and hepatotoxic potential compared to the parent mixture of LNA oligonucleotides.

Materials and methods:

Experimental design:

Table 2

Dosing C57BL/6JBom female animals, approximately 20 g at arrival, were dosed intravenously with 10 ml of compound formulated in saline or saline alone per kg body weight (based on body weight on day 0) according to Table 2.

Liver and kidney tissue sampling : Animals were anesthetized with 70% _CO2-30 % _O2 and sacrificed by cervical dislocation according to Table 2. Half of the liver and one kidney were frozen and used for tissue analysis.

Oligonucleotide levels in liver and kidney were determined by sandwich ELISA.

ALT levels are measured.

Example 7 RNase H activity of chirally constrained phosphorothioate LNA gapmers.

Using parent compound 3833:

5'-G _s ^m C _s a _s t _s t _s g _s g _s t _s a _s t _s T _s ^m C _s A-3'(SEQ ID NO 3)

Wherein capital letters denote β-D-oxy-LNA nucleosides, lowercase letters denote DNA nucleosides, the subscript s denotes a random s or r phosphorothioate bond (not chirally restricted during oligonucleotide synthesis), and the superscript m before C denotes a 5-methylcytosine LNA nucleoside.

A series of fully chirally defined variants of 3833 were designed, in which each of the 12 internucleoside positions had a unique pattern of R and S, as indicated by S or R. RNase H recruitment activity and cleavage patterns were determined using human RNase H and compared to the parent compound 3833 (chiral mixture), as well as fully phosphodiester-linked variants of 3833 (fully PO), and the 3833 compound (which contains a phosphodiester bond within the central DNA gap region and random PS bonds in the LNA flanks) (PO gap).

Compound:

All compounds were evaluated in a single experiment and are marked * only when separate experiments were performed.

experiment:

LNA oligonucleotide-mediated cleavage of RNA by RNase H1 (recombinant human).

15 pmol of LNA oligonucleotide and 45 pmol of 5' fam-labeled RNA were added to 13 μL of water. 6 μL of annealing buffer (200 mM KCl, 2 mM EDTA, pH 7.5) was added, and the temperature was raised to 90°C for 2 minutes. The sample was allowed to reach room temperature, and RNase H enzyme (0.15 U) was added to 3 μL of 750 mM KCl, 500 mM Tris-HCl, 30 _mM MgCl₂, 100 mM dithiothreitol, pH 8.3). The sample was incubated at 37°C for 30 minutes, and the reaction was stopped by adding 4 μL of 0.25 M EDTA solution.

AIE-HPLC of cleaved RNA samples

15 μL of sample was added to 200 μL of buffer A (10 mM NaClO 4 , 1 mM EDTA, 20 mM TRIS-HCl pH 7.8). The sample was injected into an A1E-HPLC with a volume of 50 μL (Collumn DNA pac 100 2x250, gradient 0 min 0.25 mL/min, 100% A, 22 min 22% B (1 mM NaClO 4 , 1 mM EDTA, 20 mM TRIS-HCl pH 7.8), 25 min. 0.25 mL/min. 100% B, 30 min. 0.25 mL/min. 100% B, 31 min. 0.5 mL/min. 0% B, 35 min. 0.25 mL/min. 0% B, 40 min. 0.25 mL/min. 0% B. Signal detection was fluorescence emission at 518 nm and excitation at 494 nm.

result

All phosphorus bonds of the LNA ^{oligonucleotide} with the sequence ^{GmCattggtatTmCA} were thiolated. The specific chirality of the phosphorothioate bonds was noted. The chirality was not noted, as it was a mixture of R and S. The peak area is listed as a percentage of the sum of all peak areas at the AIE-HPLC retention time. The ranking of the activities of the different LNA oligonucleotides was calculated by dividing the percentage of full-length RNA remaining after the enzymatic reaction of the chirally mixed LNA oligonucleotide 3833 by the percentage of RNA remaining for the remaining LNA oligonucleotides.

in conclusion

The chirality of the phosphorothioate bonds of the LNA oligonucleotides was randomly selected, with the exception of the final 5'-coupling, which was selected for S chirality, and LNA oligonucleotides 17298-17301, which were selected for point chirality. The fully formed diester and diester forms of the LNA oligonucleotides alone exhibited less activity than the mixed chiral form 3833. Chiral sequences enhance RNA activation and cleavage. For most specific chiral LNA oligonucleotides, RNase H1 activation was more effective than the chirally mixed 3833. The best sequence among the specific sequences initiated substantially more RNA cleavage than 3833 (98.4% vs. 47.7% after 30 minutes). Each specific LNA oligonucleotide is characterized by its unique RNA cleavage pattern, which can vary from one to several cleavage points.

