COMPOSITIONS AND USES FOR TREATMENT THEREOF

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (T083370051US02-SEQ-CBD.xml; Size: 782,419 bytes; and Date of Creation: Oct. 25, 2024) are herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to compositions for the treatment of DNA repeat expansion diseases. In one embodiment, the present disclosure relates to oligonucleotides directed to subunits of the DNA mismatch repair system. In one embodiment, the present disclosure relates to oligonucleotides directed at skipping of MLH3 exon 7 to slow the progression of repeat expansion disorders.

BACKGROUND OF THE INVENTION

Genomic instability underlies an increasing number of disorders, plays a major role in cancer and contributes to aging. DNA mismatch repair (MMR) is essential for maintaining genome integrity. However, when it comes to certain types of repetitive DNA, MMR actually contributes to genome instability. MMR has been implicated in repeat expansions of numerous disorders including Huntington's disease (HD) and myotonic dystrophy (DM). Friedreich ataxia (FRDA), the most common inherited ataxia, is a progressive neurodegenerative disorder caused by GAA·TTC repeat expansion in the first intron of the frataxin (FXN) gene. Currently there is no treatment and no cure for Friedreich ataxia or any of the many other DNA repeat expansion diseases. While each of the individual repeat expansion diseases is rare or not necessarily common, in aggregate, the victims of the currently known repeat expansion diseases number over 100,000 in the United States alone.

SUMMARY OF THE INVENTION

The invention is directed to therapeutics useful to slow the expansion rate in repeat expansion diseases. In one embodiment, a central mechanism is likely shared by all repeat expansion diseases thus useful in the treatment of many, if not all of the diseases in this class. For progressive repeat expansion diseases such as Friedreich ataxia or Huntington's disease MLH3 exon skipping may make it possible to delay or even prevent the onset of symptoms if treatment is started early. Chemically similar morpholino splice switching oligonucleotides (SSOs) are currently in human trials for exon skipping in Duchenne muscular dystrophy.

An aspect of the invention is directed to an isolated nuclease-resistant oligonucleotide comprising a nucleic acid sequence that hybridizes to a complementary target nucleic acid sequence of a gene or gene product encoding a component of a mismatch repair (MMR) complex. For example, the oligonucleotide comprises a sequence that specifically hybridizes in a human cell with a nucleic acid sequence encoding a subunit of the MMR system (e.g., MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2). In one embodiment, the nucleic acid sequence is a complementary sequence for a human MLH3 gene or gene product. In one embodiment, the nuclease-resistant oligonucleotide is useful for inducing exon skipping. For example, oligonucleotide(s) can induce skipping of MSH2 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or a combination of MSH2 exons. Oligonucleotide(s) can induce skipping of MSH3 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or a combination of MSH3 exons. Oligonucleotide(s) can induce skipping of MSH6 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or a combination of MSH6 exons. Oligonucleotide(s) can induce skipping of MLH1 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or a combination of MLH1 exons. Oligonucleotide(s) can induce skipping of MLH3 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or a combination of MLH3 exons. Oligonucleotide(s) can induce skipping of PMS1 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or a combination of PMS1 exons. Oligonucleotide(s) can induce skipping of PMS2 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or a combination of PMS2 exons. In one embodiment, the oligonucleotide directs skipping of one or more exons of MSH2, MSH3, MSH6, PMS1, PMS2, MLH1, or MLH3. In one embodiment, one or more oligonucleotides can induce skipping of MLH3 exon 7. In one embodiment, the oligonucleotide is decreases the rate of DNA repeat expansion. In one embodiment, the oligonucleotide is useful in treating a DNA repeat expansion disease. In one embodiment, the target nucleic acid sequence is located on human chromosome 2, 3, 5, 7, or 14. In one embodiment, the target nucleic acid sequence (or target complementary nucleic acid sequence) comprises: a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-16 described herein for GenBank Accession No. NG_007110.2, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007110.2; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-24 described herein for GenBank Accession No. NG_016607.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_016607.1; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-10 described herein for GenBank Accession No. NG_007111.1 or SEQ ID NO: 33, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007111.1 or SEQ ID NO: 33; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-19 described herein for GenBank Accession No. NG_007109.2, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007109.2; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-13 described herein for GenBank Accession No. NG_008648.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008648.1; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-13 described herein for GenBank Accession No. NG_008649.1 or SEQ ID NO: 1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008649.1 or SEQ ID NO: 1; or a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-15 described herein for GenBank Accession No. NG_008466.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008466.1. In one embodiment, the component of the MMR complex comprises MutS or MutL. In one embodiment, MutS comprises a subunit selected from the group consisting of MSH2, MSH3, and MSH6. In one embodiment, MutL comprises a subunit selected from the group consisting of MLH1, MLH3, PMS1, and PMS2. In one embodiment, MLH3 comprises SEQ ID NO: 1. In one embodiment, the oligonucleotide hybridizes to the target complementary nucleic acid sequence comprising SEQ ID NO: 2. In one embodiment, the oligonucleotide has at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence comprising SEQ ID NO: 3 or SEQ ID NO: 4. In one embodiment, the oligonucleotide has at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence depicted in Table 4. In one embodiment, the oligonucleotide comprises 15 to 30 nucleotide bases in length. In one embodiment, the oligonucleotide comprises one or more morpholino subunits, one or more locked nucleic acid subunits, one or more 2-O-methyl moieties, or one or more peptide moieties.

An aspect of the invention is directed to a pharmaceutical composition comprising a nuclease-resistant oligonucleotide comprising a nucleic acid sequence that hybridizes to a complementary target nucleic acid sequence of a gene or gene product encoding a component of a mismatch repair (MMR) complex, and a pharmaceutically acceptable carrier. For example, the oligonucleotide comprises a sequence that specifically hybridizes in a human cell with a nucleic acid sequence encoding a subunit of the MMR system (e.g., MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2). In one embodiment, the nucleic acid sequence is a complementary sequence for a human MLH3 gene or gene product. In one embodiment, the nuclease-resistant oligonucleotide is useful for inducing exon skipping. For example, oligonucleotide(s) can induce skipping of MSH2 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or a combination of MSH2 exons. Oligonucleotide(s) can induce skipping of MSH3 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or a combination of MSH3 exons. Oligonucleotide(s) can induce skipping of MSH6 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or a combination of MSH6 exons. Oligonucleotide(s) can induce skipping of MLH1 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or a combination of MLH1 exons. Oligonucleotide(s) can induce skipping of MLH3 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or a combination of MLH3 exons. Oligonucleotide(s) can induce skipping of PMS1 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or a combination of PMS1 exons. Oligonucleotide(s) can induce skipping of PMS2 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or a combination of PMS2 exons. In one embodiment, the oligonucleotide directs skipping of one or more exons of MSH2, MSH3, MSH6, PMS1, PMS2, MLH1, or MLH3. In one embodiment, one or more oligonucleotides can induce skipping of MLH3 exon 7. In one embodiment, the oligonucleotide decreases the rate of DNA repeat expansion. In one embodiment, the oligonucleotide is useful in treating a DNA repeat expansion disease. In one embodiment, the target nucleic acid sequence is located on human chromosome 2, 3, 5, 7, or 14. In one embodiment, the target nucleic acid sequence (or target complementary nucleic acid sequence) comprises: a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-16 described herein for GenBank Accession No. NG_007110.2, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007110.2; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-24 described herein for GenBank Accession No. NG_016607.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_016607.1; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-10 described herein for GenBank Accession No. NG_007111.1 or SEQ ID NO: 33, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007111.1 or SEQ ID NO: 33; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-19 described herein for GenBank Accession No. NG_007109.2, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007109.2; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-13 described herein for GenBank Accession No. NG_008648.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008648.1; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-13 described herein for GenBank Accession No. NG_008649.1 or SEQ ID NO: 1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008649.1 or SEQ ID NO: 1; or a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-15 described herein for GenBank Accession No. NG_008466.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008466.1. In one embodiment, the component of the MMR complex comprises MutS or MutL. In one embodiment, MutS comprises a subunit selected from the group consisting of MSH2, MSH3, and MSH6. In one embodiment, MutL comprises a subunit selected from the group consisting of MLH1, MLH3, PMS1, and PMS2. In one embodiment, MLH3 comprises SEQ ID NO: 1. In one embodiment, the oligonucleotide hybridizes to the target complementary nucleic acid sequence comprising SEQ ID NO: 2. In one embodiment, the oligonucleotide has at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence comprising SEQ ID NO: 3 or SEQ ID NO: 4. In one embodiment, the oligonucleotide has at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence depicted in Table 4. In one embodiment, the oligonucleotide comprises 15 to 30 nucleotide bases in length. In one embodiment, the oligonucleotide comprises one or more morpholino subunits, one or more locked nucleic acid subunits, one or more 2-O-methyl moieties, or one or more peptide moieties.

An aspect of the invention is directed to a pharmaceutical composition comprising a nuclease-resistant oligonucleotide 15 to 30 nucleotide bases in length targeted to a complementary nucleic acid sequence of a gene or gene product encoding a MutS or MutL subunit, wherein the oligonucleotide hybridizes with and decreases the expression of the human MutS or MutL subunit by at least 20%, and wherein the oligonucleotide comprises at least one modification. In one embodiment, the modification comprises a phosphorothioate backbone. In one embodiment, the modification comprises a phosphorodiamidate morpholino nucleotide. In one embodiment, the modification results in a charge-negative oligonucleotide. In one embodiment, the modification results in a charge-neutral oligonucleotide. In one embodiment, the modification comprises a phosphorodiamidate morpholino nucleotide, or a 2-aminoethylglycine functionalized nucleotide. In one embodiment, the modification comprises a phosphorothioate backbone, a 5-methylcytosine nucleotide, a 2′-O-methoxyethyl sugar moiety, a locked nucleic acid subunit, an ethylene-bridged nucleic acid subunit, or a combination thereof. In one embodiment, MutS comprises a subunit selected from the group consisting of MSH2, MSH3, and MSH6. In one embodiment, MutL comprises a subunit selected from the group consisting of MLH1, MLH3, PMS1, and PMS2. In one embodiment, MLH3 comprises SEQ ID NO: 1. In one embodiment, the oligonucleotide of the composition hybridizes to the target complementary nucleic acid sequence comprising SEQ ID NO: 2. In one embodiment, the oligonucleotide has at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence comprising SEQ ID NO: 3 or SEQ ID NO: 4. In one embodiment, the oligonucleotide has at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence depicted in Table 4. In one embodiment, the nuclease-resistant oligonucleotide of the composition is useful for inducing exon skipping. For example, oligonucleotide(s) can induce skipping of MSH2 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or a combination of MSH2 exons. Oligonucleotide(s) can induce skipping of MSH3 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or a combination of MSH3 exons. Oligonucleotide(s) can induce skipping of MSH6 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or a combination of MSH6 exons. Oligonucleotide(s) can induce skipping of MLH1 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or a combination of MLH1 exons. Oligonucleotide(s) can induce skipping of MLH3 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or a combination of MLH3 exons. Oligonucleotide(s) can induce skipping of PMS1 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or a combination of PMS1 exons. Oligonucleotide(s) can induce skipping of PMS2 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or a combination of PMS2 exons. In one embodiment, the oligonucleotide of the composition directs skipping of one or more exons of MSH2, MSH3, MSH6, PMS1, PMS2, MLH1, or MLH3. In one embodiment, the oligonucleotide(s) of the composition can induce skipping of MLH3 exon 7. In one embodiment, the oligonucleotide of the composition hybridizes with and decreases the expression of the human MutS or MutL subunit by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.

An aspect of the invention provides for an oligonucleotide complex for modulating the expression or activity of a gene or gene product encoding a component of a mismatch repair (MMR) system, the complex comprising a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide comprises a sequence complementary to an acceptor region of an exon of a gene encoding a MutS or MutL subunit, and wherein the nucleic acid sequence of the first oligonucleotide comprises a nuclease-resistant modification, and wherein the second oligonucleotide comprises a sequence complementary to a donor region of an exon of a gene encoding a MutS or MutL subunit, and wherein the nucleic acid sequence of the second oligonucleotide comprises a nuclease-resistant modification. An aspect of the invention is directed to an oligonucleotide complex for modulating the expression or activity of a gene or gene product encoding a component of a mismatch repair (MMR) system, the complex comprising a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide comprises a sequence complementary to an acceptor region of an exon of a gene encoding a MutS or MutL subunit, and wherein the nucleic acid sequence of the first oligonucleotide comprises a nuclease-resistant modification, and wherein the second oligonucleotide comprises a sequence complementary to a donor region of an exon of a gene encoding a MutS or MutL subunit. An aspect of the invention provides for an oligonucleotide complex for modulating the expression or activity of a gene or gene product encoding a component of a mismatch repair (MMR) system, the complex comprising a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide comprises a sequence complementary to an acceptor region of an exon of a gene encoding a MutS or MutL subunit, and wherein the second oligonucleotide comprises a sequence complementary to a donor region of an exon of a gene encoding a MutS or MutL subunit, and wherein the nucleic acid sequence of the second oligonucleotide comprises a nuclease-resistant modification. In one embodiment, the nuclease-resistant modification comprises one or more morpholino subunits, one or more locked nucleic acid subunits, one or more 2-O-methyl moieties, one or more peptide moieties, or a combination thereof. In one embodiment, the first oligonucleotide comprises a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence comprising SEQ ID NO: 3 or SEQ ID NO: 4. In one embodiment, the first oligonucleotide comprises a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence depicted in Table 4. In one embodiment, the first oligonucleotide is directed to a target nucleic acid sequence (or target complementary nucleic acid sequence) comprising: a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-16 described herein for GenBank Accession No. NG_007110.2, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007110.2; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-24 described herein for GenBank Accession No. NG_016607.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_016607.1; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-10 described herein for GenBank Accession No. NG_007111.1 or SEQ ID NO: 33, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007111.1 or SEQ ID NO: 33; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-19 described herein for GenBank Accession No. NG_007109.2, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007109.2; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-13 described herein for GenBank Accession No. NG_008648.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008648.1; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-13 described herein for GenBank Accession No. NG_008649.1 or SEQ ID NO: 1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008649.1 or SEQ ID NO: 1; or a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-15 described herein for GenBank Accession No. NG_008466.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008466.1. In one embodiment, the second oligonucleotide comprises a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence comprising SEQ ID NO: 3 or SEQ ID NO: 4. In one embodiment, the second oligonucleotide comprises a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence depicted in Table 4. In one embodiment, the second oligonucleotide is directed to a target nucleic acid sequence (or target complementary nucleic acid sequence) comprising: a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-16 described herein for GenBank Accession No. NG_007110.2, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007110.2; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-24 described herein for GenBank Accession No. NG_016607.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_016607.1; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-10 described herein for GenBank Accession No. NG_007111.1 or SEQ ID NO: 33, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007111.1 or SEQ ID NO: 33; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-19 described herein for GenBank Accession No. NG_007109.2, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007109.2; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-13 described herein for GenBank Accession No. NG_008648.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008648.1; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-13 described herein for GenBank Accession No. NG_008649.1 or SEQ ID NO: 1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008649.1 or SEQ ID NO: 1; or a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-15 described herein for GenBank Accession No. NG_008466.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008466.1. In one embodiment, MutS comprises a subunit selected from the group consisting of MSH2, MSH3, and MSH6. In one embodiment, MutL comprises a subunit selected from the group consisting of MLH1, MLH3, PMS1, and PMS2. In one embodiment, MLH3 comprises SEQ ID NO: 1. In one embodiment, modulation of expression or activity is a decrease in the expression or activity of the human MutS or MutL subunit by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.

An aspect of the invention provides for a kit for the treatment of a DNA Repeat Expansion Disease (DRED). In one embodiment, the kit comprises an oligonucleotide complex described herein and instructions for use. In one embodiment, the oligonucleotides of the complex comprise a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence comprising SEQ ID NO: 3 or SEQ ID NO: 4. In one embodiment, the oligonucleotides if the complex comprise a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence depicted in Table 4. In one embodiment, the oligonucleotides of the complex can be directed to a target nucleic acid sequence (or target complementary nucleic acid sequence) comprising: a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-16 described herein for GenBank Accession No. NG_007110.2, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007110.2; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-24 described herein for GenBank Accession No. NG_016607.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_016607.1; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-10 described herein for GenBank Accession No. NG_007111.1 or SEQ ID NO: 33, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007111.1 or SEQ ID NO: 33; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-19 described herein for GenBank Accession No. NG_007109.2, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007109.2; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-13 described herein for GenBank Accession No. NG_008648.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008648.1; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-13 described herein for GenBank Accession No. NG_008649.1 or SEQ ID NO: 1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008649.1 or SEQ ID NO: 1; or a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-15 described herein for GenBank Accession No. NG_008466.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008466.1.

An aspect of the invention provides for a kit for the treatment of a DNA Repeat Expansion Disease (DRED). In one embodiment, the kit comprises a nuclease-resistant oligonucleotide compound as described herein and instructions for use. In one embodiment, the oligonucleotide comprises a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence comprising SEQ ID NO: 3 or SEQ ID NO: 4. In one embodiment, the oligonucleotide comprises a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence depicted in Table 4. In one embodiment, the oligonucleotide can be directed to a target nucleic acid sequence (or target complementary nucleic acid sequence) comprising: a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-16 described herein for GenBank Accession No. NG_007110.2, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007110.2; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-24 described herein for GenBank Accession No. NG_016607.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_016607.1; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-10 described herein for GenBank Accession No. NG_007111.1 or SEQ ID NO: 33, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007111.1 or SEQ ID NO: 33; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-19 described herein for GenBank Accession No. NG_007109.2, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007109.2; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-13 described herein for GenBank Accession No. NG_008648.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008648.1; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-13 described herein for GenBank Accession No. NG_008649.1 or SEQ ID NO: 1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008649.1 or SEQ ID NO: 1; or a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-15 described herein for GenBank Accession No. NG_008466.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008466.1. In one embodiment, the DRED is selected from those diseases listed in Table 1 or Table 2.

An aspect of the invention provides for a kit for monitoring the efficacy of treatment of a DNA Repeat Expansion Disease (DRED) in a subject. In one embodiment, the kit comprises at least one primer and instructions for use. In one embodiment, the kit comprises a second primer. In one embodiment, the primer comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence comprising SEQ ID NO: 5. In one embodiment, the primer comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence comprising SEQ ID NO: 29. In one embodiment, the primer comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence comprising SEQ ID NO: 30. In one embodiment, the primer comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence comprising SEQ ID NO: 6. In one embodiment, the primer comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence comprising SEQ ID NO: 31. In one embodiment, the primer comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence comprising SEQ ID NO: 32. In one embodiment, the DRED is selected from those diseases listed in Table 1 or Table 2.

An aspect of the invention also provides for a kit for monitoring the progression of a DNA Repeat Expansion Disease (DRED). In one embodiment, the kit comprises a primer directed to a complementary target nucleic acid sequence of a gene or gene product encoding MLH3 and instructions for use. In one embodiment, the kit comprises a second primer. In one embodiment, the primer comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence comprising SEQ ID NO: 5. In one embodiment, the primer comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence comprising SEQ ID NO: 29. In one embodiment, the primer comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence comprising SEQ ID NO: 30. In one embodiment, the primer comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence comprising SEQ ID NO: 6. In one embodiment, the primer comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence comprising SEQ ID NO: 31. In one embodiment, the primer comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence comprising SEQ ID NO: 32. In one embodiment, the DRED is selected from those diseases listed in Table 1 or Table 2.

An aspect of the invention provides for a method for treating a DNA Repeat Expansion Disease (DRED) in a subject in need thereof. In one embodiment, the method comprises administering to the subject an effective amount of a nuclease-resistant oligonucleotide compound as described herein. In one embodiment, the DRED is selected from those diseases listed in Table 1 or Table 2.

An aspect of the invention provides for a method for treating a DNA Repeat Expansion Disease (DRED) in a subject in need thereof. In one embodiment, the method comprises administering to the subject an effective amount of a pharmaceutical composition comprising a nuclease-resistant oligonucleotide compound as described herein. In one embodiment, the DRED is selected from those diseases listed in Table 1 or Table 2.

An aspect of the invention provides for a method for treating a DNA Repeat Expansion Disease (DRED) in a subject in need thereof wherein the method comprises administering to the subject an effective amount of an oligonucleotide complex described herein. In one embodiment, the DRED is selected from those diseases listed in Table 1 or Table 2.

An aspect of the invention provides for a method for treating a subject in need comprising administering a nuclease-resistant oligonucleotide compound that promotes the skipping of region(s) of a gene product. For example, the oligonucleotide comprises a sequence that specifically hybridizes in a human cell with a nucleic acid sequence encoding a subunit of the MMR system (e.g., MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2). In one embodiment, the nucleic acid sequence is a complementary sequence for a human MLH3 gene or gene product. In one embodiment, the nuclease-resistant oligonucleotide is useful for inducing exon skipping. For example, oligonucleotide(s) can induce skipping of MSH2 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or a combination of MSH2 exons. Oligonucleotide(s) can induce skipping of MSH3 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or a combination of MSH3 exons. Oligonucleotide(s) can induce skipping of MSH6 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or a combination of MSH6 exons. Oligonucleotide(s) can induce skipping of MLH1 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or a combination of MLH1 exons. Oligonucleotide(s) can induce skipping of MLH3 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or a combination of MLH3 exons. Oligonucleotide(s) can induce skipping of PMS1 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or a combination of PMS1 exons. Oligonucleotide(s) can induce skipping of PMS2 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or a combination of PMS2 exons. In one embodiment, the oligonucleotide directs skipping of one or more exons of MSH2, MSH3, MSH6, PMS1, PMS2, MLH1, or MLH3. In one embodiment, one or more oligonucleotides can induce skipping of MLH3 exon 7. In one embodiment, the oligonucleotide is decreases the rate of DNA repeat expansion. In one embodiment, the oligonucleotide is useful in treating a DNA repeat expansion disease. In one embodiment, the target nucleic acid sequence is located on human chromosome 2, 3, 5, 7, or 14. In one embodiment, the target nucleic acid sequence (or target complementary nucleic acid sequence) comprises: a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-16 described herein for GenBank Accession No. NG_007110.2, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007110.2; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-24 described herein for GenBank Accession No. NG_016607.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_016607.1; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-10 described herein for GenBank Accession No. NG_007111.1 or SEQ ID NO: 33, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007111.1 or SEQ ID NO: 33; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-19 described herein for GenBank Accession No. NG_007109.2, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007109.2; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-13 described herein for GenBank Accession No. NG_008648.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008648.1; a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-13 described herein for GenBank Accession No. NG_008649.1 or SEQ ID NO: 1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008649.1 or SEQ ID NO: 1; or a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-15 described herein for GenBank Accession No. NG_008466.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008466.1. In one embodiment, the component of the MMR complex comprises MutS or MutL. In one embodiment, MutS comprises a subunit selected from the group consisting of MSH2, MSH3, and MSH6. In one embodiment, MutL comprises a subunit selected from the group consisting of MLH1, MLH3, PMS1, and PMS2. In one embodiment, MLH3 comprises SEQ ID NO: 1. In one embodiment, the oligonucleotide hybridizes to the target complementary nucleic acid sequence comprising SEQ ID NO: 2. In one embodiment, the oligonucleotide has at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence comprising SEQ ID NO: 3 or SEQ ID NO: 4. In one embodiment, the oligonucleotide has at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a nucleic acid sequence depicted in Table 4. In one embodiment, the oligonucleotide comprises 15 to 30 nucleotide bases in length. In one embodiment, the oligonucleotide comprises one or more morpholino subunits, one or more locked nucleic acid subunits, one or more 2-O-methyl moieties, or one or more peptide moieties. In one embodiment, the subject in need is diagnosed with a repeat expansion disorder (e.g., a DRED). In one embodiment, the DRED is selected from those diseases listed in Table 1 or Table 2.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed in color.

FIGS. 1A-1F show a working model for transcription initiated DNA repeat expansion via mismatch repair. FIG. 1A shows part of a GAA·TTC repeat (SEQ ID NO: 41) depicted with the purine (or R) strand in red, and the pyrimidine (or Y) strand in yellow. The numbered bases show alignment in register. FIG. 1B shows that during transcription, the two strands are separated and a variety of structures can form. One likely example, an RNA·DNA hybrid, is shown. FIG. 1C shows resolution of a structure that can lead to an out-of-register re-annealing within the repeat. Figure discloses SEQ ID NOS 42 and 43, respectively, in order of appearance. FIG. 1D shows the small loop that is formed becomes a target for binding by mismatch repair complex, MutSβ. Figure discloses SEQ ID NO: 44. FIG. 1E depicts MutSβ in turn recruiting MutLγ, an endonuclease. Figure discloses SEQ ID NOS 44 and 43, respectively, in order of appearance. FIG. 1F shows that repeat expansion has occurred with the addition of two trinucleotides (*) after repair initiated by MutSβ and facilitated by MutLγ. Figure discloses SEQ ID NO: 42.

FIGS. 2A-2B show the knockdown of MLH1 or MLH3 significantly reduces GAA·TTC expansion rate in FRDA model cells. Four independent lines were transduced with the indicated knockdown lentiviral pools. FIG. 2A is a representative gel image of PCR products measuring GAA·TTC lengths. FIG. 2B is a bar graph showing mean expansion rates. Compared to the empty vector control virus (pLKO) MLH1sh was significantly different (p=0.0009) as was MLH3sh (p=0.00045) whereas PMS2sh did not reach significance (p=0.053).

FIG. 3 shows that MLH3 knockdown does not affect MutLα expression. Western blot probed for PMS2, MLH1 and ACTB (0-actin) shows long-term lentiviral-mediated shRNA knockdown of MLH1, PMS2 and MLH3 protein in the cells used for DNA in FIGS. 2A-2B. Knockdown of MLH1 concurrently depletes PMS2 (lanes MLH1sh). Knockdown of PMS2 halves MLH1 levels (lanes PMS2sh). Knockdown of MLH3 has a little effect on MLH1 protein levels, and no effect on PMS2 levels (lanes MLH3sh). None of the MLH3 antibodies tried were effective for western blots.

FIG. 4 is a schematic showing that human MLH3, a component of MutLγ, has 2 isoforms. MLH3 isoform 1 includes exon 7, which contains a highly conserved portion of an endonuclease domain, while MLH3 isoform 2 lacks this 72 base exon. Excluding exon 7 would approximate a functional knock out if the endonuclease activity of MLH3 is critical to repeat expansion.

FIG. 5 is a schematic showing that splice switching oligos (SSOs) were designed to bind the acceptor or donor region of MLH3 exon 7 in pre-mRNA. These SSOs were used to induce skipping of exon 7 and preferential production of MLH3 splice isoform 2 (iso2).

FIG. 6 is a photographic image of a gel showing that RT-PCR demonstrates exon skipping in MLH3 mRNA from SSO treated cells. Acceptor, donor or both SSOs were given twice a week to FRDA rapid expansion model cells in culture. Cells were assessed for MLH3 isoform expression with RT-PCR. The combination of acceptor and donor SSOs at 500 nM effectively excluded exon 7 as shown in lane 6 (arrow). M: A1 kb plus size standard showing 650 bp, 500 bp, 400 bp, 300 bp and 200 bp.

FIG. 7 is a photographic image of a PCR analytic gel showing that preferential expression of MLH3 isoform 2 leads to slower GAA·TTC repeat expansion. PCR analysis of GAA·TTC expansion at week 3 with indicated treatments. Repeat size assay PCR product equals 500 bp flanking sequence+3×(repeats). Sample 6 with 250 nM each of both acceptor and donor SSOs slowed expansion (arrow). M: A1 kb plus size standard showing 3,000 bp, 2,000 bp, 1,650 bp, and 1,000 bp.

FIG. 8 is a photographic image of a gel showing that single treatment with MLH3 SSOs slows expansion in non-dividing FRDA cells. PCR products show long (L) and short (S) alleles from FRDA patient fibroblasts.

FIG. 9A is a photographic image of gel showing detection of MLH3 isoforms 1 and 2. Defined templates containing or excluding MLH3 exon 7 were mixed in 10:1, 1:1 and 1:10 ratios. Primer pairs, MLH3 L3324 and MLH3 R3757 detected MLH3 isoforms 1 and 2. M: A 1 kb plus size standard showing 500 bp, 400 bp, and 300 bp.

FIGS. 9B-9D show a schematic showing that MLH3, a component of a MutL complex, has 2 isoforms due to alternative splicing. MLH3 iso1 has exon 7 and MLH3 iso2 lacks exon 7.

FIG. 10 is a schematic illustrating that MLH3 iso1 is required for expansion by following expansion in a human cell model. Single genomic construct, rather than 2 alleles simplifies analysis. Because the GAA·TTC repeat is not in the FXN gene in the model cell, repeat expansion is freed from the negative effect of insufficient FXN. Primers targeted for unique flanking sequences. GAA·TTC repeats expand incrementally and continuously in these model cells.

FIG. 11 is a UCSC Genome Brower image illustrating concordance of mouse and human MLH3 gene structures. The red oval indicates the variable exon in hMLH3 that is missing in hMLH3 isoform 2. This variable exon contains a conserved endonuclease domain.

FIG. 12 is a schematic illustrating that splice switching oligonucleotides target a subset of mMLH3 exons. SSOs function by tightly binding pre-mRNA at splice junctions thereby excluding splicing factors.

FIG. 13 shows individual and paired SSO activity in mouse Neuro-2A cells. Cultured cells treated with the indicated morpholino oligomers at the indicated concentrations were lysed after 48 hours and RNA was isolated for cDNA synthesis. PCR analysis of the cDNA using oligonucleotides flanking the region of interest in the mMlh3 mRNA resulted in specific bands corresponding to no skipping (720 bp), skipping exon 5 or exon 6 (˜648 bp), or skipping both exons 5 and 6 (575 bp). Lane M is the 1 Kb plus DNA ladder showing bands of 1000, 850, 650, 500 and 400 base pairs.

FIG. 14 shows Mlh3 exon skipping is evident in adult mice 48 hours after a single injection. Adult C57BL/6J mice were given a single dose (5 g/g) of a mixture of mMlh3ac5 and mMlh3dr7 cell penetrating “vivo” morpholino SSOs in the tail vein. Tissues were collected after 48 hours and RNA was isolated to identify the splice variants. In this experiment, 3 of 4 mice injected with SSOs exhibited the desired mMlh3 exon skipping in kidney (green arrows). Controls (C) were injected with saline.

FIG. 15 shows Mlh3 exon skipping is robust in adult mice 48 hours after a single high dose injection. Adult C57BL/6J mice were given a single dose (50 g/g) of a mixture of mMlh3ac5 and mMlh3dr7 cell penetrating “vivo” morpholino SSOs in the tail vein. Tissues were collected after 48 hours and RNA was isolated to identify the splice variants. In this experiment, 2 of 2 mice injected with SSOs exhibited robust mMlh3 exon skipping in kidney (green arrows). In these mice the treatment did not penetrate the blood brain barrier and mMlh3 remained as isoform 1 in brain (Br).

DETAILED DESCRIPTION OF THE INVENTION
Abbreviations and Definitions

The singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

As used herein the term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).

The terms “animal,” “subject,” and “patient” as used herein includes all members of the animal kingdom including, but not limited to, mammals, animals (e.g., cats, dogs, horses, swine, etc.) and humans.

Repeat Expansion Diseases

DNA repeat expansion disorders are a family of genetic disorders characterized by the pathogenic expansion of a repeat region within a genomic region (1,2). In such disorders, the number of repeats exceeds that of a gene's normal, stable threshold, expanding into a diseased range. In most cases, the length of repeat expansion is negatively correlated with prognosis, i.e. longer repeats are correlated with an earlier age of onset and worsened disease severity. DNA repeat expansion disorders are often called trinucleotide repeat (TNR) expansion disorders because trinucleotide based disorders were the first discovered and are the most widely known form of expansion diseases. However, expansions of up to twelve base repeat units have been found to cause disease (see Table 1).

TABLE 1

Repeat Expansion Diseases

Disease, repeat and

gene affected
Estimated Prevalence

1
Friedreich Ataxia (FRDA)
2-4/100,000 (~1% of

GAA expansion in FXN
European descent

carry)

2
Fragile X Syndrome (FXS)
14/100,000 males

CGG expansion in FMR1

3
Huntington′s Disease (HD)
2.71/100,000

CAG expansion in HTT

4
Amyotrophic lateral
24%-46% of Familial

sclerosis/frontotemporal
ALS

dementia (ALS/FTD) CCGGGG

expansion in C9orf72

5
Myotonic dystrophy type 1
5/100,000

(DM1) CTG expansion in

DMPK

6
Myotonic dystrophy type 2
12.5/100,000

(DM2) CCTG expansion in

ZNF9

7
Spinal & Bulbar muscular
0.75/100,000 males

atrophy (SBMA) CAG

expansion in AR

8
Spinocerebellar Ataxia
1.5/100,000

1/SCA1 CAG expansion in

ATXN1

9
SCA2
?

CAG expansion in ATXN2

10
SCA3/Machado-Joseph
?

disease (MJD)

CAG expansion in ATXN3

11
SCA6
0.31/100,000

CAG expansion in CACNA1A

12
SCA7
2% of SCA′s

CAG expansion in ATXN7

13
SCA8
2-5% autosomal

CAG expansion in ATXN8
dominant ataxias

14
SCA10
?

ATTCT expansion in ATXN10

15
SCA12
?

CAG expansion in PPP2R2B

16
SCA17
?

CAG/CAA expansion in TBP

17
SCA31
?

TGGAA expansion

in TK2

18
SCA36
?

GGCCTG in NOP56

19
Dentatorubral-
0.48/100,000 of

pallidoluysian atrophy
Japanese

(DRPLA) CAG
population

expansion in DRPLA

20
Oculopharyngeal muscular

dystrophy (OPMD) CGG

expansion in PABPN1

21
Progressive myoclonus
5/100,000 Finnish

epilepsy (EPM1)
births

CCCCGCCCCGCG (SEQ ID NO:

34) expansion in CSTB

The repeat expansion disorders that were discovered first are predominantly dominant diseases, such as Huntington's disease. However, recessive DNA repeat expansion disorders are a rapidly growing subclass. For example, the progressive, neurodegenerative disease Friedreich ataxia (FRDA) is caused by a repeat expansion in the FXN gene from the normal range of 6 to 36 repeats to the diseased range of approximately 600 to 1600 repeats (3). The GAA·TTC repeat expanded from an Alu element in the FXN first intron (4,5). Disease severity correlates to the length of the expanded repeats and the consequent reduction of FXN gene expression. Over a million Alu elements together constitute about 11% of the human genome (6), suggesting a vast reservoir for other such expansions. Indeed, the CCTG expansion responsible for Myotonic dystrophy type 2 (DM2) and the ATTCT expansion causing spinocerebellar ataxia type 10 (SCA10) also expanded from Alu elements (7,8).

To effectively treat a relentlessly progressive and lethal disease like Friedreich ataxia, the underlying DNA repeat expansion must be addressed. Currently there is no effective treatment and no cure for any of the DNA repeat expansion diseases (see Table 2). In one embodiment, the invention is directed to treatment of DNA repeat expansion diseases (DREDs) using oligonucleotide compositions discussed herein.

TABLE 2

Exemplary DNA repeat expansion diseases.

Friedreich’s Ataxia
Spinocerebellar ataxia type 3

Blepharophimosis-ptosis-epicanthus
Spinocerebellar ataxia type 6

inversus

Cleidocranial dysplasia
Spinocerebellar ataxia type 7

Congenital central hypoventilation
Spinocerebellar ataxia type 8

Dentatorubralpallidoluysian atrophy
Spinocerebellar ataxia type 10

Fragile X syndrome
Spinocerebellar ataxia type 12

FRAXE mental retardation
Spinocerebellar ataxia type 17

Hand-foot-genital
Spincocerebellar ataxia type 31

Holoprosencephaly
Spinocerebellar ataxia type 36

Myoclonus epilepsy type 1
Huntington’s disease like 2

Myotonic dystrophy type 1
Spinal and Bulbar Muscular atrophy

Myotonic dystrophy type 2
Huntington’s disease

Oculopharyngeal muscular dystrophy
Synpolydactyly

Spinocerebellar ataxia type 1
Fragile X-associated tremor/ataxia

syndrome

Spinocerebellar ataxia type 2
Syndromic and nonsyndromic X-linked

mental retardation

Amyotyophic Lateral Sclerosis/
Dentatorubral pallidoluysian atrophy

Frontotemporal Dementia

Progressive myoclonus epilepsy

Sufficiently long DNA repeats such as those seen in DNA repeat expansion disorders are characterized by genomic instability of the repeated region. Often, the repeated regions frequently change in length during intergenerational transmission and within somatic cells. This fact holds both clinical and emotional relevance. Clinically, longer repeat lengths are associated with increased disease severity and earlier age of onset. Additionally, the progressive repeat expansion may actually cause development of the disease that otherwise would not develop, as the expansion of only 1 additional repeat can result in an individual's repeat region expanding into the diseased range. Emotionally, this possibility may result in increased anxiety and depression in the ‘at risk’ individual. Unfortunately, there are no therapeutics to slow disease repeat expansion.

DNA Mismatch Repair (MMR)

The molecular mechanism underlying repeat expansion largely remains unclear. Without being bound by theory, DNA mismatch repair contributes to the genomic instability observed in trinucleotide repeat expansion disorders. DNA mismatch repair (MMR) is a pathway that normally recognizes and repairs DNA errors made during replication. However, when it comes to certain types of repetitive DNA, MMR actually can contribute to genome instability. For example, a contribution by the MMR pathway has been established in several repeat expansion diseases including myotonic dystrophy, Huntington's disease, and Friedreich ataxia. For example, the inventors' laboratory has contributed to the understanding of the role of MMR in Friedreich ataxia. MMR requires the sequential action of the protein complexes MutS and then MutL. Humans have two different MutS complexes, alpha and beta, and three different MutL complexes, alpha, beta, and gamma. In human cells, DNA mismatches are initially recognized by a MutS protein heterodimer prior to recruitment of a MutL complex. MutSalpha, a heterodimer of MSH2 and MSH6, is the dominant MutS complex that recognizes base-base mismatches and short insertion/deletion loops. MutSbeta, a complex of MSH2 and MSH3, is less abundant than MutSalpha in most cell types, and appears to be functionally redundant to MutSalpha. In MMR, MutS heterodimers recognize a mismatch but a MutL heterodimer is required as the next step in the mismatch repair process. In humans there are four identified MutL homologues: MLH1, MLH3, PMS1 and PMS2 (9-12). MLH1 is the master subunit, much like MSH2 in the MutS system. MLH1 combines with PMS2 to form MutLalpha, with PMS1 to form MutLbeta and with MLH3 to form MutLgamma, respectively. Also like MSH2 and its partners, MLH1 and its partners are more stable as heterodimers (11). MutLalpha is the dominant species, being about ten-fold more abundant than MutLbeta (11), similar to the ratio between MutSalpha and MutSbeta (13). MutLgamma is less abundant still: about 2% the level of MutLalpha (14). MLH3 expression levels are considerably lower than the other binding partners of MLH1 and MLH3 is currently considered to be a minor player in MMR processes, as it has mostly redundant functions. Without being bound by theory, MLH3, while a minor player in canonical MMR, is a major force in DNA repeat expansion. In one embodiment, the present invention utilizes oligonucleotides (for example, antisense oligonucleotides resistant to nuclease digestion), for use in modulating the expression and/or function of subunits of the MMR system that are encoded by nucleic acid molecules discussed herein, ultimately modulating the amount of a MMR system subunit that is expressed and/or produced. In one embodiment, the MMR system subunit comprises MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2. In one embodiment, the MMR system subunit comprises MSH2, MSH3, or MSH6. In one embodiment, the MMR system subunit comprises MLH1, MLH3, PMS1, or PMS2. In one embodiment, the he MMR system subunit comprises MLH1 or MLH3. In one embodiment, the MMR system subunit comprises MLH1. In one embodiment, the MMR system subunit comprises MLH3. In one embodiment, the MMR system subunit comprises PMS1. In one embodiment, the MMR system subunit comprises PMS2. In one embodiment, the MMR system subunit comprises MSH2. In one embodiment, the MMR system subunit comprises MSH3. In one embodiment, the MMR system subunit comprises MSH6. In one embodiment, the expression and/or function of a subunit of the MMR system is decreased.

The genomic sequence for human MSH2 (found on human chromosome 2) has GenBank Accession No. NG_007110.2. Sequence information related to human MSH2 (isoform 1) is accessible in public databases by GenBank Accession numbers NP_000242.1 (protein) and NM_000251.2 (nucleic acid). Sequence information related to human MSH2 (isoform 2) is accessible in public databases by GenBank Accession numbers NP_001245210.1 (protein) and NM_001258281.1 (nucleic acid). The genomic sequence for human MSH2 (GenBank Accession No. NG_007110.2; 166,188 bp in length), is found at nucleotide no. 4,944 and terminates at nucleotide no. 85,105, wherein exon 1 is located between nucleotides 4,944 and 5,279; exon 2 is located between nucleotides 10,278 and 10,432; exon 3 is located between nucleotides 11,971 and 12,249; exon 4 is located between nucleotides 14,291 and 14,437; exon 5 is located between nucleotides 16,146 and 16,295; exon 6 is located between nucleotides 18,173 and 18,306; exon 7 is located between nucleotides 31,619 and 31,818; exon 8 is located between nucleotides 47,425 and 47,534; exon 9 is located between nucleotides 64,908 and 65,031; exon 10 is located between nucleotides 68,535 and 68,685; exon 11 is located between nucleotides 72,842 and 72,939; exon 12 is located between nucleotides 76,902 and 77,147; exon 13 is located between nucleotides 78,244 and 78,448; exon 14 is located between nucleotides 80,149 and 80,396; exon 15 is located between nucleotides 82,573 and 82,748; and exon 16 is located between nucleotides 84,656 and 85,105. It is understood that intron sequence precedes and follows the denoted nucleotide regions comprising the exon sequences listed herein. Oligonucleotide compounds (e.g., exon skipping or intron retaining SSOs) can be directed to the nucleic acid sequence corresponding to the region of interest for each of the exons 1-16 described herein, and intron-exon junctions, or exon-intron junctions listed with GenBank Accession No. NG_007110.2.

The genomic sequence for human MSH3 (found on human chromosome 5) has GenBank Accession No. NG_016607.1. Sequence information related to human MSH3 is accessible in public databases by GenBank Accession numbers NP_002430.3 (protein) and NM_002439.4 (nucleic acid). The genomic sequence for human MSH3 (GenBank Accession No. NG_016607.1; 229,341 bp in length), is found at nucleotide no. 5,174 and terminates at nucleotide no. 227,341, wherein exon 1 is located between nucleotides 5,174 and 5,490; exon 2 is located between nucleotides 6,937 and 7,057; exon 3 is located between nucleotides 15,669 and 15,889; exon 4 is located between nucleotides 20,623 and 20,835; exon 5 is located between nucleotides 22,770 and 22,886; exon 6 is located between nucleotides 23,267 and 23,384; exon 7 is located between nucleotides 25,509 and 25,654; exon 8 is located between nucleotides 29,453 and 29,619; exon 9 is located between nucleotides 75,979 and 76,091; exon 10 is located between nucleotides 79,377 and 79,491; exon 11 is located between nucleotides 91,990 and 92,074; exon 12 is located between nucleotides 95,032 and 95,141; exon 13 is located between nucleotides 112,072 and 112,204; exon 14 is located between nucleotides 118,459 and 118,646; exon 15 is located between nucleotides 119,361 and 119,529; exon 16 is located between nucleotides 126,220 and 126,284; exon 17 is located between nucleotides 129,246 and 129,362; exon 18 is located between nucleotides 138,091 and 138,198; exon 19 is located between nucleotides 143,259 and 143,370; exon 20 is located between nucleotides 164,110 and 164,267; exon 21 is located between nucleotides 204,656 and 204,842; exon 22 is located between nucleotides 215,339 and 215,468; exon 23 is located between nucleotides 223,642 and 223,813; and exon 24 is located between nucleotides 226,277 and 237,341. It is understood that intron sequence precedes and follows the denoted nucleotide regions comprising the exon sequences listed herein. Oligonucleotide compounds (e.g., exon skipping or intron retaining SSOs) can be directed to the nucleic acid sequence corresponding to the region of interest for each of the exons 1-24 described herein, and intron-exon junctions, or exon-intron junctions listed with GenBank Accession No. NG_016607.1.

The genomic sequence for human MSH6 (found on human chromosome 2) has GenBank Accession No. NG_007111.1. Sequence information related to human MSH6 (isoform 1) is accessible in public databases by GenBank Accession numbers NP_000170.1 (protein) and NM_000179.2 (nucleic acid). Sequence information related to human MSH6 (isoform 2) is accessible in public databases by GenBank Accession numbers NP_001268421.1 (protein) and NM_001281492.1 (nucleic acid). Sequence information related to human MSH6 (isoform 3) is accessible in public databases by GenBank Accession numbers NP_001268422.1 (protein) and NM_001281493.1 (nucleic acid). The genomic sequence for human MSH6 (GenBank Accession No. NG_007111.1; 30,807 bp in length; SEQ ID NO: 33), is found at nucleotide no. 4,936 and terminates at nucleotide no. 28,807, wherein exon 1 is located between nucleotides 4,936 and 5,347; exon 2 is located between nucleotides 12,781 and 12,977; exon 3 is located between nucleotides 17,748 and 17,917; exon 4 is located between nucleotides 20,465 and 23,009; exon 5 is located between nucleotides 25,274 and 25,539; exon 6 is located between nucleotides 26,764 and 26,881; exon 7 is located between nucleotides 27,472 and 27,561; exon 8 is located between nucleotides 28,058 and 28,212; exon 9 is located between nucleotides 28,306 and 28,505; and exon 10 is located between nucleotides 28,633 and 28,807. It is understood that intron sequence precedes and follows the denoted nucleotide regions comprising the exon sequences listed herein. Oligonucleotide compounds (e.g., exon skipping or intron retaining SSOs) can be directed to the nucleic acid sequence corresponding to the region of interest for each of the exons 1-10 described herein, and intron-exon junctions, or exon-intron junctions listed with GenBank Accession No. NG_007111.1 or described in SEQ ID NO: 33.

1
cactatgttg ctagctggtc ttgaactcct ggcctcaagt catccttctg tcttggcctc

61
ccaaagtgtt gggattgtaa gtgtgagcca ctgtccctgg ccagttggtg atttatttgt

121
ataactgtcc aatttattga atacgtatgg cgtgccaagc actgggctga gggcttcata

181
atgccctttc actcaatgct tagcacaacc catgaagaag gtagtgttaa tatcatccct

241
gttttacaga tgtagaaact gaggcacagg ctaaataact tgcccaacaa gctcgtgcag

301
tttagtaagc agctcagctg ggatgtgaac acaaactttg aatacagagc tcttaaccag

361
taggccagag gttcccaaac acctagtata tttactacct tagtactttc ctgccacatc

421
tcctaggcca aaacaactcc attgattgtt atgcaattta ctaagtgtag ccatttgaaa

481
aaaaaataca tataaaagaa aaatattttt attcagtttt caaaataacc catatatagt

541
catggaatgt atgtgttggt tgggcactgc agactaaaga cagtttaggg ccgggcacgg

601
tggctcaggc ctgtaatcct agcactttgg gaggccaagt agagagaagg gcttgagccc

661
aagagttgga gaccggcctg ggcaacatag caagacccag tctctacaga aaataaaatt

721
atcagggtgt gaggacgcac acctgcagtc ttagctgctt gggaggctga ggctggagga

781
tcagcctggg caacagagtg agaccctgtc tcagaaaaaa aaaaaaaaaa agacagactt

841
ttattcagat atgcatgcag gagttcacag aaaaaaaaag tgagtccagg aggctgttat

901
ttggcattta tacaactttt tttttcttga atctcgaaat ctactttata tataccattt

961
aataggggaa gaggagggag aaaaagcctt ccatgggaag aacaaatagg tttctggggg

1021
aacaaaaggg agataagaat gtttgttttt gcaggtgcaa gtggtctttg tctttttttc

1081
tggccactaa aactccccta gagaggagat ttacggcagc ttcactccca gaaatttctg

1141
ctgttagtcg cataagggaa gctttgaaac ggcatctttc tgcatctgtt ggctctcaaa

1201
tgtcttcagt tccaagtaac attcatgcca attctggggg tctgagtgtc cccacataat

1261
acatgtgttc tcttgtcttt taatgaagtt tgtgggaggc atctaactgt agcctccaaa

1321
atttggccca taggtactac tgtccttatc aaagacgagg aaacaagttc agaaaagtat

1381
taattgctcc gagttatctg cttggctagc taggatcaga gctcagttct ccatttaacc

1441
caaagcccag gctcttaacc tcttacaact ggcgcatccc ctctgaacct ccatttcctc

1501
cctgtaaaag aataacatcg gccgggcgca gtggctcaca tctataatcc cagcactttg

1561
ggaggcagag atgggcggat cacgaggtca ggagtttgag accagcctgg ccaacatggt

1621
gaaaccccat ctctactaaa aatacaaaac ttagctgggt gtgttggtgc ctgtaacccc

1681
agctactcag gagactgagg caggagaatt gccttaacct gggaggcgga ggttgtggtg

1741
agccaagatc gtgccattgc actccagcct cggtgacaga gcaagactcc atccccaaaa

1801
aaacaaacaa caacaacaaa aagagaataa cgttatattc agttgaacca aaatgaatta

1861
aatattaata tttgtacttc aaaaacggtc cagcttggct gggcgcagtg gctcccgcct

1921
gtaatcccaa cattttggga ggccgaggca ggaggatcat ttgaggtcag gagtttgaga

1981
ccagcctggc caacatggtg aaatcctgtc tctactaaaa atacaaaaat tagctgggca

2041
gtagtagcgc gtgccggtaa tcccagctat tcaggaggct gaggaaggag aattgcttga

2101
gcttgggagg tgaaagttgt ggtgagctga gactgcacta ctgcacacca gtctgggaga

2161
cagagtaaga ccctgtctca aaacaaaaca accaaaaaac caaaaaggtc cagcttgggc

2221
aacatagtga aacttcgtct ctacagaaaa tttttaaaat actagcaggg caccgggcac

2281
agtggctcat acctgtaatc ccagcacttt gggaggctga ggcaggcggg tcacttgtgg

2341
tcaggagttt gggatcaggc aggccaacat ggtgaaaccg tgtctctact aaaaaacaaa

2401
aattagctgg gcatggtggt aggcaccagt aatcctagca ctcaggaggc tgaggcatga

2461
gaattgcctg aacccgcaaa gcaggggttg cagtgaacca agatggcgtc actgtactcc

2521
agcctgggtg acagaataag actcctcaat taaaaaaaaa aaaaattagc tgggcatggt

2581
gttgcgggcc tgtggtccca ggtactcagg aggctgaggt gagaggatta cttaagcctg

2641
ggaggttgag gctacagtaa gccaagatca cgccactata ctccagcctc tgtgacagag

2701
ccagaccctg tctcaaaaaa attttaaaaa gggcaaattt tggcaatttc acatagttca

2761
acctagtata aggtggttgt aataactaaa tgagataaaa tggtgttaaa ttggaagtat

2821
tatagtattt ctgttaacaa catagggctc cagaaccagc ttccttgagt ttaaatccag

2881
gctccaccac ttcctagcta tgcagtcatg ggcaagttac ttgacccaac tgtgcctcag

2941
cttcatccat gatatggaga tacaggataa ccagcctctt acgtgcaatt ctgaaatcca

3001
aaaagctctg taaaccaaaa gtttgggggt aaactcattt ggtagcaaat tttgacctga

3061
ggctatttat agtctatatt ctgtattctt tctacttagt atgaataagc atgtaagttt

3121
tactgcatgt ttgatttcag catgttcccc cagactctct gggggtgttt acgtatgccg

3181
gtgggggaaa gagaccaact ctcaaatatt atctcaaaca gttggtttca ctgtgcttgc

3241
ttgggtagca catataccaa aattggaatg acccctgcac agggatgaaa tgcaaattcg

3301
tgaagcatac tgtatttttc ttagcacata ccacctttgg caatattctt tttttttttt

3361
tgagagggag tcttgctctg tcgcccaggc tggagtgcag aggcgcgatc tcggctcact

3421
gcaagctccg cctcccgggt tcacaccatt ctcctacctc agcctcccca gtagctggga

3481
ctacaggcgt gtgctaccac gccaggctaa ttttttgtat ttttagtaga ggcggggttt

3541
cactgtgtta gccaggatgg tctcgatctc ctgacctcgt gatccgccca cctccgcccc

3601
ccccccgaag tgccgagtgc tgggactaca ggcgtgagcc actgcgcccg gcccccgcct

3661
ttttttttta gattgatttt attacttgcc tagcaaagga gaaccttctg gcagaacagt

3721
ctccaagaac aaggcaaaca actaatttta cataggtttt taccaatgta cagctgttga

3781
ttgtgactgg tttccggcaa tctggatttc acaatctgga taaggggaca aacaattgtc

3841
tgtcttccac tatctttctt gaatttgaat agaacctttt tattctcata gcctcttagc

3901
tttctttctt ttttttttga gacggagttt cgctcttgtc gcccaggctg gagtgcagtg

3961
gcgcgacctt ggctcactgc aaacgctgcc tcccaggttc aagttattct cctgcctcag

4021
cctcccaagt agctgggatt acaggcgcat gccaccacgc ccggctaatt tttggatttt

4081
tagtagagac gggggtttca ccatgttgac taