OLIGONUCLEOTIDES FOR MODULATING MYH7 EXPRESSION

Abstract

The present invention relates to antisense oligonucleotides that are capable of modulating expression of MYH7 in a target cell. The oligonucleotides hybridize to MYH7 mRNA. The present invention further relates to conjugates of the oligonucleotide and pharmaceutical compositions and methods for treatment of hypertrophic cardiomyopathy using the oligonucleotide.

Description

FIELD OF INVENTION

The present invention relates to antisense oligonucleotides which target human myosin heavy chain 7 (MYH7) transcript. In some aspects, the oligonucleotides of the invention may be used to selectively inhibit the expression a disease associate allele of MYH7. Inhibition of MYH7 expression is beneficial for a range of medical disorders, including hypertrophic cardiomyopathy.

BACKGROUND

Familial hypertrophic cardiomyopathy (HCM) is a monogenic disease clinically characterized by asymmetrical ventricular hypertrophy, arrhythmias, and progressive heart failure. HCM has a prevalence of 1:500 and about 40% of cases are due to autosomal dominant mutations in the MYH7 gene. MYH7 encodes the β-myosin heavy chain protein that acts as a molecular motor to drive active contraction during cardiac systole. More than 300 missense mutations in MYH7 have been linked to HCM pathology, and these mutations are distributed throughout the gene. There is no common mechanism that links each MYH7 mutation to the HCM phenotype; mutations can affect filament sliding velocity, ATPase rate, force, and calcium sensitivity of activation. Regardless of the exact mutation and its specific effect on actomyosin dynamics, the link between MYH7 mutation and HCM derives from mutant myosin protein that is expressed, stable, and exerts dominant negative effects.

Hundreds of dominant negative myosin mutations have been identified that lead to hypertrophic cardiomyopathy (HCM), and the biomechanical link between mutation and disease is heterogeneous across this patient demographic. This represents a major challenge for therapeutic intervention for the treatment or prevention of hypertrophic cardiomyopathy.

WO2015/042581 discloses a method of preventing or treating hypertrophic cardiomyopathy (HCM) in a subject having in their genome a first MYH7 allele comprising an HCM-causing mutation and a second MYH7 allele that does not comprise the HCM-causing mutation, the method comprising administering to the subject an interfering RNA molecule that selectively inactivates the transcript encoded by the first MYH7 allele compared to the transcript encoded by the second MYH7 allele. siRNAs targeting the T403Q mutation are disclosed.

WO 2015/113004 discloses a method for treating a subject having hypertrophic cardiomyopathy comprising administering a siRNA which selectively down-regulates expression of myosin heavy chain-403Q.

WO2016/149684 discloses a method for down-regulating disease causing alleles using RNAi therapeutics system, where subject samples are sequences to identify deleterious mutation on a particular allele as a common variant in phase with the deleterious mutation, and selecting a RNAi therapeutic targeting the common variant using the RNAi therapeutics system, and applying the selected RNAi therapeutics system utilizing a vector and the RNAi therapeutics system. The RNAi therapeutics system may include a 2′-O-methylated antisense nucleic acid phosphorothioate compound complementary to common variants of the Myh7 gene.

OBJECTIVE OF THE INVENTION

The present invention identifies novel oligonucleotides which modulate MYH7, which may be used for allelic selective inhibition of MYH7.

SUMMARY OF INVENTION

The present invention relates to oligonucleotides targeting a MYH7 nucleic acid which are capable of inhibiting the expression of MYH7.

The invention provides oligonucleotides which target the expression of a MYH7 allelic variant selected from a MYH7 allelic variant which comprises a single nucleotide polymorphism at a position selected from rs2239578, rs2069540, and rs7157716 (RefSNP see dbSNP, NCBI Homo sapiens Annotation Release 109, 2018-03-27, hereby incorporated by reference). These three common SNPs are found in intron 2, exon 3, and exon 24 of MYH7 pre-mRNA respectively, and are referred to as rs223, rs206, and rs715 herein.

In some embodiments the oligonucleotide of the invention selectively inhibits a MYH7 allelic variant, such as an allelic variant at a position of the human MYH7 transcript selected from rs223, rs206 and rs715.

The invention provides an antisense oligonucleotide targeting human myosin heavy chain 7 (Myh7) transcript, wherein said oligonucleotide comprises a contiguous nucleotide sequence of 10-30 nucleotides in length which are at least 90% complementary to a sequence selected from the group consisting of SEQ ID NOs 3-10.

The invention provides an antisense oligonucleotide targeting human myosin heavy chain 7 (Myh7) transcript, wherein said oligonucleotide comprises a contiguous nucleotide sequence of 10-30 nucleotides in length which are at least 90% complementary to a sequence selected from the group consisting of SEQ ID NOs 3 & 4, or SEQ ID NOs 5-10.

The invention provides an antisense oligonucleotide 10-40 nucleotides in length, targeting human myosin heavy chain 7 (Myh7) transcript, wherein said oligonucleotide comprises a contiguous nucleotide sequence of 10-30 nucleotides in length which are at least 90% complementary to a sequence selected from the group consisting of SEQ ID NOs 3 & 4, or SEQ ID NOs 5-10.

In some embodiments, the antisense oligonucleotide is complementary to a region of the sequence selected from SEQ ID NOs 3-10 wherein the region of complementarity comprises the 20^th nucleotide from the 5′ end of the sequence selected from SEQ ID NOs 3-10.

In some embodiments, the antisense oligonucleotide is a LNA modified oligonucleotide, such as an LNA gapmer.

The invention provides for a conjugate comprising the oligonucleotide according to the invention and at least one conjugate moiety covalently attached to said oligonucleotide.

The invention provides for a pharmaceutically acceptable salt of the antisense oligonucleotide or the conjugate according to the invention,

The invention provides for a pharmaceutical composition comprising the antisense oligonucleotide or the conjugate of the invention and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.

The invention provides for a method for modulating human myosin heavy chain 7 (Myh7) expression in a target cell which is expressing Myh7, said method comprising administering an oligonucleotide of the invention or the conjugate of the invention or the pharmaceutically acceptable salt of the invention or the pharmaceutical composition of the invention in an effective amount to said cell. In some embodiments the method is in vivo. In some embodiments the method is in vitro.

The invention provides for a method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of an oligonucleotide of the invention or the conjugate of the invention or the pharmaceutically acceptable salt of the invention or the pharmaceutical composition of the invention to a subject suffering from or susceptible to the disease.

In some embodiments the disease is hypertrophic cardiomyopathy.

The invention provides for an oligonucleotide of the invention or the conjugate of the invention or the pharmaceutically acceptable salt of the invention or the pharmaceutical composition of the invention for use in medicine.

The invention provides for an oligonucleotide of the invention or the conjugate of the invention or the pharmaceutically acceptable salt of the invention or the pharmaceutical composition of the invention for use in the treatment or prevention of hypertrophic cardiomyopathy.

The invention provides for the use of the oligonucleotide of the invention or the conjugate of the invention or the pharmaceutically acceptable salt of the invention or the pharmaceutical composition of the invention, for the preparation of a medicament for treatment or prevention of hypertrophic cardiomyopathy.

The invention provides for a method for treatment of a human subject in need to treatment for hypertrophic cardiomyopathy, said treatment comprising the step of:

a. Taking a biological sample from the human subject

b. Sequencing the Myh7 nucleic acid alleles present in the sample of the human subject;

c. Determine the presence of a disease associated Myh7 allelic variant of the Myh7 nucleic acid;

d. Administer a therapeutically effective amount of an antisense oligonucleotide to the human subject which is selective for the disease associated Myh7 allelic variant as compared to a non-disease associate allele, such as the oligonucleotide of the invention or the conjugate of the invention or the pharmaceutically acceptable salt of the invention or the pharmaceutical composition of the invention.

BRIEF DESCRIPTION OF FIGURES

FIG. 1. SNP Targeting strategy a) SNP heterozygosity across five genetic super populations. See http://www.internationalgenome.org/category/population/ for details on population descriptions. b) Developing ASOs for individual HCM mutations is not currently a feasible therapeutic strategy. By targeting SNPs, multiple MYH7 disease-causing mutations can be targeted with a single ASO.

FIG. 2. Evaluation of SNP-selective ASOs from initial library in skeletal muscle myoblast cell lines a) Example of concentration response curves for ASO A181 from the rs223-C sub-library showing high SNP-selectivity. Selectivity is defined as the IC₅₀ in SNP-mismatched cells divided by the IC₅₀ in SNP-matched cells. The vertical dashed lines indicate potencies estimated at 0.27 and 19 μM on matched and mismatched alleles, respectively, resulting in 70-fold selectivity. b-d) Potency and selectivity evaluated at day 10 are plotted for rs715, rs223, and rs206 ASOs from the initial library. ASOs targeting the C and T allele of a given SNP are shown as red and black dots, respectively. ASOs with selectivities >50-fold were all fixed at the same level on the y-axis. In the rs715 ASO plot b), the three diamond symbols indicate the ASOs selected for redesigns.

FIG. 3. Evaluation of SNP-selective ASOs from redesign library targeting the rs715 SNP. Potency and selectivity evaluated in a) human myoblast CC-2580 cells and b) human iPSC-derived cardiomyocytes. ASOs targeting the C and T allele of a given SNP are shown as red and black dots, respectively. Dots in pink and grey are parent ASOs from the initial library. The five diamond symbols indicate the ASOs selected for evaluation in mice. c) Correlation between potencies in CC-2580 cells and iPSC-CM cells. Significance of the correlation was determined by Spearman's rank correlation test.

FIG. 4. Time course study of SNP-selective knockdown. mRNA knockdown in human iPSC-derived cardiomyocytes was evaluated at six time points over a two-week period using allele-specific droplet digital PCR. At day 0, 250 nM of rs715-T targeting ASO A259 was added via gymnosis. SNP-matched knockdown is seen for up to two weeks, while the SNP-mismatched allele does not show knockdown. Data were normalized to the no ASO day 2 time point for each allele. Mean+/−SD from three independent experiments is shown. Significance between T alleles (no ASO vs ASO) was determined by two-way ANOVA followed by Sidak's multiple comparisons test (*p<0.05, ***p<0.001).

FIG. 5. Study of SNP-selective knockdown in mice. a) Allele-specific mRNA quantitation from mouse LV one week following ASO dosing (*p<0.05, **p<0.01, ***p<0.001 comparing C and T allele abundance within a group as determined by t-test). All five compounds significantly reduce the C allele compared to the T allele. Two compounds give significant knockdown of the C allele compared to the C allele in the saline group (^###p<0.001 comparing to saline C allele as determined by one-way ANOVA followed by Dunnett's multiple comparisons test). b) ASO concentrations in heart (LV), liver, and kidney. On average, ASO concentration is 37× higher in kidney and 16× higher in liver compared to heart.

FIG. 6. a) 46 LNA gapmer ASOs were designed to various regions of the human MYH7 transcript (i.e. not SNP targeting). A subset of ASOs show robust knockdown at a concentration of 5 uM in 8220 myoblasts, establishing proof of concept that ASOs could be used to reduce MYH7 mRNA levels in vitro. A positive control ASO (S17) was identified from this initial dataset. b) The S17 positive control ASO shows similar knockdown at 5 uM in both 8220 and NH10 human skeletal muscle myoblasts, the two SNP homozygous cell lines used in the QuantiGene screen. This result suggests similar ASO uptake between the cell lines.

FIG. 7a. 102 ASOs were redesigned based on the A250 sequence, which targets the rs715-C allele (TCagcttggcgatgATCT; LNA uppercase, DNA lowercase). The primary sequence was maintained, but the distribution of LNA and DNA bases was varied. SNP-matched (C allele) and SNP-mismatched (T allele) knockdown at 0.5 uM is shown. Data points lying above the dotted line indicate stronger C allele knockdown. A250 data is shown with a larger black circle.

FIG. 7b. 162 ASOs were redesigned based on the A270 sequence, which targets the rs715-T allele (CTtggcaatgatctcATCC; LNA uppercase, DNA lowercase). The primary sequence was maintained, but the distribution of LNA and DNA bases was varied. SNP-matched (T allele) and SNP-mismatched (C allele) knockdown at 0.5 uM is shown. Data points lying below the dotted line indicate stronger T allele knockdown. A270 data is shown with a larger black circle.

FIG. 7c. ASOs were redesigned based on the A249 sequence, which targets the rs715-C allele (CAGcttggcgatgatCT; LNA uppercase, DNA lowercase). The primary sequence was maintained, but the distribution of LNA and DNA bases was varied. SNP-matched (C allele) and SNP-mismatched (T allele) knockdown at 0.5 uM is shown. Data points lying above the dotted line indicate stronger C allele knockdown. A249 data is shown with a larger black circle.

FIG. 8. Quantification of β-MHC in iCell2 hiPSC-CM with and without addition of A259 (rs715-T targeting ASO). β-MHC is not reduced at any timepoint, suggesting protein compensation by the SNP-mismatched allele. Bar graphs from n=3 independent experiments. Protein levels were normalized to the no ASO group at each timepoint.

FIG. 9. A small section of human MYH7 containing the rs715-C SNP and flanking sequence was inserted into one allele of the mouse Myh6 gene. The SNP base is shown in green. The other Myh6 allele was unchanged. This insertion of human sequence did not affect amino acid sequence. Five ASOs targeting the rs715-C allele were tested in these partially humanized mice. Because of the presence of an additional mismatch between human MYH7 and mouse Myh6 near the SNP position, all ASOs have two basepair mismatches between their template sequence and the WT Myh6 sequence.

FIG. 10. Weights and clinical chemistry following MYH7 ASO administration. No differences were seen in body weight change or heart weight/body weight. No difference was seen in the kidney injury marker BUN, but ASO B44 caused increased creatinine compared to vehicle. Liver injury markers (ALT, AST, AlkPhos) were increased following B44 and B56 dosing. *p<0.05, **p<0.01, ***p<0.001 compared to vehicle using one-way ANOVA and Dunnett's multiple comparisons test.

DEFINITIONS

Oligonucleotide

The term “oligonucleotide” as used herein is defined as it is generally understood by the skilled person as a molecule comprising two or more covalently linked nucleosides. Such covalently bound nucleosides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are commonly made in the laboratory by solid-phase chemical synthesis followed by purification and isolation. When referring to a sequence of the oligonucleotide, reference is made to the sequence or order of nucleobase moieties, or modifications thereof, of the covalently linked nucleotides or nucleosides. The oligonucleotide of the invention is man-made, and is chemically synthesized, and is typically purified or isolated. The oligonucleotide of the invention may comprise one or more modified nucleosides or nucleotides, such as 2′ sugar modified nucleosides.

Antisense Oligonucleotides

The term “Antisense oligonucleotide” as used herein is defined as oligonucleotides capable of modulating expression of a target gene by hybridizing to a target nucleic acid, in particular to a contiguous sequence on a target nucleic acid. The antisense oligonucleotides are not essentially double stranded and are therefore not siRNAs or shRNAs. Preferably, the antisense oligonucleotides of the present invention are single stranded. It is understood that single stranded oligonucleotides of the present invention can form hairpins or intermolecular duplex structures (duplex between two molecules of the same oligonucleotide), as long as the degree of intra or inter self complementarity is less than 50% across of the full length of the oligonucleotide.

Advantageously, the single stranded antisense oligonucleotide of the invention does not contain RNA nucleosides, since this will decrease nuclease resistance.

Advantageously, the antisense oligonucleotide of the invention comprises one or more modified nucleosides or nucleotides, such as 2′ sugar modified nucleosides. Furthermore, it is advantageous that the nucleosides which are not modified are DNA nucleosides.

Contiguous Nucleotide Sequence

The term “contiguous nucleotide sequence” refers to the region of the oligonucleotide which is complementary to the target nucleic acid. The term is used interchangeably herein with the term “contiguous nucleobase sequence” and the term “oligonucleotide motif sequence”. In some embodiments all the nucleotides of the oligonucleotide constitute the contiguous nucleotide sequence. In some embodiments the oligonucleotide comprises the contiguous nucleotide sequence, such as a F-G-F′ gapmer region, and may optionally comprise further nucleotide(s), for example a nucleotide linker region which may be used to attach a functional group to the contiguous nucleotide sequence. The nucleotide linker region may or may not be complementary to the target nucleic acid.

Nucleotides

Nucleotides are the building blocks of oligonucleotides and polynucleotides, and for the purposes of the present invention include both naturally occurring and non-naturally occurring nucleotides. In nature, nucleotides, such as DNA and RNA nucleotides comprise a ribose sugar moiety, a nucleobase moiety and one or more phosphate groups (which is absent in nucleosides). Nucleosides and nucleotides may also interchangeably be referred to as “units” or “monomers”.

Modified Nucleoside

The term “modified nucleoside” or “nucleoside modification” as used herein refers to nucleosides modified as compared to the equivalent DNA or RNA nucleoside by the introduction of one or more modifications of the sugar moiety or the (nucleo)base moiety. In a preferred embodiment the modified nucleoside comprise a modified sugar moiety. The term modified nucleoside may also be used herein interchangeably with the term “nucleoside analogue” or modified “units” or modified “monomers”. Nucleosides with an unmodified DNA or RNA sugar moiety are termed DNA or RNA nucleosides herein. Nucleosides with modifications in the base region of the DNA or RNA nucleoside are still generally termed DNA or RNA if they allow Watson Crick base pairing.

Modified Internucleoside Linkages

The term “modified internucleoside linkage” is defined as generally understood by the skilled person as linkages other than phosphodiester (PO) linkages, that covalently couples two nucleosides together. The oligonucleotides of the invention may therefore comprise modified internucleoside linkages. In some embodiments, the modified internucleoside linkage increases the nuclease resistance of the oligonucleotide compared to a phosphodiester linkage. For naturally occurring oligonucleotides, the internucleoside linkage includes phosphate groups creating a phosphodiester bond between adjacent nucleosides. Modified internucleoside linkages are particularly useful in stabilizing oligonucleotides for in vivo use, and may serve to protect against nuclease cleavage at regions of DNA or RNA nucleosides in the oligonucleotide of the invention, for example within the gap region of a gapmer oligonucleotide, as well as in regions of modified nucleosides, such as region F and F′.

In an embodiment, the oligonucleotide comprises one or more internucleoside linkages modified from the natural phosphodiester, such one or more modified internucleoside linkages that is for example more resistant to nuclease attack. Nuclease resistance may be determined by incubating the oligonucleotide in blood serum or by using a nuclease resistance assay (e.g. snake venom phosphodiesterase (SVPD)), both are well known in the art. Internucleoside linkages which are capable of enhancing the nuclease resistance of an oligonucleotide are referred to as nuclease resistant internucleoside linkages. In some embodiments at least 50% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are modified, such as at least 60%, such as at least 70%, such as at least 80 or such as at least 90% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are nuclease resistant internucleoside linkages. In some embodiments all of the internucleoside linkages of the oligonucleotide, or contiguous nucleotide sequence thereof, are nuclease resistant internucleoside linkages. It will be recognized that, in some embodiments the nucleosides which link the oligonucleotide of the invention to a non-nucleotide functional group, such as a conjugate, may be phosphodiester.

A preferred modified internucleoside linkage is phosphorothioate.

Phosphorothioate internucleoside linkages are particularly useful due to nuclease resistance, beneficial pharmacokinetics and ease of manufacture. In some embodiments at least 50% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are phosphorothioate, such as at least 60%, such as at least 70%, such as at least 80% or such as at least 90% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are phosphorothioate. In some embodiments all of the internucleoside linkages of the oligonucleotide, or contiguous nucleotide sequence thereof, are phosphorothioate.

Nuclease resistant linkages, such as phosphorothioate linkages, are particularly useful in oligonucleotide regions capable of recruiting nuclease when forming a duplex with the target nucleic acid, such as region G for gapmers. Phosphorothioate linkages may, however, also be useful in non-nuclease recruiting regions and/or affinity enhancing regions such as regions F and F′ for gapmers. Gapmer oligonucleotides may, in some embodiments comprise one or more phosphodiester linkages in region F or F′, or both region F and F′, which the internucleoside linkage in region G may be fully phosphorothioate.

Advantageously, all the internucleoside linkages in the contiguous nucleotide sequence of the oligonucleotide are phosphorothioate linkages.

It is recognized that, as disclosed in EP2 742 135, antisense oligonucleotide may comprise other internucleoside linkages (other than phosphodiester and phosphorothioate), for example alkyl phosphonate/methyl phosphonate internucleosides, which according to EP2 742 135 may for example be tolerated in an otherwise DNA phosphorothioate the gap region.

Nucleobase

The term nucleobase includes the purine (e.g. adenine and guanine) and pyrimidine (e.g. uracil, thymine and cytosine) moiety present in nucleosides and nucleotides which form hydrogen bonds in nucleic acid hybridization. In the context of the present invention the term nucleobase also encompasses modified nucleobases which may differ from naturally occurring nucleobases, but are functional during nucleic acid hybridization. In this context “nucleobase” refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine and hypoxanthine, as well as non-naturally occurring variants. Such variants are for example described in Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1.

In a some embodiments the nucleobase moiety is modified by changing the purine or pyrimidine into a modified purine or pyrimidine, such as substituted purine or substituted pyrimidine, such as a nucleobased selected from isocytosine, pseudoisocytosine, 5-methyl cytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil 5-thiazolo-uracil, 2-thio-uracil, 2′thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine and 2-chloro-6-aminopurine.

The nucleobase moieties may be indicated by the letter code for each corresponding nucleobase, e.g. A, T, G, C or U, wherein each letter may optionally include modified nucleobases of equivalent function. For example, in the exemplified oligonucleotides, the nucleobase moieties are selected from A, T, G, C, and 5-methyl cytosine. Optionally, for LNA gapmers, 5-methyl cytosine LNA nucleosides may be used.

Modified Oligonucleotide

The term modified oligonucleotide describes an oligonucleotide comprising one or more sugar-modified nucleosides and/or modified internucleoside linkages. The term chimeric” oligonucleotide is a term that has been used in the literature to describe oligonucleotides with modified nucleosides.

Complementarity

The term “complementarity” describes the capacity for Watson-Crick base-pairing of nucleosides/nucleotides. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)—thymine (T)/uracil (U). It will be understood that oligonucleotides may comprise nucleosides with modified nucleobases, for example 5-methyl cytosine is often used in place of cytosine, and as such the term complementarity encompasses Watson Crick base-paring between non-modified and modified nucleobases (see for example Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1).

The term “% complementary” as used herein, refers to the proportion of nucleotides (in percent) of a contiguous nucleotide sequence in a nucleic acid molecule (e.g. oligonucleotide) which across the contiguous nucleotide sequence, are complementary to a reference sequence (e.g. a target sequence or sequence motif). The percentage of complementarity is thus calculated by counting the number of aligned nucleobases that are complementary (from Watson Crick base pair) between the two sequences (when aligned with the target sequence 5′-3′ and the oligonucleotide sequence from 3′-5′), dividing that number by the total number of nucleotides in the oligonucleotide and multiplying by 100. In such a comparison a nucleobase/nucleotide which does not align (form a base pair) is termed a mismatch. Insertions and deletions are not allowed in the calculation of % complementarity of a contiguous nucleotide sequence. It will be understood that in determining complementarity, chemical modifications of the nucleobases are disregarded as long as the functional capacity of the nucleobase to form Watson Crick base pairing is retained (e.g. 5′-methyl cytosine is considered identical to a cytosine for the purpose of calculating % identity).

The term “fully complementary”, refers to 100% complementarity.

Identity

The term “Identity” as used herein, refers to the proportion of nucleotides (expressed in percent) of a contiguous nucleotide sequence in a nucleic acid molecule (e.g. oligonucleotide) which across the contiguous nucleotide sequence, are identical to a reference sequence (e.g. a sequence motif). The percentage of identity is thus calculated by counting the number of aligned nucleobases that are identical (a Match) between two sequences (in the contiguous nucleotide sequence of the compound of the invention and in the reference sequence), dividing that number by the total number of nucleotides in the oligonucleotide and multiplying by 100. Therefore, Percentage of Identity=(Matches×100)/Length of aligned region (e.g. the contiguous nucleotide sequence). Insertions and deletions are not allowed in the calculation the percentage of identity of a contiguous nucleotide sequence. It will be understood that in determining identity, chemical modifications of the nucleobases are disregarded as long as the functional capacity of the nucleobase to form Watson Crick base pairing is retained (e.g. 5-methyl cytosine is considered identical to a cytosine for the purpose of calculating % identity).

Hybridization

The term “hybridizing” or “hybridizes” as used herein is to be understood as two nucleic acid strands (e.g. an oligonucleotide and a target nucleic acid) forming hydrogen bonds between base pairs on opposite strands thereby forming a duplex. The affinity of the binding between two nucleic acid strands is the strength of the hybridization. It is often described in terms of the melting temperature (T_m) defined as the temperature at which half of the oligonucleotides are duplexed with the target nucleic acid. At physiological conditions T_m is not strictly proportional to the affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515-537). The standard state Gibbs free energy ΔG° is a more accurate representation of binding affinity and is related to the dissociation constant (K_d) of the reaction by ΔG°=−RTIn(K_d), where R is the gas constant and T is the absolute temperature. Therefore, a very low ΔG° of the reaction between an oligonucleotide and the target nucleic acid reflects a strong hybridization between the oligonucleotide and target nucleic acid. ΔG° is the energy associated with a reaction where aqueous concentrations are 1M, the pH is 7, and the temperature is 37° C. The hybridization of oligonucleotides to a target nucleic acid is a spontaneous reaction and for spontaneous reactions ΔG° is less than zero. ΔG° can be measured experimentally, for example, by use of the isothermal titration calorimetry (ITC) method as described in Hansen et al., 1965, Chem. Comm. 36-38 and Holdgate et al., 2005, Drug Discov Today. The skilled person will know that commercial equipment is available for ΔG° measurements. ΔG° can also be estimated numerically by using the nearest neighbor model as described by SantaLucia, 1998, Proc Natl Acad Sci USA. 95: 1460-1465 using appropriately derived thermodynamic parameters described by Sugimoto et al., 1995, Biochemistry 34:11211-11216 and McTigue et al., 2004, Biochemistry 43:5388-5405. In order to have the possibility of modulating its intended nucleic acid target by hybridization, oligonucleotides of the present invention hybridize to a target nucleic acid with estimated ΔG° values below −10 kcal for oligonucleotides that are 10-30 nucleotides in length. In some embodiments the degree or strength of hybridization is measured by the standard state Gibbs free energy ΔG°. The oligonucleotides may hybridize to a target nucleic acid with estimated ΔG° values below the range of −10 kcal, such as below −15 kcal, such as below −20 kcal and such as below −25 kcal for oligonucleotides that are 8-30 nucleotides in length. In some embodiments the oligonucleotides hybridize to a target nucleic acid with an estimated ΔG° value of −10 to −60 kcal, such as −12 to −40, such as from −15 to −30 kcal or −16 to −27 kcal such as −18 to −25 kcal.

Target Nucleic Acid

According to the present invention, the target nucleic acid is a nucleic acid which encodes human MYH7 and may for example be a gene, a RNA, a mRNA, and pre-mRNA, a mature mRNA or a cDNA sequence. The target may therefore be referred to as an MYH7 target nucleic acid. In some embodiments, the target nucleic acid is selected from the group consisting of SEQ ID NO: 1, and SEQ ID NO 2, or naturally occurring variants thereof (e.g. sequences encoding a human MYH7 protein. In some embodiments, the target nucleic acid is an allelic variant of the human MYH7 transcript. In some embodiment

In some embodiments the target nucleic acid is a MYH7 allelic variant which comprises a polymorphism in at a position of the human MYH7 transcript selected from rs223, rs206 and rs715.

In some embodiments the polymorphism is selected from rs223T or rs223C.

In some embodiments the polymorphism is selected from rs206C or rs206T.

In some embodiments the polymorphism is selected from rs715C or rs715T.

In some embodiments the oligonucleotide on the invention selectively inhibits the target nucleic acid as compared to an alternative allelic variant of the target nucleic acid. The target nucleic acid and the alternative allelic variant comprise a single nucleotide polymorphism within the region which is complementary to the oligonucleotide of the invention or contiguous nucleotide sequence thereof. Selective inhibition refers to a higher inhibitory activity (higher potency) against the target nucleic acid as compared to the allelic variant. Selective inhibition can be determined in vitro (IC50) or in vivo (e.g. ED50).

If employing the oligonucleotide of the invention in research or diagnostics the target nucleic acid may be a cDNA or a synthetic nucleic acid derived from DNA or RNA.

For in vivo or in vitro application, the oligonucleotide of the invention is typically capable of inhibiting the expression of the MYH7 target nucleic acid in a cell which is expressing the MYH7 target nucleic acid. The contiguous sequence of nucleobases of the oligonucleotide of the invention is typically complementary to the MYH7 target nucleic acid, as measured across the length of the oligonucleotide, optionally with the exception of one or two mismatches, and optionally excluding nucleotide based linker regions which may link the oligonucleotide to an optional functional group such as a conjugate, or other non-complementary terminal nucleotides (e.g. region D′ or D″). The target nucleic acid may, in some embodiments, be a RNA or DNA, such as a messenger RNA, such as a mature mRNA or a pre-mRNA. In some embodiments the target nucleic acid is a RNA or DNA which encodes mammalian MYH7 protein, such as human MYH7, e.g. the human MYH7 mRNA sequence, such as that disclosed as SEQ ID NO 1 or 2.

TABLE 1
Genome and assembly information for MYH7.
	NCBI reference
	Genomic coordinates		sequence* accession
Species	Chr.	Strand	Start	End	Assembly	number for mRNA
Human	14	Rv	23412738	23435718	GRCh38	NM_000257
Fwd = forward strand. Rv = reverse strand. The genome coordinates provide the pre-mRNA sequence (genomic sequence). The NCBI reference provides the mRNA sequence (cDNA sequence).
*The National Center for Biotechnology Information reference sequence database is a comprehensive, integrated, non-redundant, well-annotated set of reference sequences including genomic, transcript, and protein. It is hosted at www.ncbi.nlm.nih.gov/refseq.

TABLE 2
Sequence details for human MYH7.
	Species	RNA type	Length (nt)	SEQ ID NO
	Human	premRNA	1
	Human	mRNA	2

Target Sequence

The term “target sequence” as used herein refers to a sequence of nucleotides present in the target nucleic acid which comprises the nucleobase sequence which is complementary to the oligonucleotide of the invention. In some embodiments, the target sequence consists of a region on the target nucleic acid with a nucleobase sequence that is complementary to the contiguous nucleotide sequence of the oligonucleotide of the invention. This region of the target nucleic acid may interchangeably be referred to as the target nucleotide sequence, target sequence or target region. In some embodiments the target sequence is longer than the complementary sequence of a single oligonucleotide, and may, for example represent a preferred region of the target nucleic acid which may be targeted by several oligonucleotides of the invention.

In some embodiments the target sequence is a sequence selected from the group consisting of SEQ ID NO 3-10.

TABLE 3
Human MYH7 SNP regions.
SEQ ID NO	SNP ID	Sequence	Allele
3	rs223-t	AGAAAAGCTGAAGCTAGAGTGTTGAAAATCTAGTAAGAC	REF
4	rs223-c	AGAAAAGCTGAAGCTAGAGCGTTGAAAATCTAGTAAGAC	ALT
5	rs206-c	GCAAAGTCACTGCCGAGACCGAGTATGGCAAGACAGTGA	REF
6	rs206-t	GCAAAGTCACTGCCGAGACTGAGTATGGCAAGACAGTGA	ALT
7	rs206-c-pre	GCAAAGTCACTGCCGAGACCGAGTATGGCAAGGTGGGTG	REF
8	rs206-t-pre	GCAAAGTCACTGCCGAGACTGAGTATGGCAAGGTGGGTG	ALT
9	rs715-t	CTGGGCTGGATGAGATCATTGCCAAGCTGACCAAGGAGA	REF
10	rs715-c	CTGGGCTGGATGAGATCATCGCCAAGCTGACCAAGGAGA	ALT
The SNP positions are underlined.
REF = Reference. ALT = Alternative.

The bold underlined residue identifies a single nucleotide polymorphism (SNP) which the oligonucleotides of the invention may target (either the REF or ALT may be present in the target nucleic acid) REF refers to the designated wildtype allele of the highlighted SNP, ALT refers to an allelic variant. The respective location of SEQ ID NO 3-10 on the human MYH7 transcript sequences SEQ ID NO 1 or 2 are illustrated in table 4:

TABLE 4
Positions of human MYH7 SNP regions in the MYH7 mRNA and pre-mRNA
		SEQ ID NO 1	SEQ ID NO 1	SEQ ID NO 2	SEQ ID NO 2
SEQ ID NO	SNP ID	start	end	start	end
3	rs223-t	1529	1567
4	rs223-c	1529	1567
5	rs206-c			301	339
6	rs206-t			301	339
7	rs206-c-pre	2156	2194
8	rs206-t-pre	2156	2194
9	rs715-t	12021	12059	3079	3117
10	rs715-c	12021	12059	3079	3117

The oligonucleotide of the invention comprises a contiguous nucleotide sequence which is complementary to or hybridizes to the target nucleic acid, such as a target sequence described herein, such as a sequence selected from the group consisting of SEQ ID NO 1-10.

The oligonucleotide of the invention comprises a contiguous nucleotide sequence which is complementary to or hybridizes to the target nucleic acid, such as a target sequence described herein, such as a sequence selected from the group consisting of SEQ ID NOs 3 and 4.

The oligonucleotide of the invention comprises a contiguous nucleotide sequence which is complementary to or hybridizes to the target nucleic acid, such as a target sequence described herein, such as a sequence selected from the group consisting of SEQ ID NOs 5 and 6.

The oligonucleotide of the invention comprises a contiguous nucleotide sequence which is complementary to or hybridizes to the target nucleic acid, such as a target sequence described herein, such as a sequence selected from the group consisting of SEQ ID NOs 7-10.

The target sequence to which the oligonucleotide is complementary or hybridizes to generally comprises a contiguous nucleobases sequence of at least 10 nucleotides. The contiguous nucleotide sequence is between 10 to 40 nucleotides, such as 12 to 30, such as 14 to 20, such as 15 to 18 contiguous nucleotides.

Target Cell

The term a “target cell” as used herein refers to a cell which is expressing the target nucleic acid. In some embodiments the target cell may be in vivo or in vitro. In some embodiments the target cell is a mammalian cell such as a human cell. For experimental purposes, the target call may be an animal cell such as a mouse cell which is heterologously expressing the target nucleic acid.

In preferred embodiments the target cell expresses the target nucleic acid MYH7 mRNA, such as the MYH7 pre-mRNA or MYH7 mature mRNA. The poly A tail of MYH7 mRNA is typically disregarded for antisense oligonucleotide targeting.

Naturally Occurring Variant

The term “naturally occurring variant” refers to variants of MYH7 gene or transcripts which originate from the same genetic loci as the target nucleic acid, but may differ for example, by virtue of degeneracy of the genetic code causing a multiplicity of codons encoding the same amino acid, or due to alternative splicing of pre-mRNA, or the presence of polymorphisms, such as single nucleotide polymorphisms (SNPs), and allelic variants. Based on the presence of the sufficient complementary sequence to the oligonucleotide, the oligonucleotide of the invention may therefore target the target nucleic acid and naturally occurring variants thereof.

In some embodiments, the naturally occurring variants have at least 95% such as at least 98% or at least 99% homology or 100% homologous to a mammalian MYH7 target nucleic acid, such as a target nucleic acid selected form the group consisting of SEQ ID NO 1 or SEQ ID NO 2, or a target nucleic acid sequence selected from the group consisting of SEQ ID No 3-10. In some embodiments the naturally occurring variants have at least 99% homology to the human MYH7 target nucleic acid of SEQ ID NO: 1. In some embodiments the naturally occurring variants are the polymorphisms listed in table 3 or 4.

Selectivity

In some aspects it is advantageous that the compounds of the invention have a higher or lower potency against the expression of one allelic variant of MYH7 as compared to the wildtype MYH7 (e.g. SEQ ID NO 1 or 2), for example the allelic variants listed in table 3.

In some aspects it is advantageous that the compounds of the invention have a higher or lower potency against the expression of one allelic variant of MYH7 rs206T as compared to the wildtype MYH7 rs206C.

In some aspects it is advantageous that the compounds of the invention have a higher or lower potency against the expression of one allelic variant of MYH7 rs223C as compared to the wildtype MYH7 rs223T.

In some aspects it is advantageous that the compounds of the invention have a higher or lower potency against the expression of one allelic variant of MYH7 rs715C as compared to the wildtype MYH7 rs715T.

As illustrated in the examples selective inhibition may be determined in vitro in cell lines which are expressing both MYH7 alleles, or in separate cell lines which are each expressing one of the MYH7 allele variants.

Modulation of Expression

The term “modulation of expression” as used herein is to be understood as an overall term for an oligonucleotide's ability to alter the amount of MYH7 when compared to the amount of MYH7 before administration of the oligonucleotide. Alternatively modulation of expression may be determined by reference to a control experiment. It is generally understood that the control is an individual or target cell treated with a saline composition or an individual or target cell treated with a non-targeting oligonucleotide (mock). It may however also be an individual treated with the standard of care.

One type of modulation is the ability of an oligonucleotide to inhibit, down-regulate, reduce, suppress, remove, stop, block, prevent, lessen, lower, avoid or terminate expression of MYH7, e.g. by degradation of mRNA or blockage of transcription.

High Affinity Modified Nucleosides

A high affinity modified nucleoside is a modified nucleotide which, when incorporated into the oligonucleotide enhances the affinity of the oligonucleotide for its complementary target, for example as measured by the melting temperature (T^m). A high affinity modified nucleoside of the present invention preferably result in an increase in melting temperature between +0.5 to +12° C., more preferably between +1.5 to +10° C. and most preferably between +3 to +8° C. per modified nucleoside. Numerous high affinity modified nucleosides are known in the art and include for example, many 2′ substituted nucleosides as well as locked nucleic acids (LNA) (see e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213).

Sugar Modifications

The oligomer of the invention may comprise one or more nucleosides which have a modified sugar moiety, i.e. a modification of the sugar moiety when compared to the ribose sugar moiety found in DNA and RNA.

Numerous nucleosides with modification of the ribose sugar moiety have been made, primarily with the aim of improving certain properties of oligonucleotides, such as affinity and/or nuclease resistance.

Such modifications include those where the ribose ring structure is modified, e.g. by replacement with a hexose ring (HNA), or a bicyclic ring, which typically have a biradicle bridge between the C2 and C4 carbons on the ribose ring (LNA), or an unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons (e.g. UNA). Other sugar modified nucleosides include, for example, bicyclohexose nucleic acids (WO2011/017521) or tricyclic nucleic acids (WO2013/154798). Modified nucleosides also include nucleosides where the sugar moiety is replaced with a non-sugar moiety, for example in the case of peptide nucleic acids (PNA), or morpholino nucleic acids.

Sugar modifications also include modifications made via altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2′-OH group naturally found in DNA and RNA nucleosides. Substituents may, for example be introduced at the 2′, 3′, 4′ or 5′ positions.

2′ Sugar Modified Nucleosides

A 2′ sugar modified nucleoside is a nucleoside which has a substituent other than H or —OH at the 2′ position (2′ substituted nucleoside) or comprises a 2′ linked biradicle capable of forming a bridge between the 2′ carbon and a second carbon in the ribose ring, such as LNA (2′-4′ biradicle bridged) nucleosides.

Indeed, much focus has been spent on developing 2′ sugar substituted nucleosides, and numerous 2′ substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides. For example, the 2′ modified sugar may provide enhanced binding affinity and/or increased nuclease resistance to the oligonucleotide. Examples of 2′ substituted modified nucleosides are 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA (MOE), 2′-amino-DNA, 2′-Fluoro-RNA, and 2′-F-ANA nucleoside. For further examples, please see e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, and Deleavey and Damha, Chemistry and Biology 2012, 19, 937. Below are illustrations of some 2′ substituted modified nucleosides.

In relation to the present invention 2′ substituted sugar modified nucleosides does not include 2′ bridged nucleosides like LNA.

Locked Nucleic Acids (LNA)

A “LNA nucleoside” is a 2′-modified nucleoside which comprises a biradical linking the C2′ and C4′ of the ribose sugar ring of said nucleoside (also referred to as a “2′-4′ bridge”), which restricts or locks the conformation of the ribose ring. These nucleosides are also termed bridged nucleic acid or bicyclic nucleic acid (BNA) in the literature. The locking of the conformation of the ribose is associated with an enhanced affinity of hybridization (duplex stabilization) when the LNA is incorporated into an oligonucleotide for a complementary RNA or DNA molecule. This can be routinely determined by measuring the melting temperature of the oligonucleotide/complement duplex.

Non limiting, exemplary LNA nucleosides are disclosed in WO 99/014226, WO 00/66604, WO 98/039352 , WO 2004/046160, WO 00/047599, WO 2007/134181, WO 2010/077578, WO 2010/036698, WO 2007/090071, WO 2009/006478, WO 2011/156202, WO 2008/154401, WO 2009/067647, WO 2008/150729, Morita et al., Bioorganic & Med. Chem. Lett. 12, 73-76, Seth et al. J. Org. Chem. 2010, Vol 75(5) pp. 1569-81, and Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238, and Wan and Seth, J. Medical Chemistry 2016, 59, 9645-9667. Further non limiting, exemplary LNA nucleosides are disclosed in Scheme 1.

Scheme 1:

Particular LNA nucleosides are beta-D-oxy-LNA, 6′-methyl-beta-D-oxy LNA such as (S)-6′-methyl-beta-D-oxy-LNA (ScET) and ENA.

A particularly advantageous LNA is beta-D-oxy-LNA.

RNase H Activity and Recruitment

The RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when in a duplex with a complementary RNA molecule. WO01/23613 provides in vitro methods for determining RNaseH activity, which may be used to determine the ability to recruit RNaseH. Typically an oligonucleotide is deemed capable of recruiting RNase H if it, when provided with a complementary target nucleic acid sequence, has an initial rate, as measured in pmol/l/min, of at least 5%, such as at least 10% or more than 20% of the of the initial rate determined when using a oligonucleotide having the same base sequence as the modified oligonucleotide being tested, but containing only DNA monomers with phosphorothioate linkages between all monomers in the oligonucleotide, and using the methodology provided by Example 91-95 of WO01/23613 (hereby incorporated by reference). For use in determining RHase H activity, recombinant human RNase H1 is available from Lubio Science GmbH, Lucerne, Switzerland.

Gapmer

The antisense oligonucleotide of the invention, or contiguous nucleotide sequence thereof may be a gapmer. The antisense gapmers are commonly used to inhibit a target nucleic acid via RNase H mediated degradation. A gapmer oligonucleotide comprises at least three distinct structural regions a 5′-flank, a gap and a 3′-flank, F-G-F′ in the ‘5→3’ orientation. The “gap” region (G) comprises a stretch of contiguous DNA nucleotides which enable the oligonucleotide to recruit RNase H. The gap region is flanked by a 5′ flanking region (F) comprising one or more sugar modified nucleosides, advantageously high affinity sugar modified nucleosides, and by a 3′ flanking region (F′) comprising one or more sugar modified nucleosides, advantageously high affinity sugar modified nucleosides. The one or more sugar modified nucleosides in region F and F′ enhance the affinity of the oligonucleotide for the target nucleic acid (i.e. are affinity enhancing sugar modified nucleosides). In some embodiments, the one or more sugar modified nucleosides in region F and F′ are 2′ sugar modified nucleosides, such as high affinity 2′ sugar modifications, such as independently selected from LNA and 2′-MOE.

In a gapmer design, the 5′ and 3′ most nucleosides of the gap region are DNA nucleosides, and are positioned adjacent to a sugar modified nucleoside of the 5′ (F) or 3′ (F′) region respectively. The flanks may further defined by having at least one sugar modified nucleoside at the end most distant from the gap region, i.e. at the 5′ end of the 5′ flank and at the 3′ end of the 3′ flank. Regions F-G-F′ form a contiguous nucleotide sequence. Antisense oligonucleotides of the invention, or the contiguous nucleotide sequence thereof, may comprise a gapmer region of formula F-G-F′.

The overall length of the gapmer design F-G-F′ may be, for example 12 to 32 nucleosides, such as 13 to 24, such as 14 to 22 nucleosides, Such as from 14 to17, such as 16 to18 nucleosides. By way of example, the gapmer oligonucleotide of the present invention can be represented by the following formulae:

F_1-8-G_5-16-F′_1-8, such as

F_1-8-G_7-16-F′_2-8

with the proviso that the overall length of the gapmer regions F-G-F′ is at least 12, such as at least 14 nucleotides in length.

Regions F, G and F′ are further defined below and can be incorporated into the F-G-F′ formula.

Gapmer—Region G

Region G (gap region) of the gapmer is a region of nucleosides which enables the oligonucleotide to recruit RNaseH, such as human RNase H1, typically DNA nucleosides. RNaseH is a cellular enzyme which recognizes the duplex between DNA and RNA, and enzymatically cleaves the RNA molecule. Suitably gapmers may have a gap region (G) of at least 5 or 6 contiguous DNA nucleosides, such as 5-16 contiguous DNA nucleosides, such as 6-15 contiguous DNA nucleosides, such as 7-14 contiguous DNA nucleosides, such as 8-12 contiguous DNA nucleotides, such as 8-12 contiguous DNA nucleotides in length. The gap region G may, in some embodiments consist of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 contiguous DNA nucleosides. One or more cytosine (C) DNA in the gap region may in some instances be methylated (e.g. when a DNA c is followed by a DNA g) such residues are either annotated as 5-methyl-cytosine (^meC). In some embodiments the gap region G may consist of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 contiguous phosphorothioate linked DNA nucleosides. In some embodiments, all internucleoside linkages in the gap are phosphorothioate linkages. Whilst traditional gapmers have a DNA gap region, there are numerous examples of modified nucleosides which allow for RNaseH recruitment when they are used within the gap region. Modified nucleosides which have been reported as being capable of recruiting RNaseH when included within a gap region include, for example, alpha-L-LNA, C4′ alkylated DNA (as described in PCT/EP2009/050349 and Vester et al., Bioorg. Med. Chem. Lett. 18 (2008) 2296-2300, both incorporated herein by reference), arabinose derived nucleosides like ANA and 2′F-ANA (Mangos et al. 2003 J. AM. CHEM. SOC. 125, 654-661), UNA (unlocked nucleic acid) (as described in Fluiter et al., Mol. Biosyst., 2009, 10, 1039 incorporated herein by reference). UNA is unlocked nucleic acid, typically where the bond between C2 and C3 of the ribose has been removed, forming an unlocked “sugar” residue. The modified nucleosides used in such gapmers may be nucleosides which adopt a 2′ endo (DNA like) structure when introduced into the gap region, i.e. modifications which allow for RNaseH recruitment). In some embodiments the DNA Gap region (G) described herein may optionally contain 1 to 3 sugar modified nucleosides which adopt a 2′ endo (DNA like) structure when introduced into the gap region.

Region G—“Gap-Breaker”

Alternatively, there are numerous reports of the insertion of a modified nucleoside which confers a 3′ endo conformation into the gap region of gapmers, whilst retaining some RNaseH activity. Such gapmers with a gap region comprising one or more 3′endo modified nucleosides are referred to as “gap-breaker” or “gap-disrupted” gapmers, see for example WO2013/022984. Gap-breaker oligonucleotides retain sufficient region of DNA nucleosides within the gap region to allow for RNaseH recruitment. The ability of gapbreaker oligonucleotide design to recruit RNaseH is typically sequence or even compound specific—see Rukov et al. 2015 Nucl. Acids Res. Vol. 43 pp. 8476-8487, which discloses “gapbreaker” oligonucleotides which recruit RNaseH which in some instances provide a more specific cleavage of the target RNA. Modified nucleosides used within the gap region of gap-breaker oligonucleotides may for example be modified nucleosides which confer a 3′endo confirmation, such 2′-O-methyl (OMe) or 2′-O-MOE (MOE) nucleosides, or beta-D LNA nucleosides (the bridge between C2′ and C4′ of the ribose sugar ring of a nucleoside is in the beta conformation), such as beta-D-oxy LNA or ScET nucleosides.

As with gapmers containing region G described above, the gap region of gap-breaker or gap-disrupted gapmers, have a DNA nucleosides at the 5′ end of the gap (adjacent to the 3′ nucleoside of region F), and a DNA nucleoside at the 3′ end of the gap (adjacent to the 5′ nucleoside of region F′). Gapmers which comprise a disrupted gap typically retain a region of at least 3 or 4 contiguous DNA nucleosides at either the 5′ end or 3′ end of the gap region. Exemplary designs for gap-breaker oligonucleotides include

F_1-8-[D_3-4-E₁-D_3-4]-F′_1-8

F_1-8-[D_1-4-E₁-D_3-4]-F′_1-8

F_1-8-[D_3-4-E₁-D_1-4]-F′_1-8

wherein region G is within the brackets [D_n-E_r-D_m], D is a contiguous sequence of DNA nucleosides, E is a modified nucleoside (the gap-breaker or gap-disrupting nucleoside), and F and F′ are the flanking regions as defined herein, and with the proviso that the overall length of the gapmer regions F-G-F′ is at least 12, such as at least 14 nucleotides in length. In some embodiments, region G of a gap disrupted gapmer comprises at least 6 DNA nucleosides, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 DNA nucleosides. As described above, the DNA nucleosides may be contiguous or may optionally be interspersed with one or more modified nucleosides, with the proviso that the gap region G is capable of mediating RNaseH recruitment.

Gapmer—Flanking Regions, F and F′

Region F is positioned immediately adjacent to the 5′ DNA nucleoside of region G. The 3′ most nucleoside of region F is a sugar modified nucleoside, such as a high affinity sugar modified nucleoside, for example a 2′ substituted nucleoside, such as a MOE nucleoside, or an LNA nucleoside.

Region F′ is positioned immediately adjacent to the 3′ DNA nucleoside of region G. The 5′ most nucleoside of region F′ is a sugar modified nucleoside, such as a high affinity sugar modified nucleoside, for example a 2′ substituted nucleoside, such as a MOE nucleoside, or an LNA nucleoside.

Region F is 1-8 contiguous nucleotides in length, such as 2-6, such as 3-4 contiguous nucleotides in length. Advantageously the 5′ most nucleoside of region F is a sugar modified nucleoside. In some embodiments the two 5′ most nucleoside of region F are sugar modified nucleoside. In some embodiments the 5′ most nucleoside of region F is an LNA nucleoside. In some embodiments the two 5′ most nucleoside of region F are LNA nucleosides. In some embodiments the two 5′ most nucleoside of region F are 2′ substituted nucleoside nucleosides, such as two 3′ MOE nucleosides. In some embodiments the 5′ most nucleoside of region F is a 2′ substituted nucleoside, such as a MOE nucleoside.

Region F′ is 2-8 contiguous nucleotides in length, such as 3-6, such as 4-5 contiguous nucleotides in length. Advantageously, embodiments the 3′ most nucleoside of region F′ is a sugar modified nucleoside. In some embodiments the two 3′ most nucleoside of region F′ are sugar modified nucleoside. In some embodiments the two 3′ most nucleoside of region F′ are LNA nucleosides. In some embodiments the 3′ most nucleoside of region F′ is an LNA nucleoside. In some embodiments the two 3′ most nucleoside of region F′ are 2′ substituted nucleoside nucleosides, such as two 3′ MOE nucleosides. In some embodiments the 3′ most nucleoside of region F′ is a 2′ substituted nucleoside, such as a MOE nucleoside.

It should be noted that when the length of region F or F′ is one, it is advantageously an LNA nucleoside.

In some embodiments, region F and F′ independently consists of or comprises a contiguous sequence of sugar modified nucleosides. In some embodiments, the sugar modified nucleosides of region F may be independently selected from 2′-O-alkyl-RNA units, 2′-O-methyl-RNA, 2′-amino-DNA units, 2′-fluoro-DNA units, 2′-alkoxy-RNA, MOE units, LNA units, arabino nucleic acid (ANA) units and 2′-fluoro-ANA units.

In some embodiments, region F and F′ independently comprises both LNA and a 2′ substituted modified nucleosides (mixed wing design).

In some embodiments, region F and F′ consists of only one type of sugar modified nucleosides, such as only MOE or only beta-D-oxy LNA or only ScET. Such designs are also termed uniform flanks or uniform gapmer design.

In some embodiments, all the nucleosides of region F or F′, or F and F′ are LNA nucleosides, such as independently selected from beta-D-oxy LNA, ENA or ScET nucleosides. In some embodiments region F consists of 1-5, such as 2-4, such as 3-4 such as 1, 2, 3, 4 or 5 contiguous LNA nucleosides. In some embodiments, all the nucleosides of region F and F′ are beta-D-oxy LNA nucleosides.

In some embodiments, all the nucleosides of region F or F′, or F and F′ are 2′ substituted nucleosides, such as OMe or MOE nucleosides. In some embodiments region F consists of 1, 2, 3, 4, 5, 6, 7, or 8 contiguous OMe or MOE nucleosides. In some embodiments only one of the flanking regions can consist of 2′ substituted nucleosides, such as OMe or MOE nucleosides. In some embodiments it is the 5′ (F) flanking region that consists 2′ substituted nucleosides, such as OMe or MOE nucleosides whereas the 3′ (F′) flanking region comprises at least one LNA nucleoside, such as beta-D-oxy LNA nucleosides or cET nucleosides. In some embodiments it is the 3′ (F′) flanking region that consists 2′ substituted nucleosides, such as OMe or MOE nucleosides whereas the 5′ (F) flanking region comprises at least one LNA nucleoside, such as beta-D-oxy LNA nucleosides or cET nucleosides.

In some embodiments, all the modified nucleosides of region F and F′ are LNA nucleosides, such as independently selected from beta-D-oxy LNA, ENA or ScET nucleosides, wherein region F or F′, or F and F′ may optionally comprise DNA nucleosides (an alternating flank, see definition of these for more details). In some embodiments, all the modified nucleosides of region F and F′ are beta-D-oxy LNA nucleosides, wherein region F or F′, or F and F′ may optionally comprise DNA nucleosides (an alternating flank, see definition of these for more details).

In some embodiments the 5′ most and the 3′ most nucleosides of region F and F′ are LNA nucleosides, such as beta-D-oxy LNA nucleosides or ScET nucleosides.

In some embodiments, the internucleoside linkage between region F and region G is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage between region F′ and region G is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkages between the nucleosides of region F or F′, F and F′ are phosphorothioate internucleoside linkages.

LNA Gapmer

An LNA gapmer is a gapmer wherein either one or both of region F and F′ comprises or consists of LNA nucleosides. A beta-D-oxy gapmer is a gapmer wherein either one or both of region F and F′ comprises or consists of beta-D-oxy LNA nucleosides.

In some embodiments the LNA gapmer is of formula: [LNA]_1-5-[region G]-[LNA]_1-5, wherein region G is as defined in the Gapmer region G definition.

MOE Gapmers

A MOE gapmers is a gapmer wherein regions F and F′ consist of MOE nucleosides. In some embodiments the MOE gapmer is of design [MOE]_1-8-[Region G]-[MOE]_1-8, such as [MOE]_2-7-[Region G]_5-16-[MOE]_2-7, such as [MOE]_3-6-[Region G]-[MOE]_3-6, wherein region G is as defined in the Gapmer definition. MOE gapmers with a 5-10-5 design (MOE-DNA-MOE) have been widely used in the art.

Mixed Wing Gapmer

A mixed wing gapmer is an LNA gapmer wherein one or both of region F and F′ comprise a 2′ substituted nucleoside, such as a 2′ substituted nucleoside independently selected from the group consisting of 2′-O-alkyl-RNA units, 2′-O-methyl-RNA, 2′-amino-DNA units, 2′-fluoro-DNA units, 2′-alkoxy-RNA, MOE units, arabino nucleic acid (ANA) units and 2′-fluoro-ANA units, such as a MOE nucleosides. In some embodiments wherein at least one of region F and F′, or both region F and F′ comprise at least one LNA nucleoside, the remaining nucleosides of region F and F′ are independently selected from the group consisting of MOE and LNA. In some embodiments wherein at least one of region F and F′, or both region F and F′ comprise at least two LNA nucleosides, the remaining nucleosides of region F and F′ are independently selected from the group consisting of MOE and LNA. In some mixed wing embodiments, one or both of region F and F′ may further comprise one or more DNA nucleosides. Mixed wing gapmer designs are disclosed in WO2008/049085 and WO2012/109395, both of which are hereby incorporated by reference.

Alternating Flank Gapmers

Oligonucleotides with alternating flanks are LNA gapmer oligonucleotides where at least one of the flanks (F or F′) comprises DNA in addition to the LNA nucleoside(s). In some embodiments at least one of region F or F′, or both region F and F′, comprise both LNA nucleosides and DNA nucleosides. In such embodiments, the flanking region F or F′, or both F and F′ comprise at least three nucleosides, wherein the 5′ and 3′ most nucleosides of the F and/or F′ region are LNA nucleosides.

In some embodiments at least one of region F or F′, or both region F and F′, comprise both LNA nucleosides and DNA nucleosides. In such embodiments, the flanking region F or F′, or both F and F′ comprise at least three nucleosides, wherein the 5′ and 3′ most nucleosides of the F or F′ region are LNA nucleosides, and there is at least one DNA nucleoside positioned between the 5′ and 3′ most LNA nucleosides of region F or F′ (or both region F and F′).

Region D′ or D″ in an Oligonucleotide

The oligonucleotide of the invention may in some embodiments comprise or consist of the contiguous nucleotide sequence of the oligonucleotide which is complementary to the target nucleic acid, such as the gapmer F-G-F′, and further 5′ and/or 3′ nucleosides. The further 5′ and/or 3′ nucleosides may or may not be fully complementary to the target nucleic acid. Such further 5′ and/or 3′ nucleosides may be referred to as region D′ and D″ herein.

The addition of region D′ or D″ may be used for the purpose of joining the contiguous nucleotide sequence, such as the gapmer, to a conjugate moiety or another functional group. When used for joining the contiguous nucleotide sequence with a conjugate moiety is can serve as a biocleavable linker. Alternatively it may be used to provide exonucleoase protection or for ease of synthesis or manufacture.

Region D′ and D″ can be attached to the 5′ end of region F or the 3′ end of region F′, respectively to generate designs of the following formulas D′-F-G-F′, F-G-F′-D″ or D′-F-G-F′-D″. In this instance the F-G-F′ is the gapmer portion of the oligonucleotide and region D′ or D″ constitute a separate part of the oligonucleotide.

Region D′ or D″ may independently comprise or consist of 1, 2, 3, 4 or 5 additional nucleotides, which may be complementary or non-complementary to the target nucleic acid. The nucleotide adjacent to the F or F′ region is not a sugar-modified nucleotide, such as a DNA or RNA or base modified versions of these. The D′ or D′ region may serve as a nuclease susceptible biocleavable linker (see definition of linkers). In some embodiments the additional 5′ and/or 3′ end nucleotides are linked with phosphodiester linkages, and are DNA or RNA. Nucleotide based biocleavable linkers suitable for use as region D′ or D″ are disclosed in WO2014/076195, which include by way of example a phosphodiester linked DNA dinucleotide. The use of biocleavable linkers in poly-oligonucleotide constructs is disclosed in WO2015/113922, where they are used to link multiple antisense constructs (e.g. gapmer regions) within a single oligonucleotide.

In one embodiment the oligonucleotide of the invention comprises a region D′ and/or D″ in addition to the contiguous nucleotide sequence which constitutes the gapmer. In some embodiments, the oligonucleotide of the present invention can be represented by the following formulae:

F-G-F′; in particular F_1-8-G_5-16-F′_2-8

D′-F-G-F′, in particular D′_1-3-F_1-8-G_5-16-F′_2-8

F-G-F′-D″, in particular F_1-8-G_5-16-F′_2-8-D″_1-3

D′-F-G-F′-D″, in particular D′_1-3-F_1-8-G_5-16-F′_2-8-D″_1-3

In some embodiments the internucleoside linkage positioned between region D′ and region F is a phosphodiester linkage. In some embodiments the internucleoside linkage positioned between region F′ and region D″ is a phosphodiester linkage.

Conjugate

The term conjugate as used herein refers to an oligonucleotide which is covalently linked to a non-nucleotide moiety (conjugate moiety or region C or third region).

Conjugation of the oligonucleotide of the invention to one or more non-nucleotide moieties may improve the pharmacology of the oligonucleotide, e.g. by affecting the activity, cellular distribution, cellular uptake or stability of the oligonucleotide. In some embodiments the conjugate moiety modify or enhance the pharmacokinetic properties of the oligonucleotide by improving cellular distribution, bioavailability, metabolism, excretion, permeability, and/or cellular uptake of the oligonucleotide. In particular the conjugate may target the oligonucleotide to a specific organ, tissue or cell type and thereby enhance the effectiveness of the oligonucleotide in that organ, tissue or cell type. A the same time the conjugate may serve to reduce activity of the oligonucleotide in non-target cell types, tissues or organs, e.g. off target activity or activity in non-target cell types, tissues or organs.

In an embodiment, the non-nucleotide moiety (conjugate moiety) is selected from the group consisting of carbohydrates, cell surface receptor ligands, drug substances, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g. bacterial toxins), vitamins, viral proteins (e.g. capsids) or combinations thereof.

Linkers

A linkage or linker is a connection between two atoms that links one chemical group or segment of interest to another chemical group or segment of interest via one or more covalent bonds.

Conjugate moieties can be attached to the oligonucleotide directly or through a linking moiety (e.g. linker or tether). Linkers serve to covalently connect a third region, e.g. a conjugate moiety (Region C), to a first region, e.g. an oligonucleotide or contiguous nucleotide sequence or gapmer region F-G-F′ (region A).

In some embodiments of the invention the conjugate or oligonucleotide conjugate of the invention may optionally, comprise a linker region (second region or region B and/or region Y) which is positioned between the oligonucleotide or contiguous nucleotide sequence complementary to the target nucleic acid (region A or first region) and the conjugate moiety (region C or third region).

Region B refers to biocleavable linkers comprising or consisting of a physiologically labile bond that is cleavable under conditions normally encountered or analogous to those encountered within a mammalian body. Conditions under which physiologically labile linkers undergo chemical transformation (e.g., cleavage) include chemical conditions such as pH, temperature, oxidative or reductive conditions or agents, and salt concentration found in or analogous to those encountered in mammalian cells. Mammalian intracellular conditions also include the presence of enzymatic activity normally present in a mammalian cell such as from proteolytic enzymes or hydrolytic enzymes or nucleases. In one embodiment the biocleavable linker is susceptible to S1 nuclease cleavage. DNA phosphodiester containing biocleavable linkers are described in more detail in WO 2014/076195 (hereby incorporated by reference)—see also region D′ or D″ herein.

Region Y refers to linkers that are not necessarily biocleavable but primarily serve to covalently connect a conjugate moiety (region C or third region), to an oligonucleotide (region A or first region). The region Y linkers may comprise a chain structure or an oligomer of repeating units such as ethylene glycol, amino acid units or amino alkyl groups. The oligonucleotide conjugates of the present invention can be constructed of the following regional elements A-C, A-B-C, A-B-Y-C, A-Y-B-C or A-Y-C. In some embodiments the linker (region Y) is an amino alkyl, such as a C2-C36 amino alkyl group, including, for example C6 to C12 amino alkyl groups. In a preferred embodiment the linker (region Y) is a C6 amino alkyl group.

Treatment

The term ‘treatment’ as used herein refers to both treatment of an existing disease (e.g. a disease or disorder as herein referred to), or prevention of a disease, i.e. prophylaxis. It will therefore be recognized that treatment as referred to herein may, in some embodiments, be prophylactic.

Pharmaceutically Acceptable Salts

The compound of the invention may be in the form of a pharmaceutically acceptable salt. The term “pharmaceutically acceptable salts” refers to those salts which retain the biological effectiveness and properties of the free bases or free acids, which are not biologically or otherwise undesirable. The salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, particularly hydrochloric acid, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcystein. In addition these salts may be prepared form addition of an inorganic base or an organic base to the free acid. Salts derived from an inorganic base include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium salts. Salts derived from organic bases include, but are not limited to salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyamine resins. The compound of formula (I) can also be present in the form of zwitterions. Particularly preferred pharmaceutically acceptable salts of compounds of formula (I) are the salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and methanesulfonic acid.

Protecting Group

The term “protecting group”, alone or in combination, signifies a group which selectively blocks a reactive site in a multifunctional compound such that a chemical reaction can be carried out selectively at another unprotected reactive site. Protecting groups can be removed. Exemplary protecting groups are amino-protecting groups, carboxy-protecting groups or hydroxy-protecting groups.

DETAILED DESCRIPTION OF THE INVENTION

The Oligonucleotides of the Invention

The invention relates to antisense oligonucleotides capable of inhibiting expression of human myosin heavy chain 7 (Myh7). The invention relates to antisense oligonucleotides which target MYH7. Described herein are antisense oligonucleotides which provide allelic-specific inhibition of polymorphic variants of myosin heavy chain 7 (Myh7). The invention provides oligonucleotides which target the expression of a MYH7 allelic variant selected from a MYH7 allelic variant which comprises a single nucleotide polymorphism at a position selected from rs2239578, rs2069540, and rs7157716. These three common SNPs are found in intron 2, exon 3, and exon 24 of MYH7 pre-mRNA respectively, and are referred to as rs223, rs206, and rs715 herein.

In some embodiments the oligonucleotide of the invention selectively inhibits a MYH7 allelic variant, such as an allelic variant at a position of the human MYH7 transcript selected from rs223, rs206 and rs715.

In some embodiments the oligonucleotide of the invention selectively inhibits a MYH7 allelic variant of the human MYH7 transcript selected from rs223T or rs223C. In some embodiments the oligonucleotide of the invention selectively inhibits the rs223T MYH7 allelic variant of the human MYH7 transcript selected as compared to the rs223C allelic variant. In some embodiments the oligonucleotide of the invention selectively inhibits the rs223C MYH7 allelic variant of the human MYH7 transcript selected as compared to the rs223T allelic variant.

In some embodiments the oligonucleotide of the invention selectively inhibits a MYH7 allelic variant of the human MYH7 transcript selected from rs206C or rs206T. In some embodiments the oligonucleotide of the invention selectively inhibits the rs206T MYH7 allelic variant of the human MYH7 transcript selected as compared to the rs206C allelic variant. In some embodiments the oligonucleotide of the invention selectively inhibits the rs206C MYH7 allelic variant of the human MYH7 transcript selected as compared to the rs206T allelic variant.

In some embodiments the oligonucleotide of the invention selectively inhibits a MYH7 allelic variant of the human MYH7 transcript selected from rs715C or rs715T. In some embodiments the oligonucleotide of the invention selectively inhibits the rs715T MYH7 allelic variant of the human MYH7 transcript selected as compared to the rs715C allelic variant. In some embodiments the oligonucleotide of the invention selectively inhibits the rs715C MYH7 allelic variant of the human MYH7 transcript selected as compared to the rs715T allelic variant.

In some embodiments the oligonucleotides of the invention targets, such as selectively inhibits, a MYH7 allelic variant found within a human MYH7 intron.

The polymorphisms at rs223, rs206, and rs715 are not considered to be disease associated or disease causing, i.e. they are considered to be silent polymorphisms. However, these three polymorphisms have high heterozygosity across broad demographics and designing oligonucleotides to these SNPs enables multiple disease-linked mutations to be targeted with the same antisense compound. Clinically, this approach requires patient haplotyping to determine if the HCM mutation is on the same allele as the SNP being targeted. The results show that ASOs targeting human SNPs can distinguish alleles containing single nucleotide mismatches with both high potency (e.g. <100 nM) and high selectivity (e.g. >20×). This strategy can be applied therapeutically when a patient harbors the pathogenic MYH7 mutation and the SNP of interest on the same transcript.

In some embodiments the antisense oligonucleotide of the invention is capable of modulating the expression of the target by inhibiting or down-regulating it. Preferably, such modulation produces an inhibition of expression of at least 20% compared to the normal expression level of the target, more preferably at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% inhibition compared to the normal expression level of the target. In some embodiments oligonucleotides of the invention may be capable of inhibiting expression levels of MYH7 mRNA by at least 50%, such as at least 60% or at least 70% in vitro using Human iPSC-derived cardiomyocytes (available from Cellular Dynamics International) or human skeletal muscle myoblasts cells, such as 8220 or NH10-637A cells (see the examples for exemplary methodology) .

An aspect of the present invention relates to an antisense oligonucleotide which comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length with at least 90% complementarity to human MYD7 mature mRNA or pre-mRNA.

In some embodiments, the oligonucleotide comprises a contiguous sequence of 10 to 30 nucleotides in length, which is at least 90% complementary, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or 100% complementary with a region of the target nucleic acid or a target sequence, such as a sequence selected from SEQ ID NO 3-10.

In a preferred embodiment the oligonucleotide of the invention, or contiguous nucleotide sequence thereof is fully complementary (100% complementary) to a region of the target nucleic acid, such as a sequence selected from SEQ ID NO 3-10.

In some embodiments the oligonucleotide comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length with at least 90% complementary, such as fully (or 100%) complementary, to a region target nucleic acid region present in SEQ ID NO: 1 or SEQ ID NO 2.

In some embodiments the oligonucleotide comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length with at least 90% complementary, such as fully (or 100%) complementary, to a region target nucleic acid region present in SEQ ID NO: 3-10.

In some embodiments, the oligonucleotide of the invention comprises or consists of 10 to 35 nucleotides in length, such as from 10 to 30, such as 11 to 24, such as from 12 to 22, such as from 14 to 20 or 14 to 18 or 15 to 19 contiguous nucleotides in length.

In some embodiments, the oligonucleotide or contiguous nucleotide sequence thereof comprises or consists of 22 or less nucleotides, such as 20 or less nucleotides, such as 19 or less or 18 or less nucleotides, such as 14, 15, 16 or 17 nucleotides. It is to be understood that any range given herein includes the range endpoints. Accordingly, if an oligonucleotide is said to include from 10 to 30 nucleotides, both 10 and 30 nucleotides are included.

In some embodiments, the contiguous nucleotide sequence comprises or consists of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 contiguous nucleotides in length.

In some embodiments, the oligonucleotide or contiguous nucleotide sequence comprises or consists of a sequence selected from the group consisting of sequences listed in table 5.

Table 5 provides the compound list of the compounds used in the examples, including reference to the SEQ IDs of the compounds, and the gapmer designs of the LNA compounds. In some embodiments, for the compounds listed in table 5, capital letters=LNA nucleosides, lower case letter=DNA nucleosides, and optionally all internucleoside linkages are phosphorothioate. In some embodiments of the listed gapmer compounds, and as used in the examples, capital letters=beta-D-oxy-LNA nucleosides, LNA cytosines=5 methyl cytosine LNA, lower case letters=DNA nucleosides, and all internucleoside linkages between the nucleosides illustrated are phosphorothioate internucleoside linkages.

In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 10 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 11-344. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 12 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 11-344. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 14 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 11-344. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 15 or at least 16 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 11-344. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises a sequence selected from the group consisting of SEQ ID NO 11-344.

Oligonucleotides targeting the rs206c myh7 allele:

In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 10 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 11-85. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 12 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 11-85. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 14 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 11-85. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 16 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 11-85. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises a sequence selected from the group consisting of SEQ ID NO 11-85.

Oligonucleotides targeting the rs206t myh7 allele:

In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 10 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 86-140. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 12 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 86-140. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 14 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 86-140. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 16 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 86-140. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises a sequence selected from the group consisting of SEQ ID NO 86-140.

Oligonucleotides targeting the rs223c myh7 allele:

In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 10 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 141-192. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 12 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 141-192. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 14 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 141-192. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 16 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 141-192. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises a sequence selected from the group consisting of SEQ ID NO 141-192.

Oligonucelotides targeting the rs233t myh7 allele:

In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 10 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 193-251. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 12 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 193-251. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 14 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 193-251. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 16 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 193-251. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises a sequence selected from the group consisting of SEQ ID NO 193-251.

Oligonucleotide targeting the rs715c myh7 allele:

In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 10 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 252-266. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 12 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 252-266. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 14 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 252-266. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 16 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 252-266. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises a sequence selected from the group consisting of SEQ ID NO 252-266.

Oligonucleotide targeting the rs715t myh7 allele:

In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 10 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 267-298. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 12 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 267-298. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 14 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 267-298. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 16 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 267-298. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises a sequence selected from the group consisting of SEQ ID NO 267-298.

Other oligonucleotides targeting human Myh7:

In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 10 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 299-344. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 12 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 299-344. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 14 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 299-344. In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises at least 16 contiguous nucleosides present in a sequence selected from the group consisting of SEQ ID NO 299-344.

In some embodiments, the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, comprises a sequence selected from the group consisting of SEQ ID NO 299-344.

In some embodiments, the oligonucleotide of the invention at least 70% of the internucleoside linkages are phosphorothioate, such as at least 90% of the internucleoside linkages are phosphorothioate. In some embodiments, all the internucleoside linkages between the nucleosides of the contiguous nucleotide sequence of the oligonucleotide of the invention are phosphorothioate internucleoside linkages.

It is understood that the contiguous nucleobase sequences (motif sequence) can be modified to for example increase nuclease resistance and/or binding affinity to the target nucleic acid.

The pattern in which the modified nucleosides (such as high affinity modified nucleosides) are incorporated into the oligonucleotide sequence is generally termed oligonucleotide design.

In some embodiments, the oligonucleotides of the invention are designed with modified nucleosides and DNA nucleosides. Advantageously, high affinity modified nucleosides are used.

In an embodiment, the oligonucleotide or contiguous nuceltoide sequence thereof, comprises at least 1 modified nucleoside, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 or at least 16 modified nucleosides. In an embodiment the oligonucleotide comprises from 1 to 10 modified nucleosides, such as from 2 to 9 modified nucleosides, such as from 3 to 8 modified nucleosides, such as from 4 to 7 modified nucleosides, such as 6 or 7 modified nucleosides. Suitable modifications are described in the “Definitions” section under “modified nucleoside”, “high affinity modified nucleosides”, “sugar modifications”, “2′ sugar modifications” and Locked nucleic acids (LNA)”.

In an embodiment, the oligonucleotide or contiguous nucleotide sequence thereof, comprises one or more sugar modified nucleosides, such as 2′ sugar modified nucleosides. Preferably the oligonucleotide of the invention comprise one or more 2′ sugar modified nucleoside independently selected from the group consisting of 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA, 2′-amino-DNA, 2′-fluoro-DNA, arabino nucleic acid (ANA), 2′-fluoro-ANA and LNA nucleosides. It is advantageous if one or more of the modified nucleoside(s) is a locked nucleic acid (LNA).

In a further embodiment the oligonucleotide comprises at least one modified internucleoside linkage. Suitable internucleoside modifications are described in the “Definitions” section under “Modified internucleoside linkage”. It is advantageous if at least 75%, such as all, the internucleoside linkages within the contiguous nucleotide sequence are phosphorothioate or boranophosphate internucleoside linkages. In some embodiments all the internucleotide linkages in the contiguous sequence of the oligonucleotide are phosphorothioate linkages.

In some embodiments, the oligonucleotide, or contiguous nuceltoide sequence thereof, of the invention comprises at least one LNA nucleoside, such as 1, 2, 3, 4, 5, 6, 7, or 8 LNA nucleosides, such as from 2 to 6 LNA nucleosides, such as from 3 to 7 LNA nucleosides, 4 to 8 LNA nucleosides or 3, 4, 5, 6, 7 or 8 LNA nucleosides. In some embodiments, at least 75% of the modified nucleosides in the oligonucleotide are LNA nucleosides, such as 80%, such as 85%, such as 90% of the modified nucleosides are LNA nucleosides. In a still further embodiment all the modified nucleosides in the oligonucleotide are LNA nucleosides. In a further embodiment, the oligonucleotide may comprise both beta-D-oxy-LNA, and one or more of the following LNA nucleosides: thio-LNA, amino-LNA, oxy-LNA, ScET and/or ENA in either the beta-D or alpha-L configurations or combinations thereof. In a further embodiment, all LNA cytosine units are 5-methyl-cytosine. It is advantageous for the nuclease stability of the oligonucleotide or contiguous nucleotide sequence to have at least 1 LNA nucleoside at the 5′ end and at least 2 LNA nucleosides at the 3′ end of the nucleotide sequence.

In some embodiments of the invention the oligonucleotide of the invention is capable of recruiting RNase H.

In the current invention an advantageous structural design is a gapmer design as described in the “Definitions” section under for example “Gapmer”, “LNA Gapmer”, “MOE gapmer” and “Mixed Wing Gapmer” “Alternating Flank Gapmer”. The gapmer design includes gapmers with uniform flanks, mixed wing flanks, alternating flanks, and gapbreaker designs. In the present invention it is advantageous if the oligonucleotide of the invention is a gapmer with an F-G-F′ design.

For some embodiments of the invention, the oligonucleotide or contiguous nucleotide sequence thereof, is selected from the group of oligonucleotide compounds with CMP-ID-NO (COMP #) 11-85.

For some embodiments of the invention, the oligonucleotide or contiguous nucleotide sequence thereof, is selected from the group of oligonucleotide compounds with CMP-ID-NO (COMP #) 86-140.

For some embodiments of the invention, the oligonucleotide or contiguous nucleotide sequence thereof, is selected from the group of oligonucleotide compounds with CMP-ID-NO (COMP #) 141-192.

For some embodiments of the invention, the oligonucleotide or contiguous nucleotide sequence thereof, is selected from the group of oligonucleotide compounds with CMP-ID-NO (COMP #) 193-251.

For some embodiments of the invention, the oligonucleotide or contiguous nucleotide sequence thereof, is selected from the group of oligonucleotide compounds with CMP-ID-NO (COMP #) 252-266.

For some embodiments of the invention, the oligonucleotide or contiguous nucleotide sequence thereof, is selected from the group of oligonucleotide compounds with CMP-ID-NO (COMP #) 259,1-259,189.

For some embodiments of the invention, the oligonucleotide or contiguous nucleotide sequence thereof, is selected from the group of oligonucleotide compounds with CMP-ID-NO (COMP #) 260,1-260,101.

For some embodiments of the invention, the oligonucleotide or contiguous nucleotide sequence thereof, is selected from the group of oligonucleotide compounds with CMP-ID-NO (COMP #) 267-298.

For some embodiments of the invention, the oligonucleotide or contiguous nucleotide sequence thereof, is selected from the group of oligonucleotide compounds with CMP-ID-NO (COMP #) 280,1-280,161.

For some embodiments of the invention, the oligonucleotide or contiguous nucleotide sequence thereof, is selected from the group of oligonucleotide compounds with CMP-ID-NO (COMP #) 299-344.

Method of Manufacture

In a further aspect, the invention provides methods for manufacturing the oligonucleotides of the invention comprising reacting nucleotide units and thereby forming covalently linked contiguous nucleotide units comprised in the oligonucleotide. Preferably, the method uses phophoramidite chemistry (see for example Caruthers et al, 1987, Methods in Enzymology vol. 154, pages 287-313). In a further embodiment the method further comprises reacting the contiguous nucleotide sequence with a conjugating moiety (ligand) to covalently attach the conjugate moiety to the oligonucleotide. In a further aspect a method is provided for manufacturing the composition of the invention, comprising mixing the oligonucleotide or conjugated oligonucleotide of the invention with a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.

Pharmaceutical Salt

The compounds according to the present invention may exist in the form of their pharmaceutically acceptable salts. The term “pharmaceutically acceptable salt” refers to conventional acid-addition salts or base-addition salts that retain the biological effectiveness and properties of the compounds of the present invention and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases. Acid-addition salts include for example those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like. Base-addition salts include those derived from ammonium, potassium, sodium and, quaternary ammonium hydroxides, such as for example, tetramethyl ammonium hydroxide. The chemical modification of a pharmaceutical compound into a salt is a technique well known to pharmaceutical chemists in order to obtain improved physical and chemical stability, hygroscopicity, flowability and solubility of compounds. It is for example described in Bastin, Organic Process Research & Development 2000, 4, 427-435 or in Ansel, In: Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th ed. (1995), pp. 196 and 1456-1457. For example, the pharmaceutically acceptable salt of the compounds provided herein may be a sodium salt.

In a further aspect the invention provides a pharmaceutically acceptable salt of the antisense oligonucleotide or a conjugate thereof. In a preferred embodiment, the pharmaceutically acceptable salt is a sodium or a potassium salt.

Pharmaceutical Composition

In a further aspect, the invention provides pharmaceutical compositions comprising any of the aforementioned oligonucleotides and/or oligonucleotide conjugates or salts thereof and a pharmaceutically acceptable diluent, carrier, salt and/or adjuvant. A pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS) and pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. In some embodiments the pharmaceutically acceptable diluent is sterile phosphate buffered saline. In some embodiments the oligonucleotide is used in the pharmaceutically acceptable diluent at a concentration of 50-300 μM solution.

Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. Fora brief review of methods for drug delivery, see, e.g., Langer (Science 249:1527-1533, 1990). WO 2007/031091 provides further suitable and preferred examples of pharmaceutically acceptable diluents, carriers and adjuvants (hereby incorporated by reference). Suitable dosages, formulations, administration routes, compositions, dosage forms, combinations with other therapeutic agents, pro-drug formulations are also provided in WO2007/031091.

Oligonucleotides or oligonucleotide conjugates of the invention may be mixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents, such as in a sealed package of tablets or capsules. The composition in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment.

In some embodiments, the oligonucleotide or oligonucleotide conjugate of the invention is a prodrug. In particular with respect to oligonucleotide conjugates the conjugate moiety is cleaved of the oligonucleotide once the prodrug is delivered to the site of action, e.g. the target cell.

Applications

The oligonucleotides of the invention may be utilized as research reagents for, for example, diagnostics, therapeutics and prophylaxis.

In research, such oligonucleotides may be used to specifically modulate the synthesis of MYH7 protein in cells (e.g. in vitro cell cultures) and experimental animals thereby facilitating functional analysis of the target or an appraisal of its usefulness as a target for therapeutic intervention. Typically the target modulation is achieved by degrading or inhibiting the mRNA producing the protein, thereby prevent protein formation or by degrading or inhibiting a modulator of the gene or mRNA producing the protein.

If employing the oligonucleotide of the invention in research or diagnostics the target nucleic acid may be a cDNA or a synthetic nucleic acid derived from DNA or RNA.

The present invention provides an in vivo or in vitro method for modulating MYH7 expression in a target cell which is expressing MYH7, said method comprising administering an oligonucleotide of the invention in an effective amount to said cell.

In some embodiments, the target cell, is a mammalian cell in particular a human cell. The target cell may be an in vitro cell culture or an in vivo cell forming part of a tissue in a mammal. In preferred embodiments the target cell is a muscle cell, a skeletal muscle cell, a heart cell, or a cardiomyocyte cell.

In diagnostics the oligonucleotides may be used to detect and quantitate MYH7 expression in cell and tissues by northern blotting, in-situ hybridisation or similar techniques.

For therapeutics, the oligonucleotides may be administered to an animal or a human, suspected of having a disease or disorder, which can be treated by modulating the expression of MYH7.

The invention provides methods for treating or preventing a disease, comprising administering a therapeutically or prophylactically effective amount of an oligonucleotide, an oligonucleotide conjugate or a pharmaceutical composition of the invention to a subject suffering from or susceptible to the disease.

The invention also relates to an oligonucleotide, a composition or a conjugate as defined herein for use as a medicament.

The oligonucleotide, oligonucleotide conjugate or a pharmaceutical composition according to the invention is typically administered in an effective amount.

The invention also provides for the use of the oligonucleotide or oligonucleotide conjugate of the invention as described for the manufacture of a medicament for the treatment of a disorder as referred to herein, or for a method of the treatment of as a disorder as referred to herein.

The disease or disorder, as referred to herein, is associated with expression of MYH7. In some embodiments disease or disorder may be associated with a mutation in the MYH7 gene or a gene whose protein product is associated with or interacts with MYH7. Therefore, in some embodiments, the target nucleic acid is a mutated form of the MYH7 sequence and in other embodiments, the target nucleic acid is a regulator of the MYH7 sequence.

The methods of the invention are preferably employed for treatment or prophylaxis against diseases caused by abnormal levels and/or activity of MYH7.

The invention further relates to use of an oligonucleotide, oligonucleotide conjugate or a pharmaceutical composition as defined herein for the manufacture of a medicament for the treatment of abnormal levels and/or activity of MYH7.

In one embodiment, the invention relates to oligonucleotides, oligonucleotide conjugates or pharmaceutical compositions for use in the treatment of

Administration

The oligonucleotides or pharmaceutical compositions of the present invention may be administered topical (such as, to the skin, inhalation, ophthalmic or otic) or enteral (such as, orally or through the gastrointestinal tract) or parenteral (such as, intravenous, subcutaneous, intra-muscular, intracerebral, intracerebroventricular or intrathecal).

In a preferred embodiment the oligonucleotide or pharmaceutical compositions of the present invention are administered by a parenteral route including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, intrathecal or intracranial, e.g. intracerebral or intraventricular, intravitreal administration. In one embodiment the active oligonucleotide or oligonucleotide conjugate is administered intravenously. In another embodiment the active oligonucleotide or oligonucleotide conjugate is administered subcutaneously.

In some embodiments, the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the invention is administered at a dose of 0.1-15 mg/kg, such as from 0.2-10 mg/kg, such as from 0.25-5 mg/kg. The administration can be once a week, every 2^nd week, every third week or even once a month.

The invention also provides for the use of the oligonucleotide or oligonucleotide conjugate of the invention as described for the manufacture of a medicament wherein the medicament is in a dosage form for intravenous or subcutaneous administration.

Combination Therapies

In some embodiments the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the invention is for use in a combination treatment with another therapeutic agent. The therapeutic agent can for example be the standard of care for the diseases or disorders described above.

Personalized Method of Treatment Using Allelic Specific Compounds Targeting Myh7

The invention provides for a method for treatment of a human subject in need to treatment for hypertrophic cardiomyopathy, said treatment comprising the step of:

a. Taking a biological sample from the human subject

b. Detecting such as sequencing the Myh7 nucleic acid alleles present in the sample of the human subject;

c. Determine the presence of a disease associated Myh7 allelic variant of the Myh7 nucleic acid;

d. Administer a therapeutically effective amount of an antisense oligonucleotide to the human subject which is selective for the disease associated Myh7 allelic variant as compared to a non-disease associate allele, such as the oligonucleotide of the invention or the conjugate of the invention or the pharmaceutically acceptable salt of the invention or the pharmaceutical composition of the invention.

EXAMPLES

Materials and Methods

Oligonucleotide Synthesis

Oligonucleotide synthesis is generally known in the art. Below is a protocol which may be applied. The oligonucleotides of the present invention may have been produced by slightly varying methods in terms of apparatus, support and concentrations used.

Oligonucleotides are synthesized on uridine universal supports using the phosphoramidite approach on an Oligomaker 48 at 1 μmol scale. At the end of the synthesis, the oligonucleotides are cleaved from the solid support using aqueous ammonia for 5-16 hours at 60° C. The oligonucleotides are purified by reverse phase HPLC (RP-HPLC) or by solid phase extractions and characterized by UPLC, and the molecular mass is further confirmed by ESI-MS.

Elongation of the Oligonucleotide:

The coupling of β-cyanoethyl-phosphoramidites (DNA-A(Bz), DNA-G(ibu), DNA-C(Bz), DNA-T, LNA-5-methyl-C(Bz), LNA-A(Bz), LNA-G(dmf), or LNA-T) is performed by using a solution of 0.1 M of the 5′-O-DMT-protected amidite in acetonitrile and DCI (4,5-dicyanoimidazole) in acetonitrile (0.25 M) as activator. For the final cycle a phosphoramidite with desired modifications can be used, e.g. a C6 linker for attaching a conjugate group or a conjugate group as such. Thiolation for introduction of phosphorthioate linkages is carried out by using xanthane hydride (0.01 M in acetonitrile/pyridine 9:1). Phosphordiester linkages can be introduced using 0.02 M iodine in THF/Pyridine/water 7:2:1. The rest of the reagents are the ones typically used for oligonucleotide synthesis.

For post solid phase synthesis conjugation a commercially available C6 aminolinker phorphoramidite can be used in the last cycle of the solid phase synthesis and after deprotection and cleavage from the solid support the aminolinked deprotected oligonucleotide is isolated. The conjugates are introduced via activation of the functional group using standard synthesis methods.

Purification by RP-HPLC:

The crude compounds are purified by preparative RP-HPLC on a Phenomenex Jupiter C18 10μ 150×10 mm column. 0.1 M ammonium acetate pH 8 and acetonitrile is used as buffers at a flow rate of 5 mL/min. The collected fractions are lyophilized to give the purified compound typically as a white solid.

Abbreviations:

DCI: 4,5-Dicyanoimidazole

DCM: Dichloromethane

DMF: Dimethylformamide

DMT: 4,4′-Dimethoxytrityl

THF: Tetrahydrofurane

Bz: Benzoyl

Ibu: Isobutyryl

RP-HPLC: Reverse phase high performance liquid chromatography

T_m Assay:

Oligonucleotide and RNA target (phosphate linked, PO) duplexes are diluted to 3 mM in 500 ml RNase-free water and mixed with 500 ml 2× Tm-buffer (200 mM NaCl, 0.2 mM EDTA, 20 mM Naphosphate, pH 7.0). The solution is heated to 95° C. for 3 min and then allowed to anneal in room temperature for 30 min. The duplex melting temperatures (T_m) is measured on a Lambda 40 UV/VIS Spectrophotometer equipped with a Peltier temperature programmer PTP6 using PE Templab software (Perkin Elmer). The temperature is ramped up from 20° C. to 95° C. and then down to 25° C., recording absorption at 260 nm. First derivative and the local maximums of both the melting and annealing are used to assess the duplex T_m.

ASO Synthesis and Purification

LNA-modified gapmers were designed with fully modified phosphorothioate backbones and were synthesized on a MerMade 192× synthesizer (Bioautomation, Texas) following standard phosphoramidite protocols. The final 5′-dimethoxytrityl (DMT) group was left on the oligonucleotide. After synthesis, the oligonucleotides were cleaved from the solid support using aqueous ammonia and subsequently deprotected at 65° C. for 5 hours. The oligonucleotides were purified by solid phase extraction in TOP DNA cartridges (Agilent, Glostrup, Denmark) using the lipophilic DMT group as a chromatographic retention probe. After eluting impurities, the DMT group was removed by treatment with dichloroacetic acid. As the last step in the purification process, the oligonucleotides were eluted from the cartridge and the eluate was evaporated to dryness. The oligonucleotides were dissolved in phosphate-buffered saline (PBS) and the oligonucleotide concentration in solution determined using Beer-Lambert's law by calculating the extinction coefficient and measuring UV-absorbance. Oligonucleotide identity and purity were determined by reversed-phase Ultra Performance Liquid Chromatography coupled to Mass Spectrometry (UPLC-MS).

Cell Culture

Human skeletal muscle myoblasts (8220 and NH10-637A [9]) were seeded in collagen-coated 96 well plates at a density of 15,000 cells/well. Cells were maintained in SKM-M growth media (ZenBio, North Carolina) until confluence, at which point SKM-D differentiation media was used. Cells were cultured for 1 week in differentiation media to allow for myoblast fusion and differentiation into myotubes, with media exchange every other day. One week after switching to differentiation media, ASOs were added to the cells in the absence of transfection reagents (i.e. gymnotic delivery); biological duplicates were used. Cells were lysed at day 3 or day 6 for single point studies and at day 6 or day 10 for concentration response curves. Human iPSC-derived cardiomyocytes were purchased from Cellular Dynamics International and cultured according to the manufacturer's instructions. Cells were seeded in collagen/fibronectin coated (0.01 mg/ml) 96 well plates at a density of 20,000 cells per well. ASOs dissolved in PBS or water were added 4 days after plating and media was changed every other day until lysis.

QuantiGene

The QuantiGene 2.0 assay (Affymetrix) was used to quantify RNA abundance of MYH7 (QG probe SA-10161) and the endogenous control (Human PPIB probe SA-10003) of each lysate following the manufacturer's protocol. The QG probes are designed to exonic regions of MYH7 and PPIB. Assay signals were background subtracted and normalized to the endogenous control to correct for cell density and lysis efficiency. MYH7 knockdown is reported relative to no ASO negative control.

RNA Purification and ddPCR

Cells were lysed by removal of media followed by addition of 125 μL PureLink©Pro 96 Lysis buffer (Invitrogen 12173.001A) and 125 μL 70% ethanol. RNA was purified according to the manufacture's instruction and eluted in a final volume of 50 μL water resulting in an RNA concentration of 10-20 ng/μl. Droplet digital PCR (ddPCR) was done using BioRad Automatic Droplet Generator (AutoDG) using Automated Droplet Generation Oil for Probes (BioRad) together with the OX200 droplet digital reader. The ddPCR™ Supermix for Probes (No dUTP) (Bio-Rad 1863024) reactions were run according to the manufacturer's instructions with an annealing temperature of 55.5° C. for the human reactions and 55° C. for the mouse reactions. The droplets were read in the OX200 droplet digital reader, and the data were analyzed and quantified using the QuantaSoft™ Analysis Pro Software 1.0.596 (BioRad). The thresholds for defining the different droplet groups in the triplex PCR reaction was set by free hand within the software according to the guidelines. Assays for human SNPs: rs715T (fw_primer CAGAGGAGATGGCTGG, rev_primer TGCAGAGCTTTCTTCTCC (SEQ ID NO 345), probe CAGCTTGGCAATGATCTC HEX_IowaBlack, (SEQ ID NO 346)); rs715C (fw_primer CAGAGGAGATGGCTGG (SEQ ID NO 347), rev_primer TGCAGAGCTTTCTTCTCC (SEQ ID NO 348), probe CAGCTTGGCGATGATCT FAM_IowaBlack (SEQ ID NO 349)); GAPDH (dHsa CPE5031596, FAM_IowaBlack) and (dHsa CPE5031597, HEX_IowaBlack) from BioRad. Assays for humanized mouse model: humanized rs715C myh6 (fw_primer CCTAACAGAGGAGATG (SEQ ID NO 350), rev_primer CTTCTTGCAGAGCTTTCTT (SEQ ID NO 351), probe TGAGATCATCGCCAAGC Hex_IowaBlack (SEQ ID NO 352)); wt myh6 (fw_primer ACCTAACAGAGGAGATG (SEQ ID NO 353), rev_primer CTTCTTGCAGAGCTTTCTT (SEQ ID NO 354), probe TGAAATCATTGCCAAGCTG FAM_IowaBlack (SEQ ID NO 355)); GAPDH (dMmuCPE5195282, FAM_IowaBlack and dMmuCPE5195283, HEX_IowaBlack) from BioRad.

Statistical Analysis of Concentration-Response Curves

Concentration-response curves of RNA levels after treatment with ASO at eight different concentrations were analyzed by nonlinear least squares fitting of the two-parameter logistic function using the R software package drc [10]. For the two-parameter logistic function the lower and upper limits are fixed at 0% and 100%, respectively, and the two parameters estimated from each curve are the IC50 value and Hill coefficient. The maximal possible IC₅₀ value was set to the maximal ASO concentration evaluated.

Mouse Model Generation and In Vivo Study

Since the predominant isoform in mouse heart is Myh6, the human MYH7 sequence (ENSG00000092054) around the rs715-C SNP was inserted into the mouse Myh6 gene (ENSMUSG00000040752) using homologous recombination in C57BL/6J mice (Figure S4). This 57 nucleotide insertion (acagaggagatggctgggctggatgagatcatCgccaagctgaccaaggagaagaaa (SEQ ID NO 356) replacing acagaggagatggctgggctggatgaaatcatTgccaagctgaccaaagagaagaaa, SEQ ID NO 357) is not predicted to affect amino acid sequence (SNP nucleotide shown as uppercase). Mice are homozygous for thymine at the base position that corresponds to the rs715 SNP in humans. Heterozygous mice (human rs715-C)^+/− lacking FLP recombinase were used for the in vivo study. Animals were dosed with ASO subcutaneously at 3*30 mg/kg on days 0, 1, and 2 with takedown on day 7. Allele-specific Myh6 mRNA knockdown was measured via droplet digital PCR from RNA isolated from half of the left ventricle. The other half of the left ventricle, in addition to one kidney and a portion of liver, was quick frozen in liquid nitrogen to determine the tissue concentrations of ASO (Oligo ELISA, Exiqon, Denmark). Blood was collected at the time of sacrifice, with subsequent serum quantification of kidney and liver injury markers.

Example 1: SNP Identification

We analyzed the Phase 3 1000 Genomes database [11] to identify SNPs in the human population that occur with high frequency, i.e. genetic coordinates in MYH7 that contain different nucleotides on each allele (i.e. heterozygous base) in a large fraction of people. We found three SNPs with high heterozygosity: rs2239578 (48%), rs2069540 (48%), and rs7157716 (38%) (FIG. 1a). These three common SNPs are found in intron 2, exon 3, and exon 24 of MYH7, respectively, and will be referred to as rs223, rs206, and rs715.

For rs206, the reference nucleotide is cytosine and the SNP is thymine, while for rs223 and rs715 the reference is thymine and the SNP is cytosine (Table 3). The designation of a SNP in these cases is somewhat arbitrary since both the reference and alternate allele are common. No other polymorphisms are found within 25 bases upstream or downstream of each SNP. To each of these SNP regions, locked nucleic acid (LNA) gapmer ASOs were designed and synthesized to selectively knockdown mRNA containing either cytosine or thymine at the SNP coordinate. This strategy depends on the ability of the ASOs to induce robust degradation of the SNP-matched RNA while minimizing degradation of the SNP-mismatched RNA. This allows for multiple disease-linked mutations to be targeted with the same ASO (FIG. 1b). Within each SNP region ASOs were tiled along the transcript, resulting in some ASOs having the position of the SNP in the 5′ end, some in the DNA gap in the middle, and some in the 3′ end. Furthermore, for each position ASOs from 15 to 20 nucleotides in length were designed, with one to four LNA nucleotides in the 5′ end and two to four in the 3′end. Varying the SNP position and ASO structure in this manner resulted in 47 ASOs targeting the rs715 SNP (15-C, 32-T), 111 ASOs targeting the rs223 SNP (52-C, 59-T), and 130 ASOs targeting the rs206 SNP (75-C, 55-T) (Table S1).

Example 2: In Vitro Knockdown

We screened the initial ASO libraries in the QuantiGene 2.0 assay to identify compounds that exhibit good knockdown of MYH7 RNA. Two human skeletal muscle myoblast cell lines were used; both lines were homozygous at each SNP position and the lines were perfectly complementary (e.g. one line had C/C at rs206 and T/T at rs223 and rs715, the other had T/T at rs206 and C/C at rs223 and rs715). ASOs were screened in both cell lines at 5 uM using gymnotic delivery to determine SNP-matched and SNP-mismatched RNA knockdown at a 3 day timepoint. A non-SNP targeting ASO (S17 in FIG. 6) was used as a positive control and showed similar activity in both cell lines (88% and 85% knockdown at 5 uM (FIG. 6). This suggests that ASO uptake is similar between the two human myoblast lines. ASOs that showed mild selectivity (>50% knockdown of MYH7 mRNA in the SNP-matched cell line as well as <25% knockdown in the SNP-mismatched cell line, Table 6) were selected for follow-up potency determination. Additional ASOs that showed good SNP-matched potency but did not meet the selectivity criteria were also progressed to concentration response curves (CRCs). ASO potency values (IC₅₀) were determined from CRCs using the QuantiGene assay in both the SNP-matched and SNP-mismatched cell lines (FIG. 2a). This allows calculation of a selectivity ratio, defined as the ratio of SNP-mismatched potency to SNP-matched potency. FIGS. 2b-2d show that ASOs can be found in all three SNP regions that show good potency and selectivity, highlighting the generalizability of this approach.

Since allele selectivity was shown at all three SNP regions, we decided to focus on ASOs targeting the rs715 SNP region due to sequence homology between human and dog and cynomolgus monkey. We also developed a droplet digital PCR (ddPCR) assay that enabled us to measure allele-specific mRNA knockdown in cells that are heterozygous at the rs715 SNP position (T on one allele, C on the other). This assay used multiplexed PCR reactions to simultaneously measure allele-specific potency in a SNP-heterozygous human myoblast cell line. We generated 450 LNA gapmer redesigns based on ASOs from the initial rs715 library that exhibited good potency and selectivity (two ASOs, A249 and A250, targeting the rs715-C SNP and one, A270, targeting rs715-T). The redesigns are also shown in Table 5. Transcript start site was maintained, but ASO lengths were varied from 17 to 19 nucleotides. Furthermore, the number and position of LNA modifications within each ASO were varied, with LNA and DNA interspersed. All ASOs had between 4 and 15 consecutive DNAs to allow for RNase H binding and cleavage, with the majority of ASOs containing between 5 and 7 consecutive DNAs. These ASOs were tested at 500 nM in human myoblasts that are heterozygous at the rs715 SNP position (CC-2580 cells, Lonza), with mRNA levels determined 6 days after compound addition (FIGS. 7a-7c and Table 7). From this single point data, a subset of ASOs were selected for follow-up potency determinations in two rs715 SNP-heterozygous cell lines: human myoblasts (CC-2580), data shown in Table 8 and human iPSC-derived cardiomyocytes (iCell², CDI), data shown in Table 9. Potencies and selectivities are summarized in FIG. 3.

To determine if allele-compensation occurs during allele-selective knockdown, we performed a time course study in iCell² iPSC-CM. These cells are heterozygous at the rs715 SNP position and were treated with 250 nM of ASO A259 (see Table 5 for sequence), a potent and selective ASO targeting the rs715-T SNP. FIG. 4 shows that the ASO does not knockdown the SNP-mismatched allele (rs715-C), but does give strong knockdown of the SNP-matched allele (rs715-T). This experiment shows in vitro allele-selective mRNA knockdown at up to two weeks following ASO addition. These experiments also included replicate cell plates for quantifying the effect of ASO addition on MYH7 protein (β-myosin heavy chain). Protein lysates at all timepoints were probed with an antibody that recognizes β-MHC but not α-MHC (iCell² cells also contain α-MHC, which is encoded by the MYH6 gene). FIG. 8 shows that reduction in β-MHC was not seen at any timepoint, suggesting compensation by the SNP-mismatched allele at the translation level.

Example 3: In Vivo Knockdown

Further, we were interested in determining if these ASOs could selectively knockdown target mRNA in vivo. We generated a genetically engineered mouse model with the human MYH7 sequence inserted at the rs715 SNP region. Since the predominant myosin isoform in mouse heart is fast α-MHC, the human rs715-C SNP region was inserted into the mouse Myh6 gene. This was a 57 basepair replacement at the rs715 SNP coordinate (i.e. the location of the SNP plus 32 nucleotides upstream and 24 nucleotides downstream; see FIG. 9). This genetic modification did not change the predicted amino acid sequence of mouse α-MHC. This mouse line was heterozygous for the humanized MYH7 fragment, as it contained wildtype Myh6 on the other allele. Five ASOs targeting the rs715-C SNP were tested in vivo in these heterozygous humanized mice. Mouse Myh6 contains a thymine base at the rs715 SNP coordinate, so the rs715-C ASOs are predicted to not target the WT allele. In addition, there is another mismatch six bases upstream of the SNP (guanine in human, adenine in mouse), which gives a two basepair mismatch between the ASO targeting sequences and the wildtype allele (see FIG. 9 for details).

Mice were dosed subcutaneous with 30 mg/kg compound (or saline) on days 0, 1, and 2, and the animals were sacrificed at day 9. Target mRNA knockdown was determined in left ventricular tissue; all five treatment groups had a significant reduction in humanized rs715-C mRNA compared to wildtype Myh6 mRNA (FIG. 5a). In addition, reduction of rs715-C mRNA was significant compared to saline rs715-C in two of the groups (ASOs B82 and B44). This data clearly shows allele-selective knockdown in cardiac tissue. ASO concentrations were determined in left ventricle, kidney, and liver (FIG. 5b) using ASO-specific ELISA assays. As expected, exposure values were significantly higher in kidney and liver. Two of the five compounds were associated with elevation of liver injury markers (AST, ALT, and alkaline phosphatase; FIG. 10).

TABLE 5
Sequences and Compounds
						mRNA	Pre-mRNA
SEQ ID NO	SEQUENCE	COMP ID NO #	Compound*	Target seqs	match.to.snp.region	start	end	start	end	Example Figure ID
11	CGGTCTCGGCAGTGAC	11	CGgtctcggcagtGAC	5, 7	rs206-c, rs206-c-pre	306	321	2161	2176	A1
12	TCGGTCTCGGCAGTGAC	12	TCGgtctcggcagtGAC	5, 7	rs206-c, rs206-c-pre	306	322	2161	2177	A2
13	TCGGTCTCGGCAGTGA	13	TCGgtctcggcagtGA	5, 7	rs206-c, rs206-c-pre	307	322	2162	2177	A3
14	CTCGGTCTCGGCAGTGA	14	CTCGgtctcggcagtGA	5, 7	rs206-c, rs206-c-pre	307	323	2162	2178	A4
15	TACTCGGTCTCGGCAGTGA	15	TActcggtctcggcagTGA	5, 7	rs206-c, rs206-c-pre	307	325	2162	2180	A5
16	ATACTCGGTCTCGGCAGTGA	16	AtactcggtctcggcagtGA	5, 7	rs206-c, rs206-c-pre	307	326	2162	2181	A6
17	ATACTCGGTCTCGGCAGTG	17	AtactcggtctcggcagTG	5, 7	rs206-c, rs206-c-pre	308	326	2163	2181	A7
18	CATACTCGGTCTCGGCAGTG	18	CatactcggtctcggcagTG	5, 7	rs206-c, rs206-c-pre	308	327	2163	2182	A8
19	ATACTCGGTCTCGGCAGT	19	ATActcggtctcggcaGT	5, 7	rs206-c, rs206-c-pre	309	326	2164	2181	A9
20	CATACTCGGTCTCGGCAGT	20	CatactcggtctcggcaGT	5, 7	rs206-c, rs206-c-pre	309	327	2164	2182	A10
21	CCATACTCGGTCTCGGCAGT	21	CcatactcggtctcggcaGT	5, 7	rs206-c, rs206-c-pre	309	328	2164	2183	A11
22	TACTCGGTCTCGGCAG	22	TACtcggtctcggcAG	5, 7	rs206-c, rs206-c-pre	310	325	2165	2180	A12
23	ATACTCGGTCTCGGCAG	23	ATActcggtctcggcAG	5, 7	rs206-c, rs206-c-pre	310	326	2165	2181	A13
24	CATACTCGGTCTCGGCAG	24	CatactcggtctcggcAG	5, 7	rs206-c, rs206-c-pre	310	327	2165	2182	A14
25	CCATACTCGGTCTCGGCAG	25	CcatactcggtctcggcAG	5, 7	rs206-c, rs206-c-pre	310	328	2165	2183	A15
26	ATACTCGGTCTCGGCA	26	ATActcggtctcggCA	5, 7	rs206-c, rs206-c-pre	311	326	2166	2181	A16
27	CATACTCGGTCTCGGCA	27	CAtactcggtctcggCA	5, 7	rs206-c, rs206-c-pre	311	327	2166	2182	A17
28	CCATACTCGGTCTCGGCA	28	CcatactcggtctcggCA	5, 7	rs206-c, rs206-c-pre	311	328	2166	2183	A18
29	ATACTCGGTCTCGGC	29	ATActcggtctcgGC	5, 7	rs206-c, rs206-c-pre	312	326	2167	2181	A19
30	CATACTCGGTCTCGGC	30	CAtactcggtctcgGC	5, 7	rs206-c, rs206-c-pre	312	327	2167	2182	A20
31	CCATACTCGGTCTCGGC	31	CcatactcggtctcgGC	5, 7	rs206-c, rs206-c-pre	312	328	2167	2183	A21
32	GCCATACTCGGTCTCGGC	32	GccatactcggtctcgGC	5, 7	rs206-c, rs206-c-pre	312	329	2167	2184	A22
33	TGCCATACTCGGTCTCGGC	33	TgccatactcggtctcgGC	5, 7	rs206-c, rs206-c-pre	312	330	2167	2185	A23
34	TTGCCATACTCGGTCTCGGC	34	TtgccatactcggtctcgGC	5, 7	rs206-c, rs206-c-pre	312	331	2167	2186	A24
35	CATACTCGGTCTCGG	35	CATActcggtctcGG	5, 7	rs206-c, rs206-c-pre	313	327	2168	2182	A25
36	CCATACTCGGTCTCGG	36	CCatactcggtctcGG	5, 7	rs206-c, rs206-c-pre	313	328	2168	2183	A26
37	GCCATACTCGGTCTCGG	37	GcCatactcggtctcGG	5, 7	rs206-c, rs206-c-pre	313	329	2168	2184	A27
38	TGCCATACTCGGTCTCGG	38	TgccatactcggtctcGG	5, 7	rs206-c, rs206-c-pre	313	330	2168	2185	A28
39	TTGCCATACTCGGTCTCGG	39	TtgccatactcggtctcGG	5, 7	rs206-c, rs206-c-pre	313	331	2168	2186	A29
40	CTTGCCATACTCGGTCTCGG	40	CTtgccatactcggtctcGG	5, 7	rs206-c, rs206-c-pre	313	332	2168	2187	A30
41	CCATACTCGGTCTCG	41	CCAtactcggtctCG	5, 7	rs206-c, rs206-c-pre	314	328	2169	2183	A31
42	GCCATACTCGGTCTCG	42	GCcatactcggtctCG	5, 7	rs206-c, rs206-c-pre	314	329	2169	2184	A32
43	TGCCATACTCGGTCTCG	43	TGccatactcggtctCG	5, 7	rs206-c, rs206-c-pre	314	330	2169	2185	A33
44	TTGCCATACTCGGTCTCG	44	TtgccatactcggtctCG	5, 7	rs206-c, rs206-c-pre	314	331	2169	2186	A34
45	CTTGCCATACTCGGTCTCG	45	CttgccatactcggtctCG	5, 7	rs206-c, rs206-c-pre	314	332	2169	2187	A35
46	TGCCATACTCGGTCTC	46	TGccatactcggtcTC	5, 7	rs206-c, rs206-c-pre	315	330	2170	2185	A36
47	TTGCCATACTCGGTCTC	47	TTgccatactcggtcTC	5, 7	rs206-c, rs206-c-pre	315	331	2170	2186	A37
48	CTTGCCATACTCGGTCTC	48	CttgccatactcggtcTC	5, 7	rs206-c, rs206-c-pre	315	332	2170	2187	A38
49	TTGCCATACTCGGTCT	49	TTGccatactcggtCT	5, 7	rs206-c, rs206-c-pre	316	331	2171	2186	A39
50	CTTGCCATACTCGGTCT	50	CttgccatactcggtCT	5, 7	rs206-c, rs206-c-pre	316	332	2171	187	A40
51	CTTGCCATACTCGGTC	51	CTTgccatactcggTC	5, 7	rs206-c, rs206-c-pre	317	332	2172	2187	A41
52	TCTTGCCATACTCGGTCTCG	52	TCTtgccatactcggtctCG	5, 7	rs206-c, rs206-c-pre	314	333	2169	2188	A42
53	TCTTGCCATACTCGGTCTC	53	TCttgccatactcggtcTC	5, 7	rs206-c, rs206-c-pre	315	333	2170	2188	A43
54	GTCTTGCCATACTCGGTCTC	54	GTCTtgccatactcggtCTC	5	rs206-c	315	334			A44
55	TCTTGCCATACTCGGTCT	55	TCttgccatactcggtCT	5, 7	rs206-c, rs206-c-pre	316	333	2171	2188	A45
56	GTCTTGCCATACTCGGTCT	56	GtcttgccatactcggtCT	5	rs206-c	316	334			A46
57	TGTCTTGCCATACTCGGTCT	57	TGtcttgccatactcggtCT	5	rs206-c	316	335			A47
58	TCTTGCCATACTCGGTC	58	TCttgccatactcggTC	5, 7	rs206-c, rs206-c-pre	317	333	2172	2188	A48
59	GTCTTGCCATACTCGGTC	59	GTCTtgccatactcggTC	5	rs206-c	317	334			A49
60	TCTTGCCATACTCGGT	60	TCTtgccatactcgGT	5, 7	rs206-c, rs206-c-pre	318	333	2173	2188	A50
61	GTCTTGCCATACTCGGT	61	GTCTtgccatactcgGT	5	rs206-c	318	334			A51
62	GTCTTGCCATACTCGG	62	GTCTtgccatactCGG	5	rs206-c	319	334			A52
63	TGTCTTGCCATACTCGG	63	TGtcttgccatacTCGG	5	rs206-c	319	335			A53
64	CTGTCTTGCCATACTCGG	64	CTgtcttgccatactCGG	5	rs206-c	319	336			A54
65	CACTGTCTTGCCATACTCGG	65	CActgtcttgccatactCGG	5	rs206-c	319	338			A55
66	CCTTGCCATACTCGGTCTCG	66	CcttgccatactcggtctCG	5, 7	rs206-c, rs206-c-pre	314	333	2169	2188	A56
67	CCTTGCCATACTCGGTCTC	67	CCttgccatactcggtcTC	5, 7	rs206-c, rs206-c-pre	315	333	2170	2188	A57
68	ACCTTGCCATACTCGGTCTC	68	AccttgccatactcggtcTC	7	rs206-c-pre			2170	2189	A58
69	CCTTGCCATACTCGGTCT	69	CcttgccatactcggtCT	5, 7	rs206-c, rs206-c-pre	316	333	2171	2188	A59
70	ACCTTGCCATACTCGGTCT	70	AccttgccatactcggtCT	7	rs206-c-pre			2171	2189	A60
71	CACCTTGCCATACTCGGTCT	71	CaccttgccatactcggtCT	7	rs206-c-pre			2171	2190	A61
72	CCTTGCCATACTCGGTC	72	CcttgccatactcggTC	5, 7	rs206-c, rs206-c-pre	317	333	2172	2188	A62
73	ACCTTGCCATACTCGGTC	73	AccttgccatactcggTC	7	rs206-c-pre			2172	2189	A63
74	CACCTTGCCATACTCGGTC	74	CaccttgccatactcggTC	7	rs206-c-pre			2172	2190	A64
75	CCACCTTGCCATACTCGGTC	75	CcaccttgccatactcggTC	7	rs206-c-pre			2172	2191	A65
76	CCTTGCCATACTCGGT	76	CCttgccatactcgGT	5, 7	rs206-c, rs206-c-pre	318	333	2173	2188	A66
77	ACCTTGCCATACTCGGT	77	ACcttgccatactcgGT	7	rs206-c-pre			2173	2189	A67
78	CACCTTGCCATACTCGGT	78	CaccttgccatactcgGT	7	rs206-c-pre			2173	2190	A68
79	CCACCTTGCCATACTCGGT	79	CcaccttgccatactcgGT	7	rs206-c-pre			2173	2191	A69
80	CCCACCTTGCCATACTCGGT	80	CccaccttgccatactcgGT	7	rs206-c-pre			2173	2192	A70
81	ACCTTGCCATACTCGG	81	ACcttgccatactCGG	7	rs206-c-pre			2174	2189	A71
82	CACCTTGCCATACTCGG	82	CAccttgccatactcGG	7	rs206-c-pre			2174	2190	A72
83	CCACCTTGCCATACTCGG	83	CcaccttgccatactcGG	7	rs206-c-pre			2174	2191	A73
84	CCCACCTTGCCATACTCGG	84	CccaccttgccatactcGG	7	rs206-c-pre			2174	2192	A74
85	ACCCACCTTGCCATACTCGG	85	AcccaccttgccatactcGG	7	rs206-c-pre			2174	2193	A75
86	TCTTGCCATACTCAGTCT	86	TCTtgccatactcagtCT	6	rs206-t	316	333			A76
87	CCATACTCAGTCTCGGCA	87	CcatactcagtctcggCA	6, 8	rs206-t, rs206-t-pre	311	328	2166	2183	A77
88	TTGCCATACTCAGTCTCG	88	TTgccatactcagtcTCG	6, 8	rs206-t, rs206-t-pre	314	331	2169	2186	A78
89	TCTTGCCATACTCAGTC	89	TCTtgccatactcagTC	6	rs206-t	317	333			A79
90	CCATACTCAGTCTCGGCAGT	90	CcatactcagtctcggcaGT	6, 8	rs206-t, rs206-t-pre	309	328	2164	2183	A80
91	CCATACTCAGTCTCGG	91	CCatactcagtctcGG	6, 8	rs206-t, rs206-t-pre	313	328	2168	2183	A81
92	CTTGCCATACTCAGTCTCG	92	CTtgccatactcagtctCG	6, 8	rs206-t, rs206-t-pre	314	332	2169	2187	A82
93	CTTGCCATACTCAGTCT	93	CTtgccatactcagtCT	6, 8	rs206-t, rs206-t-pre	316	332	2171	2187	A83
94	TTGCCATACTCAGTCTCGGC	94	TtgccatactcagtctcgGC	6, 8	rs206-t, rs206-t-pre	312	331	2167	2186	A84
95	TGCCATACTCAGTCTCGG	95	TGccatactcagtctcGG	6, 8	rs206-t, rs206-t-pre	313	330	2168	2185	A85
96	CTGTCTTGCCATACTCAG	96	CTgtcttgccatactCAG	6	rs206-t	319	336			A86
97	GCCATACTCAGTCTCGG	97	GccatactcagtctcGG	6, 8	rs206-t, rs206-t-pre	313	329	2168	2184	A87
98	CTTGCCATACTCAGTCTC	98	CTtgccatactcagtcTC	6, 8	rs206-t, rs206-t-pre	315	332	2170	2187	A88
99	CTTGCCATACTCAGTCTCGG	99	CTtgccatactcagtctcGG	6, 8	rs206-t, rs206-t-pre	313	332	2168	2187	A89
100	TTGCCATACTCAGTCTCGG	100	TtgccatactcagtctcGG	6, 8	rs206-t, rs206-t-pre	313	331	2168	2186	A90
101	CATACTCAGTCTCGGCAGT	101	CatactcagtctcggcaGT	6, 8	rs206-t, rs206-t-pre	309	327	2164	2182	A91
102	CATACTCAGTCTCGGCAGTG	102	CatactcagtctcggcagTG	6, 8	rs206-t, rs206-t-pre	308	327	2163	2182	A92
103	TGCCATACTCAGTCTCG	103	TGccatactcagtctCG	6, 8	rs206-t, rs206-t-pre	314	330	2169	2185	A93
104	ATACTCAGTCTCGGCAGTG	104	AtactcagtctcggcagTG	6, 8	rs206-t, rs206-t-pre	308	326	2163	2181	A94
105	CCATACTCAGTCTCGGCAG	105	CcatactcagtctcggcAG	6, 8	rs206-t, rs206-t-pre	310	328	2165	2183	A95
106	CCATACTCAGTCTCGGC	106	CcatactcagtctcgGC	6, 8	rs206-t, rs206-t-pre	312	328	2167	2183	A96
107	CACTGTCTTGCCATACTCAG	107	CActgtcttgccatactCAG	6	rs206-t	319	338			A97
108	ATACTCAGTCTCGGCAGT	108	ATActcagtctcggcaGT	6, 8	rs206-t, rs206-t-pre	309	326	2164	2181	A98
109	GCCATACTCAGTCTCGGC	109	GccatactcagtctcgGC	6, 8	rs206-t, rs206-t-pre	312	329	2167	2184	A99
110	TCTTGCCATACTCAGTCTCG	110	TCTtgccatactcagtctCG	6	rs206-t	314	333			A100
111	CATACTCAGTCTCGGC	111	CAtactcagtctcgGC	6, 8	rs206-t, rs206-t-pre	312	327	2167	2182	A101
112	TACTCAGTCTCGGCAG	112	TActcagtctcgGcAG	6, 8	rs206 t, rs206-t-pre	310	325	2165	2180	A102
113	GTCTTGCCATACTCAGT	113	GtCTtgccatactCAGT	6	rs206-t	318	334			A103
114	CATACTCAGTCTCGGCA	114	CAtactcagtctcggCA	6, 8	rs206-t, rs206-t-pre	311	327	2166	2182	A104
115	ATACTCAGTCTCGGCAG	115	ATActcagtctcggcAG	6, 8	rs206-t, rs206-t-pre	310	326	2165	2181	A105
116	TGTCTTGCCATACTCAG	116	TGtcttgccatactCAG	6	rs206-t	319	335			A106
117	TGCCATACTCAGTCTCGGC	117	TgccatactcagtctcgGC	6, 8	rs206-t, rs206-t-pre	312	330	2167	2185	A107
118	GCCATACTCAGTCTCG	118	GCcatactcagtctCG	6, 8	rs206-t, rs206-t-pre	314	329	2169	2184	A108
119	TTGCCATACTCAGTCTC	119	TTgccatactcagtCTC	6, 8	rs206-t, rs206-t-pre	315	331	2170	2186	A109
120	TCTTGCCATACTCAGTCTC	120	TCTtgccatactcagtcTC	6	rs206-t	315	333			A110
121	ACTGTCTTGCCATACTCAG	121	ACTgtcttgccatacTCAG	6	rs206-t	319	337			A111
122	ATACTCAGTCTCGGCA	122	ATActcagtctcggCA	6, 8	rs206-t, rs206-t-pre	311	326	2166	2181	A111
123	ATACTCAGTCTCGGCAGTGA	123	AtactcagtctcggcagtGA	6, 8	rs206-t, rs206-t-pre	307	326	2162	2181	A113
124	CATACTCAGTCTCGGCAG	124	CAtactcagtctcggcAG	6, 8	rs206-t, rs206-t-pre	310	327	2165	2182	A114
125	CCTTGCCATACTCAGTCT	125	CCttgccatactcagtCT	8	rs206-t-pre			2171	2188	A115
126	CACCTTGCCATACTCAGT	126	CAccttgccatactCAGT	8	rs206-t-pre			2173	2190	A116
127	CCTTGCCATACTCAGTC	127	CCTtgccatactcagTC	8	rs206-t-pre			2172	2188	A117
128	CCACCTTGCCATACTCAGT	128	CCaccttgccatactCAGT	8	rs206-t-pre			2173	2191	A118
129	ACCTTGCCATACTCAGT	129	ACCTtgccatactcAGT	8	rs206-t-pre			2173	2189	A119
130	CCACCTTGCCATACTCAG	130	CcaccttgccatactcAG	8	rs206-t-pre			2174	2191	A120
131	CACCTTGCCATACTCAG	131	CAccttgccatactcAG	8	rs206-t-pre			2174	2190	A121
132	ACCCACCTTGCCATACTCAG	132	AcccaccttgccatactcAG	8	rs206-t-pre			2174	2193	A122
133	ACCTTGCCATACTCAGTC	133	ACCTtgccatactcagTC	8	rs206-t-pre			2172	2189	A123
134	CACCTTGCCATACTCAGTCT	134	CAccttgccatactcagtCT	8	rs206-t-pre			2171	2190	A124
135	ACCTTGCCATACTCAGTCT	135	AccttgccatactcagtCT	8	rs206-t-pre			2171	2189	A125
136	ACCTTGCCATACTCAGTCTC	136	ACcTtgccatactcagtcTC	8	rs206-t-pre			2170	2189	A126
137	CCTTGCCATACTCAGTCTCG	137	CCTtgccatactcagtctCG	8	rs206-t-pre			2169	2188	A127
138	CCCACCTTGCCATACTCAGT	138	CCcaccttgccatactCAGT	8	rs206-t-pre			2173	2192	A128
139	CCTTGCCATACTCAGTCTC	139	CCTtgccatactcagtcTC	8	rs206-t-pre			2170	2188	A129
140	CCCACCTTGCCATACTCAG	140	CccaccttgccatactcAG	8	rs206-t-pre			2174	2192	A130
141	ATTTTCAACGCTCTAGC	141	ATTTtcaacgctctAGC	4	rs223-c			1541	1557	A131
142	GATTTTCAACGCTCTAGCT	142	GAttttcaacgctctagCT	4	rs223-c			1540	1558	A132
143	GATTTTCAACGCTCTAGCTT	143	GAttttcaacgctctagcTT	4	rs223-c			1539	1558	A133
144	CTAGATTTTCAACGCTCT	144	CTAgattttcaacgctCT	4	rs223-c			1544	1561	A134
145	GATTTTCAACGCTCTAG	145	GATTttcaacgctcTAG	4	rs223-c			1542	1558	A135
146	TCAACGCTCTAGCTTCAG	146	TCAacgctctagcttcAG	4	rs223-c			1536	1553	A136
147	TTTCAACGCTCTAGCTTCA	147	TTtcaacgctctagcttCA	4	rs223-c			1537	1555	A137
148	CTAGATTTTCAACGCTCTAG	148	CTAgattttcaacgctctAG	4	rs223-c			1542	1561	A138
149	GATTTTCAACGCTCTA	149	GATTttcaacgcTCTA	4	rs223-c			1543	1558	A139
150	TACTAGATTTTCAACGCTC	150	TACTagattttcaacgcTC	4	rs223-c			1545	1563	A140
151	CTAGATTTTCAACGCT	151	CTAGattttcaacGCT	4	rs223-c			1546	1561	A141
152	TTTTCAACGCTCTAGCTT	152	TTTtcaacgctctagCTT	4	rs223-c			1539	1556	A142
153	TTTTCAACGCTCTAGCT	153	TTttcaacgctctaGCT	4	rs223-c			1540	1556	A143
154	TAGATTTTCAACGCTCT	154	TAgattttcaacgCTCT	4	rs223-c			1544	1560	A144
155	AGATTTTCAACGCTCTA	155	AGAttttcaacgctCTA	4	rs223-c			1543	1559	A145
156	TACTAGATTTTCAACGCTCT	156	TACtagattttcaacgctCT	4	rs223-c			1544	1563	A146
157	ACTAGATTTTCAACGCTCTA	157	ACtagattttcaacgctCTA	4	rs223-c			1543	1562	A147
158	ACTAGATTTTCAACGC	158	ACTAgattttcaACGC	4	rs223-c			1547	1562	A148
159	TTTCAACGCTCTAGCTT	159	TTTCaacgctctagCTT	4	rs223-c			1539	1555	A149
160	TTACTAGATTTTCAACGCTC	160	TTACtagattttcaacgcTC	4	rs223-c			1545	1564	A150
161	TTACTAGATTTTCAACGCT	161	TTActagattttcaacGCT	4	rs223-c			1546	1564	A151
162	ATTTTCAACGCTCTAGCTTC	162	ATtttcaacgctctagctTC	4	rs223-c			1538	1557	A152
163	TTTCAACGCTCTAGCTTCAG	163	TTtcaacgctctagcttcAG	4	rs223-c			1536	1555	A153
164	AGATTTTCAACGCTCTAGC	164	AGattttcaacgctctaGC	4	rs223-c			1541	1559	A154
165	ACTAGATTTTCAACGCTC	165	ACtagattttcaacGCTC	4	rs223-c			1545	1562	A155
166	AGATTTTCAACGCTCTAG	166	AGattttcaacgctCTAG	4	rs223-c			1542	1559	A156
167	ACTAGATTTTCAACGCTCT	167	ACtagattttcaacgcTCT	4	rs223-c			1544	1562	A157
168	TTTTCAACGCTCTAGCTTC	168	TTttcaacgctctagcTTC	4	rs223-c			1538	1556	A158
169	TTTCAACGCTCTAGCTTC	169	TTTcaacgctctagcTTC	4	rs223-c			1538	1555	A159
170	TTCAACGCTCTAGCTTCA	170	TTcaacgctctagctTCA	4	rs223-c			1537	1554	A160
171	AGATTTTCAACGCTCTAGCT	171	AGattttcaacgctctagCT	4	rs223-c			1540	1559	A161
172	TACTAGATTTTCAACGC	172	TACTagattttcaaCGC	4	rs223-c			1547	1563	A162
173	TTCAACGCTCTAGCTTCAG	173	TTCaacgctctagcttcAG	4	rs223-c			1536	1554	A163
174	TTTTCAACGCTCTAGCTTCA	174	TTTtcaacgctctagcttCA	4	rs223-c			1537	1556	A164
175	TTTTCAACGCTCTAGC	175	TTttcaacgctcTAGC	4	rs223-c			1541	1556	A165
176	CTTACTAGATTTTCAACGC	176	CTtactagattttcaACGC	4	rs223-c			1547	1565	A166
177	ATTTTCAACGCTCTAGCT	177	ATTTtcaacgctctagCT	4	rs223-c			1540	1557	A167
178	TACTAGATTTTCAACGCT	178	TACtagattttcaacGCT	4	rs223-c			1546	1563	A168
179	TTACTAGATTTTCAACGC	179	TTActagattttcaACGC	4	rs223-c			1547	1564	A169
180	GATTTTCAACGCTCTAGC	180	GAttttcaacgctctAGC	4	rs223-c			1541	1558	A170
181	ATTTTCAACGCTCTAGCTT	181	ATTTtcaacgctctagcTT	4	rs223-c			1539	1557	A171
182	CTAGATTTTCAACGCTCTA	182	CTAgattttcaacgctcTA	4	rs223-c			1543	1561	A172
183	CAACGCTCTAGCTTCAG	183	CAacgctctagcttCAG	4	rs223-c			1536	1552	A173
184	TTCAACGCTCTAGCTTC	184	TTcaacgctctagCTTC	4	rs223-c			1538	1554	A174
185	ACTAGATTTTCAACGCT	185	ACtagattttcaaCGCT	4	rs223-c			1546	1562	A175
186	CTTACTAGATTTTCAACGCT	186	CTTactagattttcaacgCT	4	rs223-c			1546	1565	A176
187	TAGATTTTCAACGCTCTA	187	TAgattttcaacgcTCTA	4	rs223-c			1543	1560	A177
188	TAGATTTTCAACGCTCTAG	188	TAgattttcaacgctcTAG	4	rs223-c			1542	1560	A178
189	TAGATTTTCAACGCTCTAGC	189	TAgattttcaacgctctaGC	4	rs223-c			1541	1560	A179
190	TCTTACTAGATTTTCAACGC	190	TCTtactagattttcaacGC	4	rs223-c			1547	1566	A180
191	CTAGATTTTCAACGCTC	191	CTagattttcaacGCTC	4	rs223-c			1545	1561	A181
192	TTCAACGCTCTAGCTT	192	TTCAacgctctagCTT	4	rs223-c			1539	1554	A182
193	CACTaAGCTTCAGCTTTTC	193	CActctagcttcagctttTC	3	rs223-t			1530	1549	A183
194	CACTCTAGCTTCAGCTTT	194	CActctagcttcagCTTT	3	rs223-t			1532	1549	A184
195	CAACACTCTAGCTTCAGCTT	195	CAacactctagcttcagcTT	3	rs223-t			1533	1552	A185
196	ACACTCTAGCTTCAGCT	196	ACActctagcttcagCT	3	rs223-t			1534	1550	A186
197	AACACTCTAGCTTCAGCT	197	AACActctagcttcagCT	3	rs223-t			1534	1551	A187
198	CAACACTCTAGCTTCAGCT	198	CaacactctagcttcagCT	3	rs223-t			1534	1552	A188
199	TCAACACTCTAGCTTCAGCT	199	TCaacactctagcttcagCT	3	rs223-t			1534	1553	A189
200	AACACTCTAGCTTCAGC	200	AACActctagcttcaGC	3	rs223-t			1535	1551	A190
201	CAACACTCTAGCTTCAGC	201	CAacactctagcttcaGC	3	rs223-t			1535	1552	A191
202	TCAACACTCTAGCTTCAGC	202	TCaacactctagcttcaGC	3	rs223-t			1535	1553	A192
203	TTCAACACTCTAGCTTCAGC	203	TtcaacactctagcttcaGC	3	rs223-t			1535	1554	A193
204	TCAACACTCTAGCTTCAG	204	TCAAcactctagcttcAG	3	rs223-t			1536	1553	A194
205	TTCAACACTCTAGCTTCAG	205	TTcaacactctagcttCAG	3	rs223-t			1536	1554	A195
206	TTTCAACACTCTAGCTTCAG	206	TTtcaacactctagcttCAG	3	rs223-t			1536	1555	A196
207	TCAACACTCTAGCTTCA	207	TCaacactctagcTTCA	3	rs223-t			1537	1553	A197
208	TTCAACACTCTAGCTTCA	208	TTcaacactctagcTTCA	3	rs223-t			1537	1554	A198
209	TTTCAACACTCTAGCTTCA	209	TTtcaacactctagcTTCA	3	rs223-t			1537	1555	A199
210	TTTTCAACACTCTAGCTTCA	210	TTttcaacactctagctTCA	3	rs223-t			1537	1556	A200
211	TTCAACACTCTAGCTTC	211	TTCaacactctagCTTC	3	rs223-t			1538	1554	A201
212	TTTCAACACTCTAGCTTC	212	TTTcaacactctagCTTC	3	rs223-t			1538	1555	A202
213	TTTTCAACACTCTAGCTTC	213	TTTTcaacactctagcTTC	3	rs223-t			1538	1556	A203
214	ATTTTCAACACTCTAGCTTC	214	ATTTtcaacactctagctTC	3	rs223-t			1538	1557	A204
215	TTTCAACACTCTAGCTT	215	TTTcaacactctaGCTT	3	rs223-t			1539	1555	A205
216	TTTTCAACACTCTAGCTT	216	TTttcaacactctaGCTT	3	rs223-t			1539	1556	A206
217	ATTTTCAACACTCTAGCTT	217	ATTTtcaacactctagCTT	3	rs223-t			1539	1557	A207
218	GATTTTCAACACTCTAGCTT	218	GAttttcaacactctagCTT	3	rs223-t			1539	1558	A208
219	TTTTCAACACTCTAGCT	219	TTTTcaacactctaGCT	3	rs223-t			1540	1556	A209
220	ATTTTCAACACTCTAGCT	220	ATTttcaacactctaGCT	3	rs223-t			1540	1557	A210
221	GATTTTCAACACTCTAGCT	221	GATtttcaacactctagCT	3	rs223-t			1540	1558	A211
222	AGATTTTCAACACTCTAGCT	222	AGattttcaacactctagCT	3	rs223-t			1540	1559	A212
223	ATTTTCAACACTCTAGC	223	ATTttcaacactcTAGC	3	rs223-t			1541	1557	A213
224	GATTTTCAACACTCTAGC	224	GATTttcaacactctaGC	3	rs223-t			1541	1558	A214
225	AGATTTTCAACACTCTAGC	225	AGattttcaacactctAGC	3	rs223-t			1541	1559	A215
226	TAGATTTTCAACACTCTAGC	226	TAgattttcaacactctaGC	3	rs223-t			1541	1560	A216
227	GATTTTCAACACTCTAG	227	GATTttcaacactCTAG	3	rs223-t			1542	1558	A217
228	AGATTTTCAACACTCTAG	228	AGATtttcaacactcTAG	3	rs223-t			1542	1559	A218
229	TAGATTTTCAACACTCTAG	229	TAgattttcaacactCTAG	3	rs223-t			1542	1560	A219
230	CTAGATTTTCAACACTCTAG	230	CTagattttcaacactcTAG	3	rs223-t			1542	1561	A220
231	AGATTTTCAACACTCTA	231	AGATtttcaacactCTA	3	rs223-t			1543	1559	A221
232	TAGATTTTCAACACTCTA	232	TAGAttttcaacactCTA	3	rs223-t			1543	1560	A222
233	CTAGATTTTCAACACTCTA	233	CTagattttcaacacTCTA	3	rs223-t			1543	1561	A223
234	ACTAGATTTTCAACACTCTA	234	ACtagattttcaacacTCTA	3	rs223-t			1543	1562	A224
235	TAGATTTTCAACACTCT	235	TAGAttttcaacacTCT	3	rs223-t			1544	1560	A225
236	CTAGATTTTCAACACTCT	236	CTAGattttcaacactCT	3	rs223-t			1544	1561	A226
237	ACTAGATTTTCAACACTCT	237	ACtagattttcaacaCTCT	3	rs223-t			1544	1562	A227
238	TACTAGATTTTCAACACTCT	238	TACtagattttcaacacTCT	3	rs223-t			1544	1563	A228
239	TAGATTTTCAACACTC	239	TAGAttttcaacACTC	3	rs223-t			1545	1560	A229
240	CTAGATTTTCAACACTC	240	CTAGattttcaacACTC	3	rs223-t			1545	1561	A230
241	ACTAGATTTTCAACACTC	241	ACTAgattttcaacaCTC	3	rs223-t			1545	1562	A231
242	TACTAGATTTTCAACACTC	242	TACtagattttcaacACTC	3	rs223-t			1545	1563	A232
243	TTACTAGATTTTCAACACTC	243	TTActagattttcaacACTC	3	rs223-t			1545	1564	A233
244	ACTAGATTTTCAACACT	244	ACTAgattttcaaCACT	3	rs223-t			1546	1562	A234
245	TACTAGATTTTCAACACT	245	TACtagattttcaaCACT	3	rs223-t			1546	1563	A235
246	TTACTAGATTTTCAACACT	246	TTActagattttcaaCACT	3	rs223-t			1546	1564	A236
247	CTTACTAGATTTTCAACACT	247	CTTActagattttcaacaCT	3	rs223-t			1546	1565	A237
248	TACTAGATTTTCAACAC	248	TACTagattttcaACAC	3	rs223-t			1547	1563	A238
249	TTACTAGATTTTCAACAC	249	TTACtagattttcaACAC	3	rs223-t			1547	1564	A239
250	CTTACTAGATTTTCAACAC	250	CTTActagattttcaACAC	3	rs223-t			1547	1565	A240
251	TCTTACTAGATTTTCAACAC	251	TCTtactagattttcaACAC	3	rs223-t			1547	1566	A241
252	CAGCTTGGCGATGATCTC	252	CAGCttggcgatgatcTC	10	rs715-c	3090	3107	12032	12049	A242
253	AGCTTGGCGATGATCTCAT	253	AGcttggcgatgatctcAT	10	rs715-c	3088	3106	12030	12048	A243
254	TCAGCTTGGCGATGATC	254	TCagcttggcgatgATC	10	rs715-c	3092	3108	12034	12050	A244
255	CAGCTTGGCGATGATC	255	CAGcttggcgatgATC	10	rs715-c	3092	3107	12034	12049	A245
256	TCAGCTTGGCGATGATCTCA	256	TCAGcttggcgatgatCTCA	10	rs71S-c	3089	3108	12031	12050	A246
257	CAGCTTGGCGATGATCTCAT	257	CAgcttggcgatgatctcAT	10	rs715-c	3088	3107	12030	12049	A247
258	CTTGGCGATGATCTCAT	258	CTTGgcgatgatctcAT	10	rs715-c	3088	3104	12030	12046	A248
259	CAGCTTGGCGATGATCT	259	CAGcttggcgatgatCT	10	rs715-c	3091	3107	12033	12049	A249
260	TCAGCTTGGCGATGATCT	260	TCagcttggcgatgATCT	10	rs715-c	3091	3108	12033	12050	A250
261	TCAGCTTGGCGATGATCTC	261	TCAGcttggcgatgaTCTC	10	rs715-c	3090	3108	12032	12050	A251
262	GCTTGGCGATGATCTCA	262	GCttggcgatgatctCA	10	rs715-c	3089	3105	12031	12047	A252
263	AGCTTGGCGATGATCTC	263	AGCttggcgatgatcTC	10	rs715-c	3090	3106	12032	12048	A253
264	AGCTTGGCGATGATCTCA	264	AGCttggcgatgatctCA	10	rs715-c	3089	3106	12031	12048	A254
265	CAGCTTGGCGATGATCTCA	265	CAGCttggcgatgatctCA	10	rs715-c	3089	3107	12031	12049	A255
266	GCTTGGCGATGATCTCAT	266	GCttggcgatgatctcAT	10	rs715-c	3088	3105	12030	12047	A256
267	CAATGATCTCATCCAGC	267	CAatgatctcatcCAGC	9	rs715-t	3083	3099	12025	12041	A257
268	GCAATGATCTCATCCAGC	268	GcaatgatctcatccaGC	9	rs715-t	3083	3100	12025	12042	A258
269	GGCAATGATCTCATCCAGC	269	GgCAatgatctcatccaGC	9	rs715-t	3083	3101	12025	12043	A259
270	TGGCAATGATCTCATCCAGC	270	TgGcaatgatctcatcCaGC	9	rs715-t	3083	3102	12025	12044	A260
271	GCAATGATCTCATCCAG	271	GCaatgatctcatcCAG	9	rs715-t	3084	3100	12026	12042	A261
272	GGCAATGATCTCATCCAG	272	GGcaatgatctcatcCAG	9	rs715-t	3084	3101	12026	12043	A262
273	TGGCAATGATCTCATCCAG	273	TGGcaatgatctcatcCAG	9	rs715-t	3084	3102	12026	12044	A263
274	GGCAATGATCTCATCCA	274	GGcaatgatctcatcCA	9	rs715-t	3085	3101	12027	12043	A264
275	TGGCAATGATCTCATCCA	275	TGgcaatgatctcatcCA	9	rs715-t	3085	3102	12027	12044	A265
276	TTGGCAATGATCTCATCCA	276	TTGgcaatgatctcatcCA	9	rs715-t	3085	3103	12027	12045	A266
277	CTTGGCAATGATCTCATCCA	277	CTtggcaatgatctcaTCCA	9	rs715-t	3085	3104	12027	12046	A267
278	TGGCAATGATCTCATCC	278	TGgcaatgatctcaTCC	9	rs715-t	3086	3102	12028	12044	A268
279	TTGGCAATGATCTCATCC	279	TTggcaatgatctcATCC	9	rs715-t	3086	3103	12028	12045	A269
280	CTTGGCAATGATCTCATCC	280	CTtggcaatgatctcATCC	9	rs715-t	3086	3104	12028	12046	A270
281	GCTTGGCAATGATCTCATCC	281	GCttggcaatgatctcatCC	9	rs715-t	3086	3105	12028	12047	A271
282	TTGGCAATGATCTCATC	282	TTGGcaatgatctcATC	9	rs715-t	3087	3103	12029	12045	A272
283	CTTGGCAATGATCTCATC	283	CTtggcaatgatctCATC	9	rs715-t	3087	3104	12029	12046	A273
284	GCTTGGCAATGATCTCATC	284	GCttggcaatgatctcATC	9	rs715-t	3087	3105	12029	12047	A274
285	AGCTTGGCAATGATCTCATC	285	AGcttggcaatgatctcATC	9	rs715-t	3087	3106	12029	12048	A275
286	GCTTGGCAATGATCTCAT	286	GCTtggcaatgatctcAT	9	rs715-t	3088	3105	12030	12047	A276
287	AGCTTGGCAATGATCTCAT	287	AGcttggcaatgatctCAT	9	rs715-t	3088	3106	12030	12048	A277
288	CAGCTTGGCAATGATCTCAT	288	CAgcttggcaatgatctcAT	9	rs715-t	3088	3107	12030	12049	A278
289	GCTTGGCAATGATCTCA	289	GCttggcaatgatctCA	9	rs715-t	3089	3105	12031	12047	A279
290	AGCTTGGCAATGATCTCA	290	AGcttggcaatgatctCA	9	rs715-t	3089	3106	12031	12048	A280
291	CAGCTTGGCAATGATCTCA	291	CAgcttggcaatgatctCA	9	rs715-t	3089	3107	12031	12049	A281
292	TCAGCTTGGCAATGATCTCA	292	TCAgcttggcaatgatctCA	9	rs715-t	3089	3108	12031	12050	A282
293	AGCTTGGCAATGATCTC	293	AGcttggcaatgatCTC	9	rs715-t	3090	3106	12032	12048	A283
294	CAGCTTGGCAATGATCTC	294	CAGcttggcaatgatcTC	9	rs715-t	3090	3107	12032	12049	A284
295	TCAGCTTGGCAATGATCTC	295	TCAgcttggcaatgatCTC	9	rs715-t	3090	3108	12032	12050	A285
296	CAGCTTGGCAATGATCT	296	CAgcttggcaatgaTCT	9	rs715-t	3091	3107	12033	12049	A286
297	TCAGCTTGGCAATGATCT	297	TCAgcttggcaatgatCT	9	rs715-t	3091	3108	12033	12050	A287
298	TCAGCTTGGCAATGATC	298	TCagcttggcaatGATC	9	rs715-t	3092	3108	12034	12050	A288
260	TCAGCTTGGCGATGATCT	260,001	TcaGctTgGcgatgaTCT	10	rs715-c	3091	3108	12033	12050	B1
260	TCAGCTTGGCGATGATCT	260,002	TcaGcTtggcgaTgAtCT	10	rs715-c	3091	3108	12033	12050	B2
259	CAGCTTGGCGATGATCT	259,001	CAgcttggCgaTgAtCT	10	rs715-c	3091	3107	12033	12049	B3
259	CAGCTTGGCGATGATCT	259,002	CAGcTtggcgatgAtCT	10	rs715-c	3091	3107	12033	12049	B4
259	CAGCTTGGCGATGATCT	259,003	CAGcttggcgatgATCT	10	rs715-c	3091	3107	12033	12049	B5
259	CAGCTTGGCGATGATCT	259,004	CAgcTtggcgatgaTCT	10	rs715-c	3091	3107	12033	12049	B6
259	CAGCTTGGCGATGATCT	259,005	CaGcTTgGcgatgatCT	10	rs715-c	3091	3107	12033	12049	B7
259	CAGCTTGGCGATGATCT	259,006	CAgcTTggcgatGatCT	10	rs715-c	3091	3107	12033	12049	B8
260	TCAGCTTGGCGATGATCT	260,003	TcAgcTtGgCgatgatCT	10	rs715-c	3091	3108	12033	12050	B9
259	CAGCTTGGCGATGATCT	259,007	CagCttGgcgatgaTCT	10	rs715-c	3091	3107	12033	12049	B10
259	CAGCTTGGCGATGATCT	259,008	CaGcTtggcgaTGatCT	10	rs715-c	3091	3107	12033	12049	B11
260	TCAGCTTGGCGATGATCT	260,004	TCagCttggcgatgatCT	10	rs715-c	3091	3108	12033	12050	B12
259	CAGCTTGGCGATGATCT	259,009	CAGctTggcgatGatCT	10	rs715-c	3091	3107	12033	12049	B13
259	CAGCTTGGCGATGATCT	259,010	CAgCttggcgatgAtCT	10	rs715-c	3091	3107	12033	12049	B14
259	CAGCTTGGCGATGATCT	259,011	CagCttggcgAtGaTCT	10	rs715-c	3091	3107	12033	12049	B15
259	CAGCTTGGCGATGATCT	259,012	CAGCttggcgatgaTCT	10	rs715-c	3091	3107	12033	12049	B16
259	CAGCTTGGCGATGATCT	259,013	CaGcTTgGcgatgaTCT	10	rs715-c	3091	3107	12033	12049	B17
259	CAGCTTGGCGATGATCT	259,014	CAgcttggcgaTgAtCT	10	rs715-c	3091	3107	12033	12049	B18
259	CAGCTTGGCGATGATCT	259,015	CagCttGgcgatGaTCT	10	rs715-c	3091	3107	12033	12049	B19
259	CAGCTTGGCGATGATCT	259,016	CagCTtggCgatgaTCT	10	rs715-c	3091	3107	12033	12049	B20
260	TCAGCTTGGCGATGATCT	260,005	TCagcttGgcgatgATCT	10	rs715-c	3091	3108	12033	12050	B21
259	CAGCTTGGCGATGATCT	259,017	CaGcTTggcgatgAtCT	10	rs715-c	3091	3107	12033	12049	B22
259	CAGCTTGGCGATGATCT	259,018	CaGCttggcgatGatCT	10	rs715-c	3091	3107	12033	12049	B23
259	CAGCTTGGCGATGATCT	259,019	CAgcttggcgAtGaTCT	10	rs715-c	3091	3107	12033	12049	B24
259	CAGCTTGGCGATGATCT	259,020	CaGCTtggcgAtgatCT	10	rs715-c	3091	3107	12033	12049	B25
259	CAGCTTGGCGATGATCT	259,021	CaGCttggcgatgAtCT	10	rs71S-c	3091	3107	12033	12049	B26
260	TCAGCTTGGCGATGATCT	260,006	TCagcttggcgatgATCT	10	rs715-c	3091	3108	12033	12050	B27
259	CAGCTTGGCGATGATCT	259,022	CagCTtggcgaTGatCT	10	rs715-c	3091	3107	12033	12049	B28
260	TCAGCTTGGCGATGATCT	260,007	TcaGcTtGgCgatgatCT	10	rs715-c	3091	3108	12033	12050	B29
260	TCAGCTTGGCGATGATCT	260,008	TCagctTggcgatgATCT	10	rs715-c	3091	3108	12033	12050	B30
259	CAGCTTGGCGATGATCT	259,023	CagcttggcgATgAtCT	10	rs715-c	3091	3107	12033	12049	B31
259	CAGCTTGGCGATGATCT	259,024	CAgCttggcgatgatCT	10	rs715-c	3091	3107	12033	12049	B32
259	CAGCTTGGCGATGATCT	259,025	CAgctTggcgaTgAtCT	10	rs715-c	3091	3107	12033	12049	B33
259	CAGCTTGGCGATGATCT	259,026	CagCttggcgatGatCT	10	rs715-c	3091	3107	12033	12049	B34
260	TCAGCTTGGCGATGATCT	260,009	TcAgCttggcgATgAtCT	10	rs715-c	3091	3108	12033	12050	B35
259	CAGCTTGGCGATGATCT	259,027	CAgcttggcgATGatCT	10	rs715-c	3091	3107	12033	12049	B36
260	TCAGCTTGGCGATGATCT	260,010	TCagCttggcgatGaTCT	10	rs715-c	3091	3108	12033	12050	B37
259	CAGCTTGGCGATGATCT	259,028	CAgcttggcgaTgatCT	10	rs715-c	3091	3107	12033	12049	B38
259	CAGCTTGGCGATGATCT	259,029	CAgCttggcgAtgatCT	10	rs715-c	3091	3107	12033	12049	B39
259	CAGCTTGGCGATGATCT	259,030	CAgcttggCgatgatCT	10	rs715-c	3091	3107	12033	12049	B40
260	TCAGCTTGGCGATGATCT	260,011	TCagcttggCgatgaTCT	10	rs715-c	3091	3108	12033	12050	B41
260	TCAGCTTGGCGATGATCT	260,012	TCAGcttggcgatgATCT	10	rs715-c	3091	3108	12033	12050	B42
259	CAGCTTGGCGATGATCT	259,031	CAgcttggcgatgaTCT	10	rs715-c	3091	3107	12033	12049	B43
260	TCAGCTTGGCGATGATCT	260,013	TCAGcttggcgatgaTCT	10	rs715-c	3091	3108	12033	12050	B44
260	TCAGCTTGGCGATGATCT	260,014	TcAgcTTgGcgatgaTCT	10	rs715-c	3091	3108	12033	12050	B45
259	CAGCTTGGCGATGATCT	259,032	CaGcTtggcgatGatCT	10	rs715-c	3091	3107	12033	12049	B46
259	CAGCTTGGCGATGATCT	259,033	CagCttGgcgatgATCT	10	rs715-c	3091	3107	12033	12049	B47
260	TCAGCTTGGCGATGATCT	260,015	TCagcttggcGatgATCT	10	rs715-c	3091	3108	12033	12050	B48
259	CAGCTTGGCGATGATCT	259,034	CagCtTggcgatgAtCT	10	rs715-c	3091	3107	12033	12049	B49
260	TCAGCTTGGCGATGATCT	260,016	TCAgCttggcgatGaTCT	10	rs715-c	3091	3108	12033	12050	B50
260	TCAGCTTGGCGATGATCT	260,017	TCagcttggcGatgaTCT	10	rs715-c	3091	3108	12033	12050	B51
260	TCAGCTTGGCGATGATCT	260,018	TcaGcTTggcgatgAtCT	10	rs715-c	3091	3108	12033	12050	B52
260	TCAGCTTGGCGATGATCT	260,019	TcaGcTtggcgaTgATCT	10	rs715-c	3091	3108	12033	12050	B53
260	TCAGCTTGGCGATGATCT	260,020	TcAgcTtggcgaTgAtCT	10	rs715-c	3091	3108	12033	12050	B54
260	TCAGCTTGGCGATGATCT	260,021	TCaGcttggcgatgaTCT	10	rs715-c	3091	3108	12033	12050	B55
260	TCAGCTTGGCGATGATCT	260,022	TCAGcttggcgatgatCT	10	rs715-c	3091	3108	12033	12050	B56
260	TCAGCTTGGCGATGATCT	260,023	TcAgCttggcgaTgAtCT	10	rs715-c	3091	3108	12033	12050	B57
259	CAGCTTGGCGATGATCT	259,035	CAgCtTggcGatgatCT	10	rs715-c	3091	3107	12033	12049	B58
259	CAGCTTGGCGATGATCT	259,036	CagCTtggCGatgatCT	10	rs715-c	3091	3107	12033	12049	B59
259	CAGCTTGGCGATGATCT	259,037	CagCttGgcgatGAtCT	10	rs715-c	3091	3107	12033	12049	B60
259	CAGCTTGGCGATGATCT	259,038	CagCttGgcGatgatCT	10	rs715-c	3091	3107	12033	12049	B61
259	CAGCTTGGCGATGATCT	259,039	CAgcttggcgatGatCT	10	rs715-c	3091	3107	12033	12049	B62
260	TCAGCTTGGCGATGATCT	260,024	TcaGcttggcgAtgAtCT	10	rs715-c	3091	3108	12033	12050	B63
259	CAGCTTGGCGATGATCT	259,040	CagCttggcgaTgAtCT	10	rs715-c	3091	3107	12033	12049	B64
259	CAGCTTGGCGATGATCT	259,041	CAgcttggCgatgaTCT	10	rs715-c	3091	3107	12033	12049	B65
259	CAGCTTGGCGATGATCT	259,042	CAgCttggcgaTgatCT	10	rs715-c	3091	3107	12033	12049	B66
259	CAGCTTGGCGATGATCT	259,043	CagCttGgcgatGatCT	10	rs715-c	3091	3107	12033	12049	B67
259	CAGCTTGGCGATGATCT	259,044	CaGcTtggcgatGaTCT	10	rs715-c	3091	3107	12033	12049	B68
260	TCAGCTTGGCGATGATCT	260,025	TcaGcTtGgcgatgATCT	10	rs715-c	3091	3108	12033	12050	B69
259	CAGCTTGGCGATGATCT	259,045	CagCttGgCGatgatCT	10	rs715-c	3091	3107	12033	12049	B70
259	CAGCTTGGCGATGATCT	259,046	CaGCttGgcGatgatCT	10	rs715-c	3091	3107	12033	12049	B71
259	CAGCTTGGCGATGATCT	259,047	CaGCttggcgAtgatCT	10	rs715-c	3091	3107	12033	12049	B72
260	TCAGCTTGGCGATGATCT	260,026	TcAGcTtGgcgatgaTCT	10	rs715-c	3091	3108	12033	12050	B73
259	CAGCTTGGCGATGATCT	259,048	CagCttggCgatgatCT	10	rs715-c	3091	3107	12033	12049	B74
260	TCAGCTTGGCGATGATCT	260,027	TcAGctTggcgatgAtCT	10	rs715-c	3091	3108	12033	12050	B75
259	CAGCTTGGCGATGATCT	259,049	CAgcttggcgatGaTCT	10	rs715-c	3091	3107	12033	12049	B76
259	CAGCTTGGCGATGATCT	259,050	CagCttGgCgatgatCT	10	rs715-c	3091	3107	12033	12049	B77
259	CAGCTTGGCGATGATCT	259,051	CagCttggcgAtgatCT	10	rs715-c	3091	3107	12033	12049	B78
259	CAGCTTGGCGATGATCT	259,052	CaGcTtggcgatGAtCT	10	rs715-c	3091	3107	12033	12049	B79
259	CAGCTTGGCGATGATCT	259,052	CaGcTtggcgatGAtCT	10	rs715-c	3091	3107	12033	12049	B79
259	CAGCTTGGCGATGATCT	259,053	CAgcTtgGcgatgaTCT	10	rs715-c	3091	3107	12033	12049	B80
259	CAGCTTGGCGATGATCT	259,054	CAgcttGgcgatgAtCT	10	rs715-c	3091	3107	12033	12049	B81
260	TCAGCTTGGCGATGATCT	260,028	TCagCttggcgatgaTCT	10	rs715-c	3091	3108	12033	12050	B82
260	TCAGCTTGGCGATGATCT	260,029	TcAgCttGgcgatgaTCT	10	rs715-c	3091	3108	12033	12050	B83
259	CAGCTTGGCGATGATCT	259,055	CAGcTtggcgatgatCT	10	rs715-c	3091	3107	12033	12049	B84
260	TCAGCTTGGCGATGATCT	260,030	TCAgcttggcgatgaTCT	10	rs715-c	3091	3108	12033	12050	B85
259	CAGCTTGGCGATGATCT	259,056	CagCtTggCgatgatCT	10	rs715-c	3091	3107	12033	12049	B86
260	TCAGCTTGGCGATGATCT	260,031	TCAgcttggcgatgATCT	10	rs715-c	3091	3108	12033	12050	B87
260	TCAGCTTGGCGATGATCT	260,032	TcAgcTtGgcgatgaTCT	10	rs715-c	3091	3108	12033	12050	B88
259	CAGCTTGGCGATGATCT	259,057	CagCTtggcgAtGatCT	10	rs715-c	3091	3107	12033	12049	B89
259	CAGCTTGGCGATGATCT	259,058	CaGCttggcgaTgatCT	10	rs715-c	3091	3107	12033	12049	B90
260	TCAGCTTGGCGATGATCT	260,033	TCaGcttggcgatGaTCT	10	rs715-c	3091	3108	12033	12050	B91
259	CAGCTTGGCGATGATCT	259,059	CagCTtggcgaTgaTCT	10	rs715-c	3091	3107	12033	12049	B92
259	CAGCTTGGCGATGATCT	259,060	CaGctTggcgatgATCT	10	rs715-c	3091	3107	12033	12049	B93
259	CAGCTTGGCGATGATCT	259,061	CAgcttggcGatgAtCT	10	rs715-c	3091	3107	12033	12049	B94
259	CAGCTTGGCGATGATCT	259,062	CagCTtggcgAtgatCT	10	rs715-c	3091	3107	12033	12049	B95
259	CAGCTTGGCGATGATCT	259,063	CAgcTtGgcgatgaTCT	10	rs715-c	3091	3107	12033	12049	B96
260	TCAGCTTGGCGATGATCT	260,034	TcAgCTtGgcgatgaTCT	10	rs715-c	3091	3108	12033	12050	B97
260	TCAGCTTGGCGATGATCT	260,035	TcAgcTTggcgatGatCT	10	rs715-c	3091	3108	12033	12050	B98
260	TCAGCTTGGCGATGATCT	260,036	TCagcttggcgatGaTCT	10	rs715-c	3091	3108	12033	12050	B99
259	CAGCTTGGCGATGATCT	259,064	CAgcttggcGatGatCT	10	rs715-c	3091	3107	12033	12049	B100
259	CAGCTTGGCGATGATCT	259,065	CagCTtggcgaTgatCT	10	rs715-c	3091	3107	12033	12049	B101
259	CAGCTTGGCGATGATCT	259,066	CAgCTtggcgaTgatCT	10	rs715-c	3091	3107	12033	12049	B102
259	CAGCTTGGCGATGATCT	259,067	CagCtTggCGatgatCT	10	rs715-c	3091	3107	12033	12049	B103
259	CAGCTTGGCGATGATCT	259,068	CAgcTtggcgatgatCT	10	rs715-c	3091	3107	12033	12049	B104
259	CAGCTTGGCGATGATCT	259,069	CagCTtgGcgatgaTCT	10	rs715-c	3091	3107	12033	12049	B105
259	CAGCTTGGCGATGATCT	259,070	CAgcttggcgaTgaTCT	10	rs715-c	3091	3107	12033	12049	B106
259	CAGCTTGGCGATGATCT	259,071	CagCTTggCgatgatCT	10	rs715-c	3091	3107	12033	12049	B107
259	CAGCTTGGCGATGATCT	259,072	CaGcttggcgATgaTCT	10	rs715-c	3091	3107	12033	12049	B108
260	TCAGCTTGGCGATGATCT	260,037	TcaGctTgGcgatgatCT	10	rs715-c	3091	3108	12033	12050	B109
260	TCAGCTTGGCGATGATCT	260,038	TcaGcTTggCgatgatCT	10	rs715-c	3091	3108	12033	12050	B110
260	TCAGCTTGGCGATGATCT	260,039	TCaGctTggcgatgATCT	10	rs715-c	3091	3108	12033	12050	B111
259	CAGCTTGGCGATGATCT	259,073	CAgctTggcgatgaTCT	10	rs715-c	3091	3107	12033	12049	B112
260	TCAGCTTGGCGATGATCT	260,040	TcAgCttggcgAtGatCT	10	rs715-c	3091	3108	12033	12050	B113
259	CAGCTTGGCGATGATCT	259,074	CAgcTtgGcgatgatCT	10	rs715-c	3091	3107	12033	12049	B114
260	TCAGCTTGGCGATGATCT	260,041	TCagCttggcgAtGatCT	10	rs715-c	3091	3108	12033	12050	B115
259	CAGCTTGGCGATGATCT	259,075	CAgcttggcgAtgAtCT	10	rs715-c	3091	3107	12033	12049	B116
260	TCAGCTTGGCGATGATCT	260,042	TcAGcTtggcgatGatCT	10	rs715-c	3091	3108	12033	12050	B117
259	CAGCTTGGCGATGATCT	259,076	CAgcttggcgatgatCT	10	rs715-c	3091	3107	12033	12049	B118
259	CAGCTTGGCGATGATCT	259,077	CagCTTggcgaTgAtCT	10	rs715-c	3091	3107	12033	12049	B119
259	CAGCTTGGCGATGATCT	259,078	CAGcttggcgaTgAtCT	10	rs715-c	3091	3107	12033	12049	B120
259	CAGCTTGGCGATGATCT	259,079	CaGcttggcgAtGatCT	10	rs715-c	3091	3107	12033	12049	B121
259	CAGCTTGGCGATGATCT	259,080	CAgcTtggcgatGatCT	10	rs715-c	3091	3107	12033	12049	B122
260	TCAGCTTGGCGATGATCT	260,043	TcAgcTtggcgatGAtCT	10	rs715-c	3091	3108	12033	12050	B123
259	CAGCTTGGCGATGATCT	259,081	CAGctTggcgatgatCT	10	rs715-c	3091	3107	12033	12049	B124
259	CAGCTTGGCGATGATCT	259,082	CagCTtGgcgatgaTCT	10	rs715-c	3091	3107	12033	12049	B125
260	TCAGCTTGGCGATGATCT	260,044	TcaGctTggcgatgATCT	10	rs715-c	3091	3108	12033	12050	B126
260	TCAGCTTGGCGATGATCT	260,045	TCagcTtggcgatgaTCT	10	rs715-c	3091	3108	12033	12050	B127
259	CAGCTTGGCGATGATCT	259,083	CAgcttGgcgatgatCT	10	rs715-c	3091	3107	12033	12049	B128
260	TCAGCTTGGCGATGATCT	260,046	TcaGcTTggcgatGatCT	10	rs715-c	3091	3108	12033	12050	B129
259	CAGCTTGGCGATGATCT	259,084	CaGcTtGgCgatgatCT	10	rs715-c	3091	3107	12033	12049	B130
259	CAGCTTGGCGATGATCT	259,085	CagCtTggcgatGatCT	10	rs715-c	3091	3107	12033	12049	B131
259	CAGCTTGGCGATGATCT	259,086	CAgCttGgcgatGatCT	10	rs715-c	3091	3107	12033	12049	B132
259	CAGCTTGGCGATGATCT	259,087	CagCTtGgcgatGatCT	10	rs715-c	3091	3107	12033	12049	B133
259	CAGCTTGGCGATGATCT	259,088	CagCttggCGatgatCT	10	rs715-c	3091	3107	12033	12049	B134
259	CAGCTTGGCGATGATCT	259,089	CagCTTggcgatGatCT	10	rs715-c	3091	3107	12033	12049	B135
259	CAGCTTGGCGATGATCT	259,090	CaGcTtGgcgatgaTCT	10	rs715-c	3091	3107	12033	12049	B136
259	CAGCTTGGCGATGATCT	259,091	CAgCTtGgcGatgatCT	10	rs715-c	3091	3107	12033	12049	B137
259	CAGCTTGGCGATGATCT	259,092	CagCttGgcgatgAtCT	10	rs715-c	3091	3107	12033	12049	B138
260	TCAGCTTGGCGATGATCT	260,047	TcAgcTTggcgatgAtCT	10	rs715-c	3091	3108	12033	12050	B139
259	CAGCTTGGCGATGATCT	259,093	CAgcttggCgatgAtCT	10	rs715-c	3091	3107	12033	12049	B140
260	TCAGCTTGGCGATGATCT	260,048	TCagcttggcgaTgaTCT	10	rs715-c	3091	3108	12033	12050	B141
260	TCAGCTTGGCGATGATCT	260,049	TcaGcTTgGcgatgatCT	10	rs715-c	3091	3108	12033	12050	B142
259	CAGCTTGGCGATGATCT	259,094	CaGcTtGgcgatgAtCT	10	rs715-c	3091	3107	12033	12049	B143
259	CAGCTTGGCGATGATCT	259,095	CaGctTgGcgatgatCT	10	rs715-c	3091	3107	12033	12049	B144
259	CAGCTTGGCGATGATCT	259,096	CagCttggcgATgaTCT	10	rs715-c	3091	3107	12033	12049	B145
259	CAGCTTGGCGATGATCT	259,097	CagCttggcgAtgATCT	10	rs715-c	3091	3107	12033	12049	B146
259	CAGCTTGGCGATGATCT	259,098	CAGcttggcgatgaTCT	10	rs715-c	3091	3107	12033	12049	B147
260	TCAGCTTGGCGATGATCT	260,050	TcaGcTtGgcgatgAtCT	10	rs715-c	3091	3108	12033	12050	B148
259	CAGCTTGGCGATGATCT	259,099	CAgcttggcgatgATCT	10	rs715-c	3091	3107	12033	12049	B149
260	TCAGCTTGGCGATGATCT	260,051	TCaGcttggcgatgATCT	10	rs715-c	3091	3108	12033	12050	B150
259	CAGCTTGGCGATGATCT	259,100	CagCTtgGcgatgatCT	10	rs715-c	3091	3107	12033	12049	B151
260	TCAGCTTGGCGATGATCT	260,052	TcAgCttggcgaTgaTCT	10	rs715-c	3091	3108	12033	12050	B152
259	CAGCTTGGCGATGATCT	259,101	CAGcttggcgatgAtCT	10	rs715-c	3091	3107	12033	12049	B153
259	CAGCTTGGCGATGATCT	259,102	CaGctTggCgatgatCT	10	rs715-c	3091	3107	12033	12049	B154
259	CAGCTTGGCGATGATCT	259,103	CagCttggcgatGaTCT	10	rs715-c	3091	3107	12033	12049	B1S5
260	TCAGCTTGGCGATGATCT	260,053	TCagcttgGcgatgaTCT	10	rs715-c	3091	3108	12033	12050	B156
259	CAGCTTGGCGATGATCT	259,104	CagCTtggcgatGatCT	10	rs715-c	3091	3107	12033	12049	B157
260	TCAGCTTGGCGATGATCT	260,054	TCagCttggcgaTgAtCT	10	rs715-c	3091	3108	12033	12050	B158
259	CAGCTTGGCGATGATCT	259,105	CAgcttgGcgatgAtCT	10	rs715-c	3091	3107	12033	12049	B159
259	CAGCTTGGCGATGATCT	259,106	CagcttggcgATGAtCT	10	rs715-c	3091	3107	12033	12049	B160
259	CAGCTTGGCGATGATCT	259,107	CAgcttGgcgatgaTCT	10	rs715-c	3091	3107	12033	12049	B161
259	CAGCTTGGCGATGATCT	259,108	CAgCTtggcgAtgatCT	10	rs715-c	3091	3107	12033	12049	B162
259	CAGCTTGGCGATGATCT	259,109	CaGCTtggcgatGatCT	10	rs715-c	3091	3107	12033	12049	B163
260	TCAGCTTGGCGATGATCT	260,055	TcaGcttggcgAtgATCT	10	rs715-c	3091	3108	12033	12050	B164
260	TCAGCTTGGCGATGATCT	260,056	TCagcttggCgatgATCT	10	rs715-c	3091	3108	12033	12050	B165
259	CAGCTTGGCGATGATCT	259,110	CagCttggcgATgAtCT	10	rs715-c	3091	3107	12033	12049	B166
259	CAGCTTGGCGATGATCT	259,111	CAgCttggcgatGatCT	10	rs715-c	3091	3107	12033	12049	B167
259	CAGCTTGGCGATGATCT	259,112	CAgCttggcgatgaTCT	10	rs715-c	3091	3107	12033	12049	B168
259	CAGCTTGGCGATGATCT	259,113	CagCTtggcgATgAtCT	10	rs715-c	3091	3107	12033	12049	B169
259	CAGCTTGGCGATGATCT	259,114	CagCttggcgatgAtCT	10	rs715-c	3091	3107	12033	12049	B170
260	TCAGCTTGGCGATGATCT	260,057	TcAgcTTggCgatgatCT	10	rs715-c	3091	3108	12033	12050	B171
259	CAGCTTGGCGATGATCT	259,115	CAGCttggcgatgatCT	10	rs715-c	3091	3107	12033	12049	B172
259	CAGCTTGGCGATGATCT	259,116	CAgCttggcgaTgAtCT	10	rs715-c	3091	3107	12033	12049	B173
259	CAGCTTGGCGATGATCT	259,117	CagCttggcgaTgATCT	10	rs715-c	3091	3107	12033	12049	B174
260	TCAGCTTGGCGATGATCT	260,058	TCagcttggcgatgaTCT	10	rs715-c	3091	3108	12033	12050	B175
259	CAGCTTGGCGATGATCT	259,118	CAgcttggcGatGAtCT	10	rs715-c	3091	3107	12033	12049	B176
259	CAGCTTGGCGATGATCT	259,119	CAgcTtGgcgatgAtCT	10	rs715-c	3091	3107	12033	12049	B177
259	CAGCTTGGCGATGATCT	259,120	CagCTtGgcgatgAtCT	10	rs715-c	3091	3107	12033	12049	B178
259	CAGCTTGGCGATGATCT	259,121	CaGctTggcgatgAtCT	10	rs715-c	3091	3107	12033	12049	B179
260	TCAGCTTGGCGATGATCT	260,059	TCAgcTtggcgatGaTCT	10	rs715-c	3091	3108	12033	12050	B180
260	TCAGCTTGGCGATGATCT	260,060	TCagcttggcgAtgATCT	10	rs715-c	3091	3108	12033	12050	B181
260	TCAGCTTGGCGATGATCT	260,061	TcaGcTtggcgatGatCT	10	rs715-c	3091	3108	12033	12050	B182
259	CAGCTTGGCGATGATCT	259,122	CagCttggcgATgatCT	10	rs715-c	3091	3107	12033	12049	B183
259	CAGCTTGGCGATGATCT	259,123	CagCTTggcgatgAtCT	10	rs715-c	3091	3107	12033	12049	B184
260	TCAGCTTGGCGATGATCT	260,062	TcAgcTtGgcgatgAtCT	10	rs715-c	3091	3108	12033	12050	B185
259	CAGCTTGGCGATGATCT	259,124	CagCttggcgATGatCT	10	rs715-c	3091	3107	12033	12049	B186
260	TCAGCTTGGCGATGATCT	260,063	TcaGcTtgGcgatgaTCT	10	rs715-c	3091	3108	12033	12050	B187
259	CAGCTTGGCGATGATCT	259,125	CaGcTtggcgaTgAtCT	10	rs715-c	3091	3107	12033	12049	B188
259	CAGCTTGGCGATGATCT	259,126	CAgcttggcgATgAtCT	10	rs715-c	3091	3107	12033	12049	B189
260	TCAGCTTGGCGATGATCT	260,064	TCagcTtggcgatgATCT	10	rs715-c	3091	3108	12033	12050	B190
259	CAGCTTGGCGATGATCT	259,127	CagCtTggcgaTgAtCT	10	rs715-c	3091	3107	12033	12049	B191
259	CAGCTTGGCGATGATCT	259,128	CagCTTggcgaTgatCT	10	rs715-c	3091	3107	12033	12049	B192
260	TCAGCTTGGCGATGATCT	260,065	TCagCttggcgaTgatCT	10	rs715-c	3091	3108	12033	12050	B193
259	CAGCTTGGCGATGATCT	259,129	CAgcttggcgAtgaTCT	10	rs715-c	3091	3107	12033	12049	B194
260	TCAGCTTGGCGATGATCT	260,066	TcaGcTtGgcgatgaTCT	10	rs715-c	3091	3108	12033	12050	B195
260	TCAGCTTGGCGATGATCT	260,067	TcAgcTTgGcgatgatCT	10	rs715-c	3091	3108	12033	12050	B196
259	CAGCTTGGCGATGATCT	259,130	CAgcTtggcgaTgAtCT	10	rs715-c	3091	3107	12033	12049	B197
260	TCAGCTTGGCGATGATCT	260,068	TcAgCttGgcgatgAtCT	10	rs715-c	3091	3108	12033	12050	B198
259	CAGCTTGGCGATGATCT	259,131	CagCTtggcgatGAtCT	10	rs715-c	3091	3107	12033	12049	B199
260	TCAGCTTGGCGATGATCT	260,069	cAgcTtggcgaTgaTCT	10	rs715-c	3091	3108	12033	12050	B200
259	CAGCTTGGCGATGATCT	259,132	CagCttggcgAtgAtCT	10	rs715-c	3091	3107	12033	12049	B201
259	CAGCTTGGCGATGATCT	259,133	CAgCTtggcgatGatCT	10	rs715-c	3091	3107	12033	12049	B202
259	CAGCTTGGCGATGATCT	259,134	CAgcttgGcgatgatCT	10	rs715-c	3091	3107	12033	12049	B203
259	CAGCTTGGCGATGATCT	259,135	CagCttggcgatgatCT	10	rs715-c	3091	3107	12033	12049	B204
260	TCAGCTTGGCGATGATCT	260,070	TcaGcttggcgATgAtCT	10	rs715-c	3091	3108	12033	12050	B205
259	CAGCTTGGCGATGATCT	259,136	CaGcTtggcgaTgaTCT	10	rs715-c	3091	3107	12033	12049	B206
259	CAGCTTGGCGATGATCT	259,137	CaGCttggCgatgatCT	10	rs715-c	3091	3107	12033	12049	B207
259	CAGCTTGGCGATGATCT	259,138	CagCtTggcGatgatCT	10	rs715-c	3091	3107	12033	12049	B208
260	TCAGCTTGGCGATGATCT	260,071	TCagCttggcgatGatCT	10	rs715-c	3091	3108	12033	12050	B209
260	TCAGCTTGGCGATGATCT	260,072	TcaGctTggcgatGatCT	10	rs715-c	3091	3108	12033	12050	B210
260	TCAGCTTGGCGATGATCT	260,073	TCagCttGgcgatgATCT	10	rs715-c	3091	3108	12033	12050	B211
260	TCAGCTTGGCGATGATCT	260,074	TcaGcTTgGcgatgaTCT	10	rs715-c	3091	3108	12033	12050	B212
259	CAGCTTGGCGATGATCT	259,139	CagCttggcgaTgaTCT	10	rs715-c	3091	3107	12033	12049	B213
259	CAGCTTGGCGATGATCT	259,140	CAgcttggcgAtGatCT	10	rs715-c	3091	3107	12033	12049	B214
259	CAGCTTGGCGATGATCT	259,141	CAgcttggcGAtgAtCT	10	rs715-c	3091	3107	12033	12049	B215
259	CAGCTTGGCGATGATCT	259,142	CAgCTtggCgatgatCT	10	rs715-c	3091	3107	12033	12049	B216
259	CAGCTTGGCGATGATCT	259,143	CaGcTTggCgatgatCT	10	rs715-c	3091	3107	12033	12049	B217
259	CAGCTTGGCGATGATCT	259,144	CagCttggcgAtGAtCT	10	rs715-c	3091	3107	12033	12049	B218
259	CAGCTTGGCGATGATCT	259,145	CagCTtggcgatGaTCT	10	rs715-c	3091	3107	12033	12049	B219
260	TCAGCTTGGCGATGATCT	260,075	TcaGcTtggcgatGAtCT	10	rs715-c	3091	3108	12033	12050	B220
260	TCAGCTTGGCGATGATCT	260,076	TcAGctTggcgatGatCT	10	rs715-c	3091	3108	12033	12050	B221
259	CAGCTTGGCGATGATCT	259,146	CaGcTTggcgatGatCT	10	rs715-c	3091	3107	12033	12049	B222
259	CAGCTTGGCGATGATCT	259,147	CAgcTtggcgatgAtCT	10	rs715-c	3091	3107	12033	12049	B223
259	CAGCTTGGCGATGATCT	259,148	CaGcttggcgAtgAtCT	10	rs715-c	3091	3107	12033	12049	B224
259	CAGCTTGGCGATGATCT	259,149	CAgctTggcgatgAtCT	10	rs715-c	3091	3107	12033	12049	B225
259	CAGCTTGGCGATGATCT	259,150	CaGcttggcgATgAtCT	10	rs715-c	3091	3107	12033	12049	B226
260	TCAGCTTGGCGATGATCT	260,077	TcaGcTtggcgaTgaTCT	10	rs715-c	3091	3108	12033	12050	B227
259	CAGCTTGGCGATGATCT	259,151	CAgcttggcGaTgAtCT	10	rs715-c	3091	3107	12033	12049	B228
260	TCAGCTTGGCGATGATCT	260,078	TcaGcTtgGcgatgatCT	10	rs715-c	3091	3108	12033	12050	B229
259	CAGCTTGGCGATGATCT	259,152	CagCttggcgaTgatCT	10	rs715-c	3091	3107	12033	12049	B230
259	CAGCTTGGCGATGATCT	259,153	CAgCttggcgATgatCT	10	rs715-c	3091	3107	12033	12049	B231
259	CAGCTTGGCGATGATCT	259,154	CaGcTtgGcgatgatCT	10	rs715-c	3091	3107	12033	12049	B232
260	TCAGCTTGGCGATGATCT	260,079	TCagcttGgcgatgaTCT	10	rs715-c	3091	3108	12033	12050	B233
259	CAGCTTGGCGATGATCT	259,155	CagCTtggCgatgatCT	10	rs715-c	3091	3107	12033	12049	B234
260	TCAGCTTGGCGATGATCT	260,080	TcaGctTggCgatgatCT	10	rs715-c	3091	3108	12033	12050	B235
260	TCAGCTTGGCGATGATCT	260,081	TCAgCttGgcgatGaTCT	10	rs715-c	3091	3108	12033	12050	B236
259	CAGCTTGGCGATGATCT	259,156	CAgcttggcGAtGatCT	10	rs715-c	3091	3107	12033	12049	B237
259	CAGCTTGGCGATGATCT	259,157	CAgCttggcgAtGatCT	10	rs715-c	3091	3107	12033	12049	B238
260	TCAGCTTGGCGATGATCT	260,082	TCagcttggcgaTgATCT	10	rs715-c	3091	3108	12033	12050	B239
259	CAGCTTGGCGATGATCT	259,158	CAgcttgGcgaTgAtCT	10	rs715-c	3091	3107	12033	12049	B240
259	CAGCTTGGCGATGATCT	259,159	CagCttgGCgatgatCT	10	rs715-c	3091	3107	12033	12049	B241
259	CAGCTTGGCGATGATCT	259,160	CAgcttggcgAtgATCT	10	rs715-c	3091	3107	12033	12049	B242
259	CAGCTTGGCGATGATCT	259,161	CAgcttggCgatGatCT	10	rs715-c	3091	3107	12033	12049	B243
260	TCAGCTTGGCGATGATCT	260,083	TCAgcTtggcgatGatCT	10	rs715-c	3091	3108	12033	12050	B244
259	CAGCTTGGCGATGATCT	259,162	CagCTtGgCgatgatCT	10	rs715-c	3091	3107	12033	12049	B245
259	CAGCTTGGCGATGATCT	259,163	CaGcTtgGcgatgaTCT	10	rs715-c	3091	3107	12033	12049	B246
260	TCAGCTTGGCGATGATCT	260,084	TcAgCttggcgAtgAtCT	10	rs715-c	3091	3108	12033	12050	B247
260	TCAGCTTGGCGATGATCT	260,085	TcAgcTtgGcgatgatCT	10	rs715-c	3091	3108	12033	12050	B248
259	CAGCTTGGCGATGATCT	259,164	CaGctTgGcgatgaTCT	10	rs715-c	3091	3107	12033	12049	B249
259	CAGCTTGGCGATGATCT	259,165	CagCttggcgAtGatCT	10	rs715-c	3091	3107	12033	12049	B250
259	CAGCTTGGCGATGATCT	259,166	CagCttggcgaTGatCT	10	rs715-c	3091	3107	12033	12049	B251
259	CAGCTTGGCGATGATCT	259,167	CAgcttggcgatgAtCT	10	rs715-c	3091	3107	12033	12049	B252
259	CAGCTTGGCGATGATCT	259,168	CAgctTggcgatgatCT	10	rs715-c	3091	3107	12033	12049	B253
260	TCAGCTTGGCGATGATCT	260,086	TCagctTggcgatgaTCT	10	rs715-c	3091	3108	12033	12050	B254
259	CAGCTTGGCGATGATCT	259,169	CAgcttGgcgaTgAtCT	10	rs715-c	3091	3107	12033	12049	B255
259	CAGCTTGGCGATGATCT	259,170	CagCTtggcgaTgAtCT	10	rs715-c	3091	3107	12033	12049	B256
259	CAGCTTGGCGATGATCT	259,171	CagCttggcgatGAtCT	10	rs715-c	3091	3107	12033	12049	B257
260	TCAGCTTGGCGATGATCT	260,087	TcAGcttggcgAtGatCT	10	rs715-c	3091	3108	12033	12050	B258
260	TCAGCTTGGCGATGATCT	260,088	TcAgcTtggcgatGatCT	10	rs715-c	3091	3108	12033	12050	B259
260	TCAGCTTGGCGATGATCT	260,089	TCagcttggcgAtgaTCT	10	rs715-c	3091	3108	12033	12050	B260
259	CAGCTTGGCGATGATCT	259,172	CaGctTggcgatGatCT	10	rs715-c	3091	3107	12033	12049	B261
260	TCAGCTTGGCGATGATCT	260,090	TcAgcTtGgcgatgATCT	10	rs715-c	3091	3108	12033	12050	B262
260	TCAGCTTGGCGATGATCT	260,091	TcaGctTggcgatGAtCT	10	rs715-c	3091	3108	12033	12050	B263
259	CAGCTTGGCGATGATCT	259,173	CAGcTtggcgatGatCT	10	rs715-c	3091	3107	12033	12049	B264
260	TCAGCTTGGCGATGATCT	260,092	TCagCttggcgAtgAtCT	10	rs715-c	3091	3108	12033	12050	B265
259	CAGCTTGGCGATGATCT	259,174	CAgcttgGcgatgaTCT	10	rs715-c	3091	3107	12033	12049	B266
259	CAGCTTGGCGATGATCT	259,175	CAGcttggcgatGatCT	10	rs715-c	3091	3107	12033	12049	B267
260	TCAGCTTGGCGATGATCT	260,093	TCagCTtggcgatGatCT	10	rs715-c	3091	3108	12033	12050	B268
259	CAGCTTGGCGATGATCT	259,176	CagCTtGgcGatgatCT	10	rs715-c	3091	3107	12033	12049	B269
259	CAGCTTGGCGATGATCT	259,177	CagCTtggcgATgatCT	10	rs715-c	3091	3107	12033	12049	B270
259	CAGCTTGGCGATGATCT	259,178	CAGcTtggcgatgaTCT	10	rs715-c	3091	3107	12033	12049	B271
260	TCAGCTTGGCGATGATCT	260,094	TCagcttggcgatgatCT	10	rs715-c	3091	3108	12033	12050	B272
260	TCAGCTTGGCGATGATCT	260,095	TcaGcttggcgAtGatCT	10	rs715-c	3091	3108	12033	12050	B273
260	TCAGCTTGGCGATGATCT	260,096	TCagcttgGcgatgATCT	10	rs715-c	3091	3108	12033	12050	B274
259	CAGCTTGGCGATGATCT	259,179	CAgCttGgcGatgatCT	10	rs715-c	3091	3107	12033	12049	B275
260	TCAGCTTGGCGATGATCT	260,097	TcAgcTtgGcgatgaTCT	10	rs715-c	3091	3108	12033	12050	B276
259	CAGCTTGGCGATGATCT	259,180	CaGctTGgcgatgaTCT	10	rs715-c	3091	3107	12033	12049	B277
259	CAGCTTGGCGATGATCT	259,181	CAgcttggcGatgaTCT	10	rs715-c	3091	3107	12033	12049	B278
260	TCAGCTTGGCGATGATCT	260,098	TCAgcttggcgaTgATCT	10	rs715-c	3091	3108	12033	12050	B279
260	TCAGCTTGGCGATGATCT	260,099	TCaGcTtggcgatgATCT	10	rs715-c	3091	3108	12033	12050	B280
259	CAGCTTGGCGATGATCT	259,182	CAgcttggcGatgatCT	10	rs715-c	3091	3107	12033	12049	B281
259	CAGCTTGGCGATGATCT	259,183	CaGCttggcgatgatCT	10	rs715-c	3091	3107	12033	12049	B282
259	CAGCTTGGCGATGATCT	259,184	CagCtTggcgaTgatCT	10	rs715-c	3091	3107	12033	12049	B283
259	CAGCTTGGCGATGATCT	259,185	CaGcttggcgAtgATCT	10	rs715-c	3091	3107	12033	12049	B284
259	CAGCTTGGCGATGATCT	259,186	CAGCttggcgatgATCT	10	rs715-c	3091	3107	12033	12049	B285
259	CAGCTTGGCGATGATCT	259,187	CAgCttggCgatgatCT	10	rs715-c	3091	3107	12033	12049	B286
259	CAGCTTGGCGATGATCT	259,188	CAgcttggcgAtgatCT	10	rs715-c	3091	3107	12033	12049	B287
259	CAGCTTGGCGATGATCT	259,189	CagCTtggcGatgatCT	10	rs715-c	3091	3107	12033	12049	B288
260	TCAGCTTGGCGATGATCT	260,100	TcaGctTggcgatgAtCT	10	rs715-c	3091	3108	12033	12050	B289
260	TCAGCTTGGCGATGATCT	260,101	TCAGctTggcgatGaTCT	10	rs715-c	3091	3108	12033	12050	B290
280	CTTGGCAATGATCTCATCC	280,001	CTtgGcaatgatCtcATCC	9	rs715-t	3086	3104	12028	12046	B291
280	CTTGGCAATGATCTCATCC	280,002	CtTggCaatgatCtcAtCC	9	rs715-t	3086	3104	12028	12046	B292
280	CTTGGCAATGATCTCATCC	280,003	CttggcaaTgAtcTcAtCC	9	rs715-t	3086	3104	12028	12046	B293
280	CTTGGCAATGATCTCATCC	280,004	CTTGgcaatgatctcatCC	9	rs715-t	3086	3104	12028	12046	B294
280	CTTGGCAATGATCTCATCC	280,005	CttggCaatgatctCatCC	9	rs715-t	3086	3104	12028	12046	B295
280	CTTGGCAATGATCTCATCC	280,006	CTtggcaatgAtctCaTCC	9	rs715-t	3086	3104	12028	12046	B296
280	CTTGGCAATGATCTCATCC	280,007	CTtggcaaTgATctCatCC	9	rs715-t	3086	3104	12028	12046	B297
280	CTTGGCAATGATCTCATCC	280,008	CTTggcaatgatcTcaTCC	9	rs715-t	3086	3104	12028	12046	B298
280	CTTGGCAATGATCTCATCC	280,009	CttggCaatgatctCAtCC	9	rs715-t	3086	3104	12028	12046	B299
280	CTTGGCAATGATCTCATCC	280,010	CttGgCaatgatCtcatCC	9	rs715-t	3086	3104	12028	12046	B300
280	CTTGGCAATGATCTCATCC	280,011	CTtggcAatgatCtcAtCC	9	rs715-t	3086	3104	12028	12046	B301
280	CTTGGCAATGATCTCATCC	280,012	CttGgcaatgAtCtcatCC	9	rs715-t	3086	3104	12028	12046	B302
280	CTTGGCAATGATCTCATCC	280,013	CTtggcaatGatctCaTCC	9	rs715-t	3086	3104	12028	12046	B303
280	CTTGGCAATGATCTCATCC	280,014	CTtggcaatGaTctCatCC	9	rs715-t	3086	3104	12028	12046	B304
280	CTTGGCAATGATCTCATCC	280,015	CttgGcaatgatCtcatCC	9	rs715-t	3086	3104	12028	12046	B305
280	CTTGGCAATGATCTCATCC	280,016	CttGgCaatgatctCatCC	9	rs715-t	3086	3104	12028	12046	B306
280	CTTGGCAATGATCTCATCC	280,017	CttggcaAtgatCtcAtCC	9	rs715-t	3086	3104	12028	12046	B307
280	CTTGGCAATGATCTCATCC	280,018	CTTggcaatgATctCatCC	9	rs715-t	3086	3104	12028	12046	B308
280	CTTGGCAATGATCTCATCC	280,019	CtTggCaatgatcTcAtCC	9	rs715-t	3086	3104	12028	12046	B309
280	CTTGGCAATGATCTCATCC	280,020	CttggcaATgaTctCatCC	9	rs715-t	3086	3104	12028	12046	B310
280	CTTGGCAATGATCTCATCC	280,021	CttggcaatgAtCtcAtCC	9	rs715-t	3086	3104	12028	12046	B311
280	CTTGGCAATGATCTCATCC	280,022	CttGgcaatgATctCatCC	9	rs715-t	3086	3104	12028	12046	B312
280	CTTGGCAATGATCTCATCC	280,023	CTtggcaatGatcTcaTCC	9	rs715-t	3086	3104	12028	12046	B313
280	CTTGGCAATGATCTCATCC	280,024	CTtggcaAtgatCtcatCC	9	rs715-t	3086	3104	12028	12046	B314
280	CTTGGCAATGATCTCATCC	280,025	CttggcaatgatCtcAtCC	9	rs715-t	3086	3104	12028	12046	B315
280	CTTGGCAATGATCTCATCC	280,026	CTtggcaatgatctcatCC	9	rs715-t	3086	3104	12028	12046	B316
280	CTTGGCAATGATCTCATCC	280,027	CttggcAatgatCtcAtCC	9	rs715-t	3086	3104	12028	12046	B317
280	CTTGGCAATGATCTCATCC	280,028	CTtggcaatgatCtCaTCC	9	rs715-t	3086	3104	12028	12046	B318
280	CTTGGCAATGATCTCATCC	280,029	CttggcaatgatCtcATCC	9	rs715-t	3086	3104	12028	12046	B319
280	CTTGGCAATGATCTCATCC	280,030	CtTggcaatgatCtcAtCC	9	rs715-t	3086	3104	12028	12046	B320
280	CTTGGCAATGATCTCATCC	280,031	CttGgcAatgatCtcatCC	9	rs715-t	3086	3104	12028	12046	B321
280	CTTGGCAATGATCTCATCC	280,032	CTTGgcaatgatctcATCC	9	rs715-t	3086	3104	12028	12046	B322
280	CTTGGCAATGATCTCATCC	280,033	CttggcAatgatCtCatCC	9	rs715-t	3086	3104	12028	12046	B323
280	CTTGGCAATGATCTCATCC	280,034	CTtggcaatgatctCaTCC	9	rs715-t	3086	3104	12028	12046	B324
280	CTTGGCAATGATCTCATCC	280,035	CtTggcaatgATcTcAtCC	9	rs715-t	3086	3104	12028	12046	B325
280	CTTGGCAATGATCTCATCC	280,036	CttGgcaatgaTcTcAtCC	9	rs715-t	3086	3104	12028	12046	B326
280	CTTGGCAATGATCTCATCC	280,037	CTTggCaatgatCtcAtCC	9	rs715-t	3086	3104	12028	12046	B327
280	CTTGGCAATGATCTCATCC	280,038	CttggcaATgAtcTcAtCC	9	rs715-t	3086	3104	12028	12046	B328
280	CTTGGCAATGATCTCATCC	280,039	CttggcaaTgATcTcAtCC	9	rs715-t	3086	3104	12028	12046	B329
280	CTTGGCAATGATCTCATCC	280,040	CttggcaaTgaTctCatCC	9	rs715-t	3086	3104	12028	12046	B330
280	CTTGGCAATGATCTCATCC	280,041	CttggcaaTgATctCatCC	9	rs715-t	3086	3104	12028	12046	B331
280	CTTGGCAATGATCTCATCC	280,042	CTtggcAatgatctcATCC	9	rs715-t	3086	3104	12028	12046	B332
280	CTTGGCAATGATCTCATCC	280,043	CtTggcAatgatCtcAtCC	9	rs715-t	3086	3104	12028	12046	B333
280	CTTGGCAATGATCTCATCC	280,044	CTtggcaatgatcTcATCC	9	rs715-t	3086	3104	12028	12046	B334
280	CTTGGCAATGATCTCATCC	280,045	CTtggcAatgatCtCatCC	9	rs715-t	3086	3104	12028	12046	B335
280	CTTGGCAATGATCTCATCC	280,046	CTtggcaatGaTcTcAtCC	9	rs715-t	3086	3104	12028	12046	B336
280	CTTGGCAATGATCTCATCC	280,047	CttggcaAtgAtCtcatCC	9	rs715-t	3086	3104	12028	12046	B337
280	CTTGGCAATGATCTCATCC	280,048	CTtggcaatgAtctcATCC	9	rs715-t	3086	3104	12028	12046	B338
280	CTTGGCAATGATCTCATCC	280,049	CTtggcaAtgatctcATCC	9	rs715-t	3086	3104	12028	12046	B339
280	CTTGGCAATGATCTCATCC	280,050	CttggcaatGatctCAtCC	9	rs715-t	3086	3104	12028	12046	B340
280	CTTGGCAATGATCTCATCC	280,051	CttggcAatgAtCtcatCC	9	rs715-t	3086	3104	12028	12046	B341
280	CTTGGCAATGATCTCATCC	280,052	CTtGgcaatgatctcATCC	9	rs715-t	3086	3104	12028	12046	B342
280	CTTGGCAATGATCTCATCC	280,053	CttggcaatgaTCtcatCC	9	rs715-t	3086	3104	12028	12046	B343
280	CTTGGCAATGATCTCATCC	280,054	CttggcAAtGatCtcatCC	9	rs715-t	3086	3104	12028	12046	B344
280	CTTGGCAATGATCTCATCC	280,055	CTtggcaAtgatcTcaTCC	9	rs715-t	3086	3104	12028	12046	B345
280	CTTGGCAATGATCTCATCC	280,056	CTtgGcaatgatCtCatCC	9	rs715-t	3086	3104	12028	12046	B346
280	CTTGGCAATGATCTCATCC	280,057	CTtggcaatgatcTCaTCC	9	rs715-t	3086	3104	12028	12046	B347
280	CTTGGCAATGATCTCATCC	280,058	CttggcaatGatCtcAtCC	9	rs715-t	3086	3104	12028	12046	B348
280	CTTGGCAATGATCTCATCC	280,059	CtTgGcaatgaTctCatCC	9	rs715-t	3086	3104	12028	12046	B349
280	CTTGGCAATGATCTCATCC	280,060	CTtggcaatGAtcTcAtCC	9	rs715-t	3086	3104	12028	12046	B350
280	CTTGGCAATGATCTCATCC	280,061	CttggcAatgatCtcaTCC	9	rs715-t	3086	3104	12028	12046	B351
280	CTTGGCAATGATCTCATCC	280,062	CttGgcaatgaTcTCatCC	9	rs715-t	3086	3104	12028	12046	B352
280	CTTGGCAATGATCTCATCC	280,063	CttggcAAtgAtCtcatCC	9	rs715-t	3086	3104	12028	12046	B353
280	CTTGGCAATGATCTCATCC	280,064	CttggcaatgatctCAtCC	9	rs715-t	3086	3104	12028	12046	B354
280	CTTGGCAATGATCTCATCC	280,065	CTtggcaaTgatctcATCC	9	rs715-t	3086	3104	12028	12046	B355
280	CTTGGCAATGATCTCATCC	280,066	CTTggCaatgatcTcAtCC	9	rs715-t	3086	3104	12028	12046	B356
280	CTTGGCAATGATCTCATCC	280,067	CtTgGcaatgatCtcAtCC	9	rs715-t	3086	3104	12028	12046	B357
280	CTTGGCAATGATCTCATCC	280,068	CttggcaaTgatCtcAtCC	9	rs715-t	3086	3104	12028	12046	B358
280	CTTGGCAATGATCTCATCC	280,069	CTTGgcaatgatctcaTCC	9	rs715-t	3086	3104	12028	12046	B359
280	CTTGGCAATGATCTCATCC	280,070	CttggcAatgatCtcATCC	9	rs715-t	3086	3104	12028	12046	B360
280	CTTGGCAATGATCTCATCC	280,071	CttggcaAtgatcTCatCC	9	rs715-t	3086	3104	12028	12046	B361
280	CTTGGCAATGATCTCATCC	280,072	CTtgGcaatgatCTcAtCC	9	rs715-t	3086	3104	12028	12046	B362
280	CTTGGCAATGATCTCATCC	280,073	CttGgCaatgatcTCatCC	9	rs715-t	3086	3104	12028	12046	B363
280	CTTGGCAATGATCTCATCC	280,074	CTtggcaaTgAtcTcAtCC	9	rs715-t	3086	3104	12028	12046	B364
280	CTTGGCAATGATCTCATCC	280,075	CTTggcaatGatCtcAtCC	9	rs715-t	3086	3104	12028	12046	B365
280	CTTGGCAATGATCTCATCC	280,076	CTtGgCaatgatcTcAtCC	9	rs715-t	3086	3104	12028	12046	B366
280	CTTGGCAATGATCTCATCC	280,077	CttGgcaatgATcTcAtCC	9	rs715-t	3086	3104	12028	12046	B367
280	CTTGGCAATGATCTCATCC	280,078	CttGgcaatgAtCtcAtCC	9	rs715-t	3086	3104	12028	12046	B368
280	CTTGGCAATGATCTCATCC	280,079	CttggcaAtgAtcTcAtCC	9	rs715-t	3086	3104	12028	12046	B369
280	CTTGGCAATGATCTCATCC	280,080	CttGgcaatgatCtcAtCC	9	rs715-t	3086	3104	12028	12046	B370
280	CTTGGCAATGATCTCATCC	280,081	CttggcAatgatCtcatCC	9	rs715-t	3086	3104	12028	12046	B371
280	CTTGGCAATGATCTCATCC	280,082	CttggCaatgatCtcAtCC	9	rs715-t	3086	3104	12028	12046	B372
280	CTTGGCAATGATCTCATCC	280,083	CttgGcaAtgatctCatCC	9	rs715-t	3086	3104	12028	12046	B373
280	CTTGGCAATGATCTCATCC	280,084	CttggcaatgAtCtcatCC	9	rs715-t	3086	3104	12028	12046	B374
280	CTTGGCAATGATCTCATCC	280,085	CtTggcaatgatCTcaTCC	9	rs715-t	3086	3104	12028	12046	B375
280	CTTGGCAATGATCTCATCC	280,086	CttgGcaatgatCtcAtCC	9	rs715-t	3086	3104	12028	12046	B376
280	CTTGGCAATGATCTCATCC	280,087	CTTggcaatgatctcaTCC	9	rs715-t	3086	3104	12028	12046	B377
280	CTTGGCAATGATCTCATCC	280,088	CTtggcaaTgaTcTcAtCC	9	rs715-t	3086	3104	12028	12046	B378
280	CTTGGCAATGATCTCATCC	280,089	CTtggcaatgaTctcATCC	9	rs715-t	3086	3104	12028	12046	B379
280	CTTGGCAATGATCTCATCC	280,090	CttgGcaatgAtCtcatCC	9	rs715-t	3086	3104	12028	12046	B380
280	CTTGGCAATGATCTCATCC	280,091	CttggcAAtgatcTCatCC	9	rs715-t	3086	3104	12028	12046	B381
280	CTTGGCAATGATCTCATCC	280,092	CTtgGcaatgatcTcaTCC	9	rs715-t	3086	3104	12028	12046	B382
280	CTTGGCAATGATCTCATCC	280,093	CtTggcaatgatctCAtCC	9	rs715-t	3086	3104	12028	12046	B383
280	CTTGGCAATGATCTCATCC	280,094	CTtggcaatGatCtcAtCC	9	rs715-t	3086	3104	12028	12046	B384
280	CTTGGCAATGATCTCATCC	280,095	CttGgcaatgAtcTcAtCC	9	rs715-t	3086	3104	12028	12046	B385
280	CTTGGCAATGATCTCATCC	280,096	CTtggcaatgaTctCaTCC	9	rs715-t	3086	3104	12028	12046	B386
280	CTTGGCAATGATCTCATCC	280,097	CttggcaAtgATctCatCC	9	rs715-t	3086	3104	12028	12046	B387
280	CTTGGCAATGATCTCATCC	280,098	CTtgGcaatgaTctCatCC	9	rs715-t	3086	3104	12028	12046	B388
280	CTTGGCAATGATCTCATCC	280,099	CTtgGcaatgaTcTcAtCC	9	rs715-t	3086	3104	12028	12046	B389
280	CTTGGCAATGATCTCATCC	280,100	CtTggcaatgATctCatCC	9	rs715-t	3086	3104	12028	12046	B390
280	CTTGGCAATGATCTCATCC	280,101	CTtggcaatgatCtcATCC	9	rs715-t	3086	3104	12028	12046	B391
280	CTTGGCAATGATCTCATCC	280,102	CTtggCaatgatcTcaTCC	9	rs715-t	3086	3104	12028	12046	B392
280	CTTGGCAATGATCTCATCC	280,103	CttggcaAtGatCtcatCC	9	rs715-t	3086	3104	12028	12046	B393
280	CTTGGCAATGATCTCATCC	280,104	CTtggCaatgatctcATCC	9	rs715-t	3086	3104	12028	12046	B394
280	CTTGGCAATGATCTCATCC	280,105	CttGgcAatgatctCatCC	9	rs715-t	3086	3104	12028	12046	B395
280	CTTGGCAATGATCTCATCC	280,106	CttggcAatgaTCtcatCC	9	rs715-t	3086	3104	12028	12046	B396
280	CTTGGCAATGATCTCATCC	280,107	CttgGcAatgatCtcAtCC	9	rs715-t	3086	3104	12028	12046	B397
280	CTTGGCAATGATCTCATCC	280,108	CttggcaAtgatCtcatCC	9	rs715-t	3086	3104	12028	12046	B398
280	CTTGGCAATGATCTCATCC	280,109	CtTggcaatgAtcTcAtCC	9	rs715-t	3086	3104	12028	12046	B399
280	CTTGGCAATGATCTCATCC	280,110	CTtggcaatgaTcTcaTCC	9	rs715-t	3086	3104	12028	12046	B400
280	CTTGGCAATGATCTCATCC	280,111	CttggcAAtgatCtCatCC	9	rs715-t	3086	3104	12028	12046	B401
280	CTTGGCAATGATCTCATCC	280,112	CTtggcaAtgatctCaTCC	9	rs715-t	3086	3104	12028	12046	B402
280	CTTGGCAATGATCTCATCC	280,113	CttGgcaatgatctCAtCC	9	rs715-t	3086	3104	12028	12046	B403
280	CTTGGCAATGATCTCATCC	280,114	CTTggcaatgatctcatCC	9	rs715-t	3086	3104	12028	12046	B404
280	CTTGGCAATGATCTCATCC	280,115	CTtggcaaTgatcTcaTCC	9	rs715-t	3086	3104	12028	12046	B405
280	CTTGGCAATGATCTCATCC	280,116	CttGgCaatgatCtcAtCC	9	rs715-t	3086	3104	12028	12046	B406
280	CTTGGCAATGATCTCATCC	280,117	CttGgcaatgaTctCatCC	9	rs715-t	3086	3104	12028	12046	B407
280	CTTGGCAATGATCTCATCC	280,118	CttGgCaatgatcTcAtCC	9	rs715-t	3086	3104	12028	12046	B408
280	CTTGGCAATGATCTCATCC	280,119	CTtggcaaTgaTctCatCC	9	rs715-t	3086	3104	12028	12046	B409
280	CTTGGCAATGATCTCATCC	280,120	CTtGgcaatgAtcTcAtCC	9	rs715-t	3086	3104	12028	12046	B410
280	CTTGGCAATGATCTCATCC	280,121	CtTggcaatGaTctCatCC	9	rs715-t	3086	3104	12028	12046	B411
280	CTTGGCAATGATCTCATCC	280,122	CTtggcaatGatctcATCC	9	rs715-t	3086	3104	12028	12046	B412
280	CTTGGCAATGATCTCATCC	280,123	CttggcaatgatCtcatCC	9	rs715-t	3086	3104	12028	12046	B413
280	CTTGGCAATGATCTCATCC	280,124	CTtgGcaatgatctCaTCC	9	rs715-t	3086	3104	12028	12046	B414
280	CTTGGCAATGATCTCATCC	280,125	CttggcAatgatctCaTCC	9	rs715-t	3086	3104	12028	12046	B415
280	CTTGGCAATGATCTCATCC	280,126	CTtGgcaatgatCtcAtCC	9	rs715-t	3086	3104	12028	12046	B416
280	CTTGGCAATGATCTCATCC	280,127	CTtggcaatgatctcaTCC	9	rs715-t	3086	3104	12028	12046	B417
280	CTTGGCAATGATCTCATCC	280,128	CttggcAAtgatCtcAtCC	9	rs715-t	3086	3104	12028	12046	B418
280	CTTGGCAATGATCTCATCC	280,129	CTtggcaaTgatctCaTCC	9	rs715-t	3086	3104	12028	12046	B419
280	CTTGGCAATGATCTCATCC	280,130	CttggcAatgAtCtcAtCC	9	rs715-t	3086	3104	12028	12046	B420
280	CTTGGCAATGATCTCATCC	280,131	CttggcAATgatctCatCC	9	rs715-t	3086	3104	12028	12046	B421
280	CTTGGCAATGATCTCATCC	280,132	CttggcAatGatCtcAtCC	9	rs715-t	3086	3104	12028	12046	B422
280	CTTGGCAATGATCTCATCC	280,133	CTtgGcaatgatCtcATCC	9	rs715-t	3086	3104	12028	12046	B423
280	CTTGGCAATGATCTCATCC	280,134	CTtGgcaatgatcTcaTCC	9	rs715-t	3086	3104	12028	12046	B424
280	CTTGGCAATGATCTCATCC	280,135	CTtGgcaatgatctCaTCC	9	rs715-t	3086	3104	12028	12046	B425
280	CTTGGCAATGATCTCATCC	280,136	CTtggcaatGatCtCatCC	9	rs715-t	3086	3104	12028	12046	B426
280	CTTGGCAATGATCTCATCC	280,137	CtTggcaatGatCtcAtCC	9	rs715-t	3086	3104	12028	12046	B427
280	CTTGGCAATGATCTCATCC	280,138	CTtgGcaatgatCtcAtCC	9	rs715-t	3086	3104	12028	12046	B428
280	CTTGGCAATGATCTCATCC	280,139	CTtGgcaatgaTctCatCC	9	rs715-t	3086	3104	12028	12046	B429
280	CTTGGCAATGATCTCATCC	280,140	CttggcaaTgaTcTcAtCC	9	rs715-t	3086	3104	12028	12046	B430
280	CTTGGCAATGATCTCATCC	280,141	CttggcaAtgATcTcAtCC	9	rs715-t	3086	3104	12028	12046	B431
280	CTTGGCAATGATCTCATCC	280,142	CTTggcaatgAtcTcAtCC	9	rs715-t	3086	3104	12028	12046	B432
280	CTTGGCAATGATCTCATCC	280,143	CTtGgcaatgaTcTcAtCC	9	rs715-t	3086	3104	12028	12046	B433
280	CTTGGCAATGATCTCATCC	280,144	CTtggcaatgAtcTcaTCC	9	rs715-t	3086	3104	12028	12046	B434
280	CTTGGCAATGATCTCATCC	280,145	CttgGcaatgatctCatCC	9	rs715-t	3086	3104	12028	12046	B435
280	CTTGGCAATGATCTCATCC	280,146	CTTggcaatgatctcATCC	9	rs715-t	3086	3104	12028	12046	B436
280	CTTGGCAATGATCTCATCC	280,147	CttggcaatgatCtcaTCC	9	rs715-t	3086	3104	12028	12046	B437
280	CTTGGCAATGATCTCATCC	280,148	CTtggCaatgatctCaTCC	9	rs715-t	3086	3104	12028	12046	B438
280	CTTGGCAATGATCTCATCC	280,149	CTTggcaatgatctCaTCC	9	rs715-t	3086	3104	12028	12046	B439
280	CTTGGCAATGATCTCATCC	280,150	CttgGcaatgatctCAtCC	9	rs715-t	3086	3104	12028	12046	B440
280	CTTGGCAATGATCTCATCC	280,151	CTtggcaatgatCTcaTCC	9	rs715-t	3086	3104	12028	12046	B441
280	CTTGGCAATGATCTCATCC	280,152	CttGgcAatgatCtcAtCC	9	rs715-t	3086	3104	12028	12046	B442
280	CTTGGCAATGATCTCATCC	280,153	CttggcaatgAtCtCatCC	9	rs715-t	3086	3104	12028	12046	B443
280	CTTGGCAATGATCTCATCC	280,154	CttGgcaAtgatctCatCC	9	rs715-t	3086	3104	12028	12046	B444
280	CTTGGCAATGATCTCATCC	280,155	CTtGgcaatgatCTcaTCC	9	rs715-t	3086	3104	12028	12046	B445
280	CTTGGCAATGATCTCATCC	280,156	CttgGcAAtgatctCatCC	9	rs715-t	3086	3104	12028	12046	B446
280	CTTGGCAATGATCTCATCC	280,157	CTtggcAatgatctCaTCC	9	rs715-t	3086	3104	12028	12046	B447
280	CTTGGCAATGATCTCATCC	280,158	CttggcAAtgatCtcatCC	9	rs715-t	3086	3104	12028	12046	B448
280	CTTGGCAATGATCTCATCC	280,159	CttggCaatgatCtcatCC	9	rs715-t	3086	3104	12028	12046	B449
280	CTTGGCAATGATCTCATCC	280,160	CTtggcAatgatcTcaTCC	9	rs715-t	3086	3104	12028	12046	B450
299	TCGAGGTTAAATGGCTT	299	TCgaggttaaatgGCTT					1049	1065	S1
300	ATCGAGGTTAAATGGCTT	300	ATCGaggttaaatggCTT					1049	1066	S2
301	GGTCAGGGTAATGGTCA	301	GGtcagggtaatggtCA					3374	3390	S3
302	AAACATGAAGGGGATGGA	302	AAACatgaaggggaTGGA					3423	3440	S4
303	GGAAATGTTTCTGAAGGG	303	GGAAatgtttctgaagGG					3533	3550	SS
304	GAATGGGAAATGTTTCTG	304	GAATgggaaatgtttCTG					3538	3555	S6
305	AAGTTGGTAGGGCTGGA	305	AAgttggtagggctgGA					3557	3573	S7
306	AAAGTTGGTAGGGCTGG	306	AAAGttggtagggctGG					3558	3574	S8
307	AAAAGTTGGTAGGGCTGG	307	AAaagttggtagggcTGG					3558	3575	S9
308	AAAAGTTGGTAGGGCTG	308	AAaagttggtaggGCTG					3559	3575	S10
309	GAAAAGTTGGTAGGGCTG	309	GAAaagttggtagggCTG					3559	3576	511
310	GAAAAGTTGGTAGGGCT	310	GAAaagttggtaggGCT					3560	3576	S12
311	GGAAAAGTTGGTAGGGCT	311	GGaaaagttggtagggCT					3560	3577	S13
312	AGAGACTTAAAGAGGAGA	312	AGAgacttaaagaggAGA					4400	4417	S14
313	GGTTGTTGGTGATCAG	313	GGttgttggtgatCAG			1035	1050	5064	5079	S15
314	ATGCATAATCGTAGGG	314	ATGcataatcgtAGGG			1050	1065	5079	5094	S16
315	AAAGGATGTAAGATGCA	315	AAAGgatgtaagaTGCA					5616	5632	S17
316	AAAGGATGTAAGATGC	316	AAAGgatgtaagATGC					5617	5632	S18
317	ACAAAGGATGTAAGATG	317	ACAAaggatgtaaGATG					5618	5634	S19
318	AACAAAGGATGTAAGATG	318	AACAaaggatgtaaGATG					5618	5635	S20
319	CTATTTTGTCTATGGTGT	319	CTATtttgtctatggtGT					7269	7286	S21
320	CTATTTTGTCTATGGTG	320	CTattttgtctatGGTG					7270	7286	S22
321	CAGGTGGTTGTCAAACA	321	CAggtggttgtcaAACA			1786	1802	7901	7917	S23
322	CATTGAAGTGGTGGGGTG	322	CAttgaagtggtggggTG					8208	8225	S24
323	GATGGAGAGAATTCGAGA	323	GATGgagagaattcgaGA					8749	8766	S25
324	CGTACAAAGTGGGGATG	324	CGtacaaagtgggGATG			2127	2143	8894	8910	S26
325	ACGTACAAAGTGGGGATG	325	ACGtacaaagtggggATG			2127	2144	8894	8911	S27
326	CGTACAAAGTGGGGAT	326	CGtacaaagtggGGAT			2128	2143	8895	8910	S28
327	ACGTACAAAGTGGGGA	327	ACGtacaaagtggGGA			2129	2144	8896	8911	S29
328	AACGTACAAAGTGGGG	328	AACGtacaaagtGGGG			2130	2145	8897	8912	S30
329	AGTAGGAGGAGTCTGTGA	329	AGtaggaggagtctgtGA					9605	9622	S31
330	AAGTAGGAGGAGTCTGTG	330	AAGtaggaggagtctgTG					9606	9623	S32
331	GAAGTAGGAGGAGTCTGT	331	GAAgtaggaggagtctGT					9607	9624	S33
332	GGAAGTAGGAGGAGTCTG	332	GGaagtaggaggagtcTG					9608	9625	S34
333	GGAGGGGAAGAGTTTCAG	333	GGaggggaagagtttcAG					11413	11430	S35
334	TCTTGCAGGTAGAGGGAA	334	TCttgcaggtagagggAA					11503	11520	S36
335	GAGTGATAAGTGAGTCA	335	GAGTgataagtgagtCA					12620	12636	S37
336	TTATTAGGGGACTGTGAG	336	TTAttaggggactgtGAG					13243	13260	S38
337	GAAGGCTGTTATTTTCAT	337	GAAggctgttatttTCAT					13794	13811	S39
338	GGAAGGCTGTTATTTTCA	338	GGaaggctgttatttTCA					13795	13812	S40
339	AGGAGGGGATCTGAGAAC	339	AGgaggggatctgagAAC					15987	16004	S41
340	AAGGAGGGGATCTGAGAA	340	AAGgaggggatctgaGAA					15988	16005	S42
341	AGGCGTTCTTGAGTTTG	341	AGgcgttcttgagttTG			4577	4593	18493	18509	S43
342	AAATGATCTGTACCAGG	342	AAAtgatctgtacCAGG					21431	21447	S44
343	AAGTTCTGGAGGGTAGGG	343	AAgttctggagggtagGG					22659	22676	S45
344	TGAGAGGGGTCTGATGG	344	TGagaggggtctgatGG					22756	22772	S46
*For Compounds, capital letters = LNA nucleosides, lower case letter = DNA nucleosides, optionally all internucleoside linkages are phosphorothioate.
In the examples, capital letters = beta-D-oxy-LNA nucleosides, LNA cytosines = 5 methyl cytosine LNA, lower case letters = DNA nucleosides, all internucleoside linkages between the nucleosides illustrated are phosphorothioate internucleoside linkages.

TABLE 6
Knock down of MYH7 RNA in 8820 and NH10 cells following treatment with 5 μM oligos.
RNA was measured using the QuantiGene assay. Both cells types are homozygous for
each SNP. Data presented at % mRNA compared to the level in PBS treated cells.
	Perfect match to	Mismatch to	Perfect match to	Mismatch to
	c-allele in	t-allele in	t-allele in	c-allele in	Compound ref.
CMP ID	8820 cells	NH10 cells	NH10 cells	8820 cells	used in
NO	(% PBS)	(% PBS)	(% PBS)	(% PBS)	examples
11	112	113			A1
12	102	73			A2
13	102	264			A3
14	118	161			A4
15	132	87			A5
16	117	125			A6
17	124	92			A7
18	136	87			A8
19	106	218			A9
20	100	115			A10
21	121	121			A11
22	100	81			A12
23	101	246			A13
24	121	122			A14
25	115	90			A15
26	99	98			A16
27	99				A17
28	95	154			A18
29	90	115			A19
30	120	153			A20
31	138	85			A21
32	96	98			A22
33	94	82			A23
34	95	139			A24
35	53	137			A25
36	72	136			A26
37	83	84			A27
38	100	106			A28
39	78	150			A29
40	88	79			A30
41	45	132			A31
42	34	58			A32
43	70	75			A33
44	93	117			A34
45	84	90			A35
46	79	131			A36
47	66	70			A37
48	88	83			A38
49	66	81			A39
50	78				A40
51	36	59			A41
52	60	71			A42
53	90	68			A43
54	51	45			A44
55	55	70			A45
56	89	121			A46
57	84	100			A47
58	60	92			A48
59	34	49			A49
60	40	106			A50
61	31	42			A51
62	39	80			A52
63	40	55			A53
64	32	52			A54
65	48	87			A55
66	93	141			A56
67	100	125			A57
68	104	118			A58
69	114	331			A59
70	106	97			A60
71	90	133			A61
72	92	178			A62
73	93	145			A63
74	102	212			A64
75	105	356			A65
76	68	130			A66
77	118	135			A67
78	101	118			A68
79	121	107			A69
80	123	77			A70
81	55	128			A71
82	110	137			A72
83	124	169			A73
84	104	153			A74
85	104	189			A75
86			52	64	A76
87			101	139	A77
88			20	59	A78
89			49		A79
90			97	117	A80
91			85	83	A81
92			90	181	A82
93			74	89	A83
94			132	192	A84
95			65	137	A85
96			51	80	A86
97			124	132	A87
98			71		A88
99			100	139	A89
100			103	196	A90
101			129	146	A91
102			106	154	A92
103			55	94	A93
104			97	171	A94
105			99	131	A95
106			112	128	A96
107			51	84	A97
108			77	128	A98
109			66	116	A99
110			53	133	A100
111			148	123	A101
112			122	159	A102
113			36	56	A103
114			95	164	A104
115			95	126	A105
116			57	140	A106
117			82	128	A107
118			36	89	A108
119			33	60	A109
120			59	73	A110
121			37	109	A111
122			118	136	A112
123			85	131	A113
124			100		A114
125			83	117	A115
126			54	126	A116
127			73	114	A117
128			57	133	A118
129			101	147	A119
130			120	117	A120
131			250	142	A121
132			257	129	A122
133			81	73	A123
134			91	104	A124
135			205	165	A125
136			158	119	A126
137			95	102	A127
138			67	89	A128
139			69	127	A129
140			114	173	A130
141			79	98	A131
142			114	118	A132
143			180	108	A133
144			63	83	A134
145			83	76	A135
146			69	89	A136
147			103	116	A137
148			83	97	A138
149			69	65	A139
150			85	111	A140
151			64	76	A141
152			137	120	A142
153			144	112	A143
154			100	154	A144
155			124	131	A145
156			113	85	A146
157			133	88	A147
158			108	118	A148
159			60	78	A149
160			89	123	A150
161			76	141	A151
162			154	120	A152
163			122	126	A153
164			104	119	A154
165			58	84	A155
166			112	95	A156
167			103	139	A157
168			99	87	A158
169			69	102	A159
170			88	104	A160
171			107	89	A161
172			91	87	A162
173			58	96	A163
174			127	98	A164
175			101	93	A165
176			60	129	A166
177			83	112	A167
178			88	116	A168
179			53	94	A169
180			78	90	A170
181			101	134	A171
182			73	106	A172
183			134	106	A173
184			90	117	A174
185			67	100	A175
186			123	142	A176
187			125	114	A177
188			88	130	A178
189			95	119	A179
190			169	130	A180
191			50	88	A181
192			76	89	A182
193	100	168			A183
194	95	139			A184
195	104	125			A185
196	98	178			A186
197	58	165			A187
198	120	136			A188
199	86	91			A189
200	50	131			A190
201	103	203			A191
202	82	192			A192
203	123	133			A193
204	28	127			A194
205	83	125			A195
206	94	143			A196
207	36	85			A197
208	47	94			A198
209	53	93			A199
210	72	164			A200
211	35	82			A201
212	38	68			A202
213	26	53			A203
214	40	131			A204
215	38	49			A205
216	40	112			A206
217	52	93			A207
218	81	191			A208
219	45	84			A209
220	45	130			A210
221	70	175			A211
222	89	209			A212
223	32	66			A213
224	23	94			A214
225	57	132			A215
226	120	129			A216
227	13	43			A217
228	34	112			A218
229	114	92			A219
230	124	159			A220
231	102	134			A221
232	120	101			A222
233	87	91			A223
234	107	151			A224
235	132	154			A225
236	49	96			A226
237	78	175			A227
238	93	177			A228
239	44	133			A229
240	46	88			A230
241	50	88			A231
242	41	134			A232
243	36	96			A233
244	34	65			A234
245	66	156			A235
246	49	160			A236
247	54	116			A237
248	80	151			A238
249	80	125			A239
250	54	89			A240
251	81	130			A241
252			63	76	A242
253			138	99	A243
254			81	80	A244
255			89	70	A245
256			43	40	A246
257			120	101	A247
258			93	100	A248
259			60	76	A249
260			55	53	A250
261			55	59	A251
262			122	104	A252
263			86	71	A253
264			82	82	A254
265			57	52	A255
266			94	108	A256
267	43	117			A257
268	109	171			A258
269	42	151			A259
270	61	74			A260
271	47	164			A261
272	57	103			A262
273	67	92			A263
274	51	128			A264
275	80	146			A265
276	60	97			A266
277	48	125			A267
278	47	135			A268
279	29	86			A269
280	34	97			A270
281	66	151			A271
282	70	102			A272
283	55	202			A273
284	58	116			A274
285	67	102			A275
286	59	140			A276
287	79				A277
288	79	173			A278
289	59	134			A279
290	80	122			A280
291	72	133			A281
292	73	78			A282
293	51	141			A283
294	45	116			A284
295	33	73			A285
296	41	82			A286
297	47	136			A287
298	58	142			A288
315	15	16	17	13	S17

TABLE 7
Knock down of MYH7 RNA in CC-2580 cells following 6 days treatment
with 5 μM oligonucleotide. RNA was measured using the ddPCR
assay. The cell line is heterozygous for the Rs715 SNP. Data presented
at % mRNA compared to the level in PBS treated cells.
	Perfect match to	Mismatch to	Perfect match to	Mismatch to
	c-allele in	t-allele in	t-allele in	c-allele in	Compound ref.
CMP ID	CC-2580 cells	CC-2580 cells	CC-2580 cells	CC-2580 cells	used in
NO	(% PBS)	(% PBS)	(% PBS)	(% PBS)	examples
260.1	30	54			B1
260.2	96	107			B2
259.1	98	100			B3
259.2	66	111			B4
259.3	26	61			B5
259.4	47	91			B6
259.5	110	170			B7
259.6	74	112			B8
260.3	86	111			B9
259.7	60	107			B10
259.8	74	93			B11
260.4	52	79			B12
259.9	75	100			B13
259.1	74	130			B14
259.11	80	81			B15
259.12	42	73			B16
259.13	70	68			B17
259.14	60	78			B18
259.15	132	142			B19
259.16	99	108			B20
260.5	28	51			B21
259.17	89	116			B22
259.18	60	108			B23
259.19	68	83			B24
259.2	51	70			B25
259.21	71	140			B26
260.6	68	125			B27
259.22	91	110			B28
260.7	78	104			B29
260.8	49	80			B30
259.23	53	70			B31
259.24	41	84			B32
259.25	75	84			B33
259.26	65	82			B34
260.9	130	141			B35
259.27	61	70			B36
260.1	49	63			B37
259.28	40	63			B38
259.29	51	78			B39
259.3	61	91			B40
260.11	78	112			B41
260.12	39	75			B42
259.31	33	55			B43
260.13	30	72			B44
260.14	69	84			B45
259.32	79	97			B46
259.33	59	101			B47
260.15	52	54			B48
259.34	79	134			B49
260.16	82	98			B50
260.17	61	73			B51
260.18	82	114			B52
260.19	75	88			B53
260.2	125	142			B54
260.21	20	50			B55
260.22	24	44			B56
260.23	133	160			B57
259.35	101	107			B58
259.36	64	81			B59
259.37	105	139			B60
259.38	103	143			B61
259.39	36	57			B62
260.24	79	79			B63
259.4	86	92			B64
259.41	93	109			B65
259.42	56	84			B66
259.43	60	80			B67
259.44	91	98			B68
260.25	83	129			B69
259.45	127	145			B70
259.46	72	95			B71
259.47	59	88			B72
260.26	70	74			B73
259.48	66	83			B74
260.27	95	135			B75
259.49	51	89			B76
259.5	93	123			B77
259.51	79	96			B78
259.52	73	99			B79
259.53	91	116			B80
259.54	60	114			B81
260.28	34	67			B82
260.29	68	101			B83
259.55	20	67			B84
260.3	22	53			B85
259.56	112	160			B86
260.31	32	48			B87
260.32	70	120			B88
259.57	92	102			B89
259.58	62	80			B90
260.33	41	60			B91
259.59	81	110			B92
259.6	76	114			B93
259.61	95	108			B94
259.62	69	93			B95
259.63	55	114			B96
260.34	65	84			B97
260.35	56	84			B98
260.36	36	45			B99
259.64	81	95			B100
259.65	71	107			B101
259.66	55	69			B102
259.67	87	97			B103
259.68	35	65			B104
259.69	85	109			B105
259.7	41	46			B106
259.71	88	97			B107
259.72	54	72			B108
260.37	67	97			B109
260.38	84	102			B110
260.39	64	89			B111
259.73	79	122			B112
260.4	104	112			B113
259.74	57	101			B114
260.41	79	99			B115
259.75	63	67			B116
260.42	58	97			B117
259.76	57	86			B118
259.77	121	150			B119
259.78	72	94			B120
259.79	91	110			B121
259.8	56	90			B122
260.43	64	81			B123
259.81	25	41			B124
259.82	63	98			B125
260.44	57	97			B126
260.45	64	99			B127
259.83	48	80			B128
260.46	65	92			B129
259.84	117	115			B130
259.85	85	114			B131
259.86	68	107			B132
259.87	113	147			B133
259.88	100	124			B134
259.89	58	88			B135
259.9	63	86			B136
259.91	106	99			B137
259.92	79	125			B138
260.47	92	129			B139
259.93	116	152			B140
260.48	33	45			B141
260.49	56	93			B142
259.94	110	151			B143
259.95	80	141			B144
259.96	84	86			B145
259.97	71	77			B146
259.98	33	64			B147
260.5	75	96			B148
259.99	35	55			B149
260.51	37	80			B150
259.1	65	93			B151
260.52	67	82			B152
259.101	40	87			B153
259.102	57	91			B154
259.103	52	74			B155
260.53	53	77			B156
259.104	50	90			B157
260.54	86	100			B158
259.105	83	125			B159
259.106	101	110			B160
259.107	59	137			B161
259.108	66	84			B162
259.109	58	91			B163
260.55	57	55			B164
260.56	64	66			B165
259.11	82	95			B166
259.111	59	89			B167
259.112	31	68			B168
259.113	78	86			B169
259.114	96	136			B170
260.57	83	97			B171
259.115	34	69			B172
259.116	115	142			B173
259.117	93	116			B174
260.58	36	60			B175
259.118	70	75			B176
259.119	91	133			B177
259.12	73	112			B178
259.121	58	91			B179
260.59	58	89			B180
260.6	69	60			B181
260.61	69	91			B182
259.122	144	136			B183
259.123	70	108			B184
260.62	78	121			B185
259.124	130	130			B186
260.63	72	100			B187
259.125	95	112			B188
259.126	86	82			B189
260.64	56	89			B190
259.127	101	124			B191
259.128	92	128			B192
260.65	65	83			B193
259.129	52	71			B194
260.66	72	124			B195
260.67	69	103			B196
259.13	82	97			B197
260.68	77	106			B198
259.131	62	78			B199
260.69	50	68			B200
259.132	147	175			B201
259.133	58	96			B202
259.134	46	82			B203
259.135	73	98			B204
260.7	86	92			B205
259.136	62	71			B206
259.137	62	107			B207
259.138	88	104			B208
260.71	65	84			B209
260.72	63	89			B210
260.73	81	119			B211
260.74	64	66			B212
259.139	97	101			B213
259.14	74	95			B214
259.141	83	97			B215
259.142	69	97			B216
259.143	117	129			B217
259.144	79	87			B218
259.145	96	121			B219
260.75	97	115			B220
260.76	58	85			B221
259.146	69	83			B222
259.147	61	115			B223
259.148	61	73			B224
259.149	72	96			B225
259.15	62	66			B226
260.77	53	70			B227
259.151	99	101			B228
260.78	73	104			B229
259.152	104	111			B230
259.153	76	87			B231
259.154	72	97			B232
260.79	41	79			B233
259.155	68	98			B234
260.8	63	94			B235
260.81	100	98			B236
259.156	79	71			B237
259.157	96	120			B238
260.82	54	55			B239
259.158	80	85			B240
259.159	77	84			B241
259.16	63	65			B242
259.161	83	102			B243
260.83	56	82			B244
259.162	91	115			B245
259.163	82	97			B246
260.84	95	116			B247
260.85	66	101			B248
259.164	71	97			B249
259.165	89	85			B250
259.166	102	112			B251
259.167	34	54			B252
259.168	53	80			B253
260.86	38	70			B254
259.169	85	95			B255
259.17	72	102			B256
259.171	71	95			B257
260.87	65	76			B258
260.88	69	83			B259
260.89	60	67			B260
259.172	53	75			B261
260.9	75	113			B262
260.91	88	105			B263
259.173	66	104			B264
260.92	137	145			B265
259.174	63	114			B266
259.175	36	54			B267
260.93	74	102			B268
259.176	117	143			B269
259.177	85	88			B270
259.178	29	62			B271
260.94	44	67			B272
260.95	63	68			B273
260.96	42	54			B274
259.179	85	113			B275
260.97	100	145			B276
259.18	50	92			B277
259.181	82	105			B278
260.98	58	64			B279
260.99	89	135			B280
259.182	95	121			B281
259.183	47	88			B282
259.184	78	93			B283
259.185	60	69			B284
259.186	73	96			B285
259.187	77	101			B286
259.188	68	97			B287
259.189	77	98			B288
260.1	76	80			B289
260.101	92	106			B290
280.1			62	92	B291
280.2			75	111	B292
280.3			78	96	B293
280.4			40	55	B294
280.5			28	71	B295
280.6			32	67	B296
280.7			66	90	B297
280.8			20	36	B298
280.9			27	84	B299
280.1			58	103	B300
280.11			45	89	B301
280.12			73	113	B302
280.13			33	57	B303
280.14			37	65	B304
280.15			51	85	B305
280.16			47	97	B306
280.17			42	79	B307
280.18			29	38	B308
280.19			62	115	B309
280.2			74	95	B310
280.21			66	107	B311
280.22			93	102	B312
280.23			46	69	B313
280.24			63	124	B314
280.25			52	82	B315
280.26			55	84	B316
280.27			42	81	B317
280.28			46	91	B318
280.29			46	67	B319
280.3			32	57	B320
280.31			47	96	B321
280.32			39	50	B322
280.33			45	84	B323
280.34			29	65	B324
280.35			76	71	B325
280.36			66	83	B326
280.37			89	118	B327
280.38			114	143	B328
280.39			107	117	B329
280.4			56	73	B330
280.41			57	76	B331
280.42			31	64	B332
280.43			52	85	B333
280.44			17	51	B334
280.45			37	63	B335
280.46			81	109	B336
280.47			55	71	B337
280.48			49	83	B338
280.49			31	55	B339
280.5			55	69	B340
280.51			51	83	B341
280.52			19	40	B342
280.53			68	96	B343
280.54			70	78	B344
280.55			42	85	B345
280.56			102	145	B346
280.57			35	62	B347
280.58			51	91	B348
280.59			104	129	B349
280.6			63	93	B350
280.61			59	102	B351
280.62			44	61	B352
280.63			58	86	B353
280.64			62	108	B354
280.65			17	45	B355
280.66			70	105	B356
280.67			91	119	B357
280.68			50	66	B358
280.69			27	42	B359
280.7			34	64	B360
280.71			43	79	B361
280.72			103	110	B362
280.73			72	101	B363
280.74			60	99	B364
280.75			56	74	B365
280.76			84	104	B366
280.77			114	132	B367
280.78			103	125	B368
280.79			94	116	B369
280.8			41	61	B370
280.81			51	83	B371
280.82			94	133	B372
280.83			55	119	B373
280.84			55	78	B374
280.85			29	56	B375
280.86			78	97	B376
280.87			29	54	B377
280.88			65	95	B378
280.89			48	78	B379
280.9			88	99	B380
280.91			29	74	B381
280.92			47	72	B382
280.93			22	54	B383
280.94			41	64	B384
280.95			86	94	B385
280.96			32	62	B386
280.97			66	83	B387
280.98			154	116	B388
280.99			125	143	B389
280.1			36	53	B390
280.101			23	49	B391
280.102			44	83	B392
280.103			40	68	B393
280.104			16	43	B394
280.105			43	80	B395
280.106			64	98	B396
280.107			71	114	B397
280.108			59	81	B398
280.109			57	86	B399
280.11			48	66	B400
280.111			33	48	B401
280.112			30	64	B402
280.113			19	56	B403
280.114			35	64	B404
280.115			38	70	B405
280.116			121	162	B406
280.117			40	63	B407
280.118			97	128	B408
280.119			52	82	B409
280.12			99	99	B410
280.121			52	67	B411
280.122			51	97	B412
280.123			70	124	B413
280.124			38	74	B414
280.125			25	61	B415
280.126			62	77	B416
280.127			84	135	B417
280.128			76	100	B418
280.129			32	59	B419
280.13			69	78	B420
280.131			71	80	B421
280.132			106	121	B422
280.133			44	89	B423
280.134			36	62	B424
280.135			31	53	B425
280.136			41	66	B426
280.137			67	105	B427
280.138			74	102	B428
280.139			42	55	B429
280.14			63	88	B430
280.141			82	107	B431
280.142			40	65	B432
280.143			61	74	B433
280.144			34	69	B434
280.145			36	63	B435
280.146			11	34	B436
280.147			39	88	B437
280.148			39	77	B438
280.149			15	26	B439
280.15			33	101	B440
280.151			26	51	B441
280.152			54	87	B442
280.153			42	76	B443
280.154			68	122	B444
280.155			42	79	B445
280.156			83	118	B446
280.157			20	53	B447
280.158			43	71	B448
280.159			57	86	B449
280.16			47	96	B450
280			30	61	A270
259	23	58			A249
260	31	50			A250
315	12	11	11	12	S17

TABLE 8
Knock down of MYH7 RNA in CC-2580 cells following 6 days treatment with various concentration of
oligonucleotide. Allele specific RNA was measured using the ddPCR assay. EC50 values are calculated
for each allele and the selectivity was calculated as the ratio between the two EC50 values.
	EC50 for	EC50 for		EC50 for	EC50 for
	perfect match	mismatch to	Selectivity	perfect match	mismatch to	Selectivity
	to c-allele in	t-allele in	between c- and	to t-allele in	c-allele in	between t- and	Compound
CMP ID	CC-2580 cells	CC-2580 cells	t-allele in	CC-2580 cells	CC-2580 cells	c-allele in	ref. used in
NO	(uM)	(uM)	CC-2580 cells	(uM)	(uM)	CC-2580 cells	examples
259	0.18	3.67	20.9				A249
260	0.13	0.35	2.6				A250
280				0.12	0.86	7.2	A270
280.91				0.26	5.00	19.2	B381
280.93				0.36	1.95	5.4	B383
280.113				0.13	1.01	7.8	B403
280.15				0.63	5.00	7.9	B440
280.9				0.21	5.00	23.8	B299
280.125				0.15	2.29	15.8	B415
280.5				0.26	5.00	19.2	B295
280.146				0.09	0.29	3.1	B436
280.104				0.09	1.16	12.8	B394
280.65				0.07	0.50	7.2	B355
280.44				0.10	0.54	5.6	B334
280.149				0.07	0.17	2.4	B439
280.157				0.16	1.79	11.1	B447
280.151				0.17	0.95	5.6	B441
280.85				0.22	0.92	4.1	B375
259.112	0.25	1.76	7.0				B168
259.101	0.65	5.00	7.7				B153
259.115	0.32	2.38	7.3				B172
259.55	0.41	3.43	8.3				B84
259.178	0.29	1.45	5.1				B271
259.98	0.15	0.88	5.9				B147
259.3	0.23	1.46	6.4				B5
260.3	0.10	0.69	7.2				B85
260.21	0.22	1.98	9.1				B55
260.28	0.16	1.62	10.2				B82
260.79	0.17	3.33	20.1				B233
260.51	0.26	1.97	7.6				B150
260.5	0.24	1.47	6.1				B21
260.58	0.50	0.51	1.0				B175
260.22	0.07	0.78	11.2				B56
260.13	0.08	0.57	7.6				B44

TABLE 9
Knock down of MYH7 RNA in hIPSC cardiomyocytes (CMs) following 6 days treatment with various concentration
of oligonucleotide. Allele specific RNA was measured using the ddPCR assay. EC50 values are calculated
for each allele and the selectivity was calculated as the ratio between the two EC50 values.
	EC50 for	EC50 for		EC50 for	EC50 for
	perfect match	mismatch to	Selectivity	perfect match	mismatch to	Selectivity
	to c-allele in	t-allele in	between c- and	to t-allele in	c-allele in	between t- and	Compound
CMP ID	hIPSC-CM cells	hIPSC-CM cells	t-allele in	hIPSC-CM cells	hIPSC-CM cells	c-allele in	ref. used in
NO	(uM)	(uM)	hIPSC-CM cells	(uM)	(uM)	hIPSC-CM cells	examples
259	0.05	0.28	5.1				A249
260	0.19	0.43	2.2				A250
280				0.11	0.60	5.4	A270
280.91				0.10	0.34	3.5	B381
280.113				0.06	0.26	4.2	B403
280.15				0.09	0.86	9.2	B440
280.9				0.08	0.60	7.7	B299
280.146				0.04	0.10	2.2	B436
280.104				0.06	0.30	4.7	B394
280.65				0.09	0.42	4.5	B355
280.44				0.05	0.18	3.8	B334
259.55	0.12	0.98	7.9				B84
259.3	0.47	5.00	10.7				B5
260.3	0.05	0.21	4.1				B85
260.21	0.09	0.96	11.2				B55
260.28	0.13	1.88	14.5				B82
260.5	0.09	0.66	7.0				B21
260.22	0.03	0.16	5.2				B56
260.13	0.04	0.32	7.3				B44

Claims

1. An antisense oligonucleotide for the inhibition of a human myosin heavy chain 7 (Myh7) transcript, wherein said oligonucleotide comprises a contiguous nucleotide sequence of 10-30 nucleotides in length which are at least 90% complementary to a sequence selected from the group consisting of SEQ ID NOs 3-10.

2. The antisense oligonucleotide according to claim 1, wherein said oligonucleotide comprises a contiguous nucleotide sequence of 13-24 nucleotides in length which are fully complementary to a sequence selected from the group consisting of SEQ ID NOs 3-10.

3. The antisense oligonucleotide according to claim 1, wherein said antisense oligonucleotide is complementary to a region of the sequence selected from SEQ ID NOs 3-10 which comprises the 20th nucleotide from the 5′ end of the sequence selected from SEQ ID NOs 3-10.

4. The antisense oligonucleotide according to claim 1, wherein the contiguous nucleotide sequence of the oligonucleotide is fully complementary to a sequence selected from the group consisting of SEQ ID NO 3 or SEQ ID NO 4.

5. The antisense oligonucleotide according to claim 1, wherein the contiguous nucleotide sequence of the oligonucleotide is fully complementary to a sequence selected from the group consisting of SEQ ID NO 5 or SEQ ID NO 6.

6. The antisense oligonucleotide according to claim 1, wherein the contiguous nucleotide sequence of the oligonucleotide is fully complementary to a sequence selected from the group consisting of SEQ ID NO 7 or SEQ ID NO 8.

7. The antisense oligonucleotide according to claim 1, wherein the contiguous nucleotide sequence of the oligonucleotide is fully complementary to a sequence selected from the group consisting of SEQ ID NO 9 or SEQ ID NO 10.

8. The antisense oligonucleotide according to claim 1, wherein the Myh7 transcript is the human Myh7 mature mRNA or pre-mRNA.

9. The antisense oligonucleotide according to claim 1, wherein the Myh7 transcript originates from a disease associated allele of the human Myh7 gene.

10. The antisense oligonucleotide according to claim 1, wherein the antisense oligonucleotide is selective for Myh7 transcript originating from a disease associated allele of the human Myh7 transcript, as compared to a non-disease associated allele.

11. The antisense oligonucleotide according to claim 9, wherein the Myh7 transcript originating from a disease associated allele comprises one or more disease associated single nucleotide polymorphisms which are present in a region other than the sequence selected from the group consisting of SEQ ID NO 3-10.

12. The antisense oligonucleotide of claims 1, comprising one or more modified nucleosides.

13. The antisense oligonucleotide of claim 12, wherein the one or more modified nucleosides is a 2′ sugar modified nucleoside.

14. The antisense oligonucleotide of claim 13, wherein the one or more 2′ sugar modified nucleoside is independently selected from the group consisting of 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA, 2′-amino-DNA, 2′-fluoro-DNA, arabino nucleic acid (ANA), 2′-fluoro-ANA and LNA nucleosides.

15. The antisense oligonucleotide of claim 12, wherein the one or more modified nucleoside is a LNA nucleoside.

16. The antisense oligonucleotide of claim 1, where the oligonucleotide comprises at least one phosphorothioate internucleoside linkage within the contiguous nucleotide sequence.

17. The antisense oligonucleotide of claim 16, wherein the internucleoside linkages within the contiguous nucleotide sequence are phosphorothioate internucleoside linkages.

18. The antisense oligonucleotide of claim 1, wherein the oligonucleotide is capable of recruiting RNase H.

19. The antisense oligonucleotide of claim 1, wherein the antisense oligonucleotide, or contiguous nucleotide sequence thereof, consists or comprises a gapmer of formula 5′-F-G-F′-3′, where region F and F′ independently comprise 1-8 nucleosides, of which 1-5 are 2′ sugar modified and defines the 5′ and 3′ end of the F and F′ region, and G is a region between 5 and 16 nucleosides which are capable of recruiting RNaseH, such as a region comprising 5-16 DNA nucleosides.

20. The antisense oligonucleotide according to claim 19, wherein region F and F′ comprise at least one LNA nucleoside, and wherein region G comprises 7-14 nucleotides.

21. The antisense oligonucleotide according to claim 1, wherein the antisense oligonucleotide comprises a sequence selected from the group consisting of 11-344.

22. The antisense oligonucleotide according to claim 1, wherein the antisense oligonucleotide consists or comprises of compound ID No 11-344, wherein a capital letter represents a LNA nucleotide, LNA C are LNA 5 methyl cytosine, lower case letters are DNA nucleosides, and optionally all internucleoside linkages are phosphorothioate internucleotides linkages.

23. The antisense oligonucleotide according to claim 1, wherein the antisense oligonucleotide consists or comprises of compound ID No 11-344, wherein a capital letter represents a beta-D-oxy LNA nucleotide, LNA C are LNA 5 methyl cytosine, lower case letters are DNA nucleosides, and all internucleoside linkages are phosphorothioate internucleotides linkages.

24. A conjugate comprising the oligonucleotide according to claim 1, and at least one conjugate moiety covalently attached to said oligonucleotide.

25. A pharmaceutically acceptable salt of the antisense oligonucleotide according to claim 1.

26. A pharmaceutical composition comprising the oligonucleotide of claim 1 and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.

27. An in vivo or in vitro method for modulating human myosin heavy chain 7 (Myh7) expression in a target cell which is expressing Myh7, said method comprising administering an oligonucleotide of claim 1, in an effective amount to said cell.

28. A method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of an oligonucleotide of claim to a subject suffering from or susceptible to the disease.

29. The method of claim 28, wherein the disease is selected from the group consisting of hypertrophic cardiomyopathy.

30. The oligonucleotide of claim 1 for use in medicine.

31. The oligonucleotide of claim 1 for use in the treatment or prevention of hypertrophic cardiomyopathy.

32. Use of the oligonucleotide of claim 1 for the preparation of a medicament for treatment or prevention of hypertrophic cardiomyopathy.

33. A method for treatment of a human subject in need to treatment for hypertrophic cardiomyopathy, said treatment comprising the step of: a. Taking a biological sample from the human subjectb. Sequencing the Myh7 nucleic acid alleles present in the sample of the human subject;c. Determining the presence of a disease associated Myh7 allelic variant of the Myh7 nucleic acid;d. Administering a therapeutically effective amount of an antisense oligonucleotide to the human subject which is selective for the disease associated Myh7 allelic variant as compared to a non-disease associate allele.

34. The method according to claim 33, wherein the antisense oligonucleotide is as according to claim 1.