Example 8 In vitro toxicity screening of primary hepatocytes

Mouse liver perfusion

Primary mouse hepatocytes were isolated from 10- to 13-week-old male C57Bl6 mice by retrograde two-step collagenase liver perfusion. Briefly, chow-fed mice were anesthetized with sodium pentobarbital (120 mg/kg, intraperitoneally). The perfusion cannula was inserted into the caudal vena cava via the right ventricle. After ligation of the caudal vena cava distal to the common iliac vein, the portal vein was severed, and two-step liver perfusion and cell isolation were performed. The liver was first perfused for 5 minutes with a preperfusion solution consisting of calcium-free, EGTA (0.5 mM)-supplemented HEPES (20 mM)-buffered Hank's balanced salt solution, followed by perfusion for 12 minutes with NaHCO3 (25 mM)-supplemented Hank's solution containing CaCl2 (5 mM) and collagenase (0.2 U/ml; collagenase type II, Worthington). The flow rate was maintained at 7 ml/min, and all solutions were maintained at 37°C. After in situ perfusion, the liver was excised, the liver capsule was mechanically opened, and the cells were suspended in William's Medium E (WME) (Sigma W-1878) without phenol red and filtered through a set of nylon cell strainers (40- and 70 mesh). Dead cells were removed by a Percoll (Sigma P-4937) centrifugation step (percoll density: 1.06 g/ml, 50 g, 10 minutes) and an additional centrifugation in WME (50 x g, 3 minutes).

Compounds used

(Mother #56) (Comp#57) (Comp#58) (Comp#59) (Comp#60)

Capital letters are β-D-oxy LNA nucleosides, and lowercase letters are DNA nucleosides.

Subscript x = phosphorothioate linkage randomly incorporated from the racemic mixture of Rp and Sp monomers.

Subscript s = stereocontrolled phosphoramidite bond from the Sp monomer.

Subscript r = stereocontrolled phosphoramidite bond from the Rp monomer.

The superscript m before the capital letter C indicates 5-methylcytosine LNA nucleoside.

Hepatocyte culture

For cell culture, primary mouse hepatocytes were suspended in WME supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (0.1 mg/ml) at approximately 5 x ¹⁰ cells/ml and seeded into collagen-coated 96-well plates (Becton Dickinson AG, Allschwil, Switzerland) at a density of 0.25 x ¹⁰ cells/well. The cells were pre-cultured for 3 to 4 hours and allowed to adhere to the cell culture plates before treatment with the oligonucleotides began. Oligonucleotides dissolved in PBS were added to the cell cultures and placed on the cells for 3 days. According to the manufacturer's protocol, Cytotoxicity levels were determined by measuring the amount of lactate dehydrogenase (LDH) released into the culture medium using a cytotoxicity assay kit (Roche 11644793001, Roche Diagnostics GmbH Roche Applied Science Mannheim, Germany). To determine cellular ATP levels, we used a luminescent cell viability assay (G9242, Promega Corporation, Madison WI, USA) according to the manufacturer's protocol. Each sample was tested in triplicate.

Target knockdown analysis

mRNA was purified from mouse hepatocytes using the RNeasy 96 kit (Qiagen, Hombrechtikon, Switzerland) according to the manufacturer's instructions, which included RNase-free DNase I treatment. cDNA was synthesized using the iScript Single-Stranded cDNA Synthesis Kit (Bio-Rad Laboratories AG, Reinach, Switzerland). Quantitative real-time PCR assays (qRT-PCR) were performed using the Roche SYBR Green I PCR Kit and Light Cycler 480 (Roche Diagnostics, Rotkreuz, Switzerland) with specific DNA primers. Analysis was performed by the ΔCt threshold method to determine expression relative to RPS12 mRNA. Each analytical reaction was performed in duplicate, with two samples per condition. The results are shown in Figures 5 and 6. Compounds 58 and #60 had significantly reduced toxicity while retaining potent antisense activity against the target (Myd88). These compounds include a stereospecific phosphorothioate bond to Rp.

Example 9 Nephrotoxicity Screening Assay

The same compounds used in Examples 6 and 8 were used in the following RPTEC-TERT1 culture, oligonucleotide treatment, and viability assays:

RPTEC-TERT1 (Evercyte GmbH, Austria) were cultured in PTEC medium (containing 1% Pen/Strep, 10 mM Hepes, 5.0 μg/ml human insulin, 5.0 μg/ml human transferrin, 8.65 ng/ml sodium selenite, 0.1 μM hydrocortisone, 10 ng/ml human recombinant epidermal growth factor, 3.5 μg/ml ascorbic acid, 25 ng/ml prostaglandin E1, 3.2 pg/ml triiodo-L-thyronine, and 100 μg/ml geneticin) according to the manufacturer's instructions. For viability assays, PTEC-TERT1 were seeded at a density of 2× ¹⁰ cells/well in 96-well plates (Falcon, 353219) and grown until confluent before treatment with oligonucleotides. Oligonucleotides were dissolved in PBS and added to the cell cultures at a final concentration of 10 or 30 μM. The culture medium was replaced and fresh oligonucleotides were added every 3 days. After 9 days of oligonucleotide treatment, cell viability was determined by measuring cellular ATP levels using the Luminescent Cell Viability Assay (G7571, Promega Corporation, Madison WI, USA) according to the manufacturer's protocol. The mean ATP concentration and standard deviation were calculated for triplicate wells. PBS was used as a vehicle control.

The results are shown in Figure 8. Compound #10 showed reduced nephrotoxicity compared to non-stereospecific compound #1 and compound #14. Stereospecific compounds #57, #58, and #60 showed significantly reduced nephrotoxicity compared to the parent compound (#56).

Example 10 Mismatch specificity of chirally constrained phosphorothioate LNA gapmers.

The experimental procedure used was as described in Example 7, except that alternative RNA substrates were used that introduced mismatches at various positions compared to the parent compound 3833. RNase H activity was determined against perfectly matched and mismatched RNA substrates.

Table 3: Effects of mismatches on RNase H activity of 3833.

Mismatches are shown by using a bolder font size. RNase H cleavage was analyzed after 30 minutes. The cleavage products varied depending on the position of the mismatch.

Table 4: Effect of mispairing on RNase H activity of stereospecific variants of 3833.

For perfectly matched RNA substrates, chirality-constrained phosphorothioate oligonucleotides tend to activate RNase H-mediated RNA cleavage more profoundly than ASOs with mixed chirality. However, it was found that chirality-constrained oligonucleotides of selected phosphorothioate (ASO) configurations had significantly reduced RNase H cleavage of mismatched RNA, highlighting the ability to screen libraries of chirality-constrained variants of oligonucleotides to identify single stereospecific compounds with improved mismatch selectivity.

Example 11

Using parent compound 4358:

5′G _s ^m C _s a _s a _s g _s c _s a _s t _{s c} s c _s t _s G _s T 3'(SEQ ID NO 5)

Wherein capital letters denote β-D-oxy-LNA nucleosides, lowercase letters denote DNA nucleosides, the subscript s denotes a random s or r phosphorothioate bond (not chirally restricted during oligonucleotide synthesis), and the superscript m before C denotes a 5-methylcytosine LNA nucleoside.

A series of fully chirally defined variants of 4358 were designed with unique patterns of R and S at each of the 11 internucleoside positions, as indicated by S or R. RNase H recruitment activity and cleavage patterns were determined using human RNase H and compared with the parent compound 4358 (chiral mixture). The following results were obtained:

Claims (24)

1. A method for reducing the toxicity of an antisense oligonucleotide sequence as a parent oligonucleotide, wherein the parent oligonucleotide is 10-20 nucleotides long, comprising the following steps: a. Generate a library of stereooriented phosphate-thioester oligonucleotide variants as daughter oligonucleotides, wherein each oligonucleotide variant retains the nucleobase sequence of at least 10 consecutive nucleotides of the parent oligonucleotide and has at least one stereooriented phosphate-thioester nucleoside bond that differs from the parent oligonucleotide and other daughter oligonucleotides. b. Screening the library generated in step a for in vitro hepatotoxicity in cells. c identifies one or more stereooriented phosphate thioester variants in the library that exhibit reduced hepatotoxicity in cells compared to the parent oligonucleotide. 2. The method according to claim 1, wherein the parent oligonucleotide is an antisense interstitial oligonucleotide. 3. The method of claim 2, wherein each member of the stereooriented phosphate thioester oligonucleotide variant library is generated by inserting at least one stereooriented phosphate thioester nucleoside internucleotide bond into the interstitial region of the interstitial body. 4. The method of claim 2, wherein each member of the stereooriented phosphate thioester oligonucleotide variant library is generated by inserting at least one stereooriented phosphate thioester nucleoside internucleotide bond into one or both wing regions of the spacer. 5. The method according to any one of claims 1-4, wherein each member of the stereooriented phosphate-thioester oligonucleotide variant library comprises at least two stereooriented phosphate-thioester bonds, wherein the remaining bonds may optionally be non-stereooriented phosphate-thioester bonds. 6. The method of claim 5, wherein each member of the stereooriented phosphate thioester oligonucleotide variant library comprises at least three stereooriented phosphate thioester bonds. 7. The method of claim 5, wherein each member of the stereooriented phosphate thioester oligonucleotide variant library comprises at least four stereooriented phosphate thioester bonds. 8. The method according to any one of claims 1-4, wherein all thiophosphate bonds present in each member of the stereooriented thiophosphate oligonucleotide variant library are stereooriented thiophosphate bonds. 9. The method according to any one of claims 1-4, wherein all internucleotide bonds present in each member of the library of stereooriented phosphate-thioester oligonucleotide variants are stereooriented phosphate-thioester bonds. 10. The method according to any one of claims 2-4, wherein all internucleotide bonds present in the interstitial region of each member of the stereooriented phosphate-thioester oligonucleotide variant library are stereooriented phosphate-thioester bonds. 11. The method according to any one of claims 1-4, wherein each member of the stereooriented phosphate thioester oligonucleotide variant library retains the pattern of modified and unmodified nucleosides present in the parent oligonucleotide. 12. The method according to any one of claims 2-4, wherein each member of the stereooriented phosphate thioester oligonucleotide variant library is an antisense interstitial oligonucleotide. 13. The method according to any one of claims 2 to 4, wherein the parent interstitial oligonucleotide is an LNA interstitial oligonucleotide. 14. The method according to any one of claims 1-4, wherein the method further comprises the step of determining the IC50 of one or more stereooriented thiophosphate variants identified in step c). 15. The method of claim 14, wherein the stereooriented phosphate thioester oligonucleotide variant identified in step c) retains at least 25% of the IC50 of the parent oligonucleotide. 16. The method of claim 15, wherein the stereooriented phosphate thioester oligonucleotide variant identified in step c) retains at least 50% of the IC50 of the parent oligonucleotide. 17. The method according to any one of claims 1-4, wherein the method further comprises the step of determining the EC50 or ED50 of one or more stereooriented phosphate thioester oligonucleotide variants identified in step c). 18. The method of claim 17, wherein the stereooriented thiophosphate oligonucleotide variant identified in step c) retains an EC50 value no greater than 3 to 10 times that of the parent compound. 19. The method according to any one of claims 1-4, wherein the parent oligonucleotide and stereooriented phosphate thionucleotide variant library members comprise at least one LNA nucleoside selected from β-D-oxo-LNA and 6'-methylβ-D-oxo-LNA nucleoside. 20. The method of claim 19, wherein the LNA nucleoside is S(cET). 21. The method according to any one of claims 1-4, wherein the parent oligonucleotide is hepatotoxic, and one or more stereooriented phosphate thionucleotide variants identified in step c) have lower hepatotoxicity than the parent oligonucleotide. 22. The method according to any one of claims 1-4, wherein the library screening performed in step b) comprises screening for in vitro hepatotoxicity in primary hepatocytes. 23. The method of claim 22, wherein the primary hepatocytes are primary mammalian hepatocytes. 24. The method of claim 23, wherein the mammal is a mouse or a rat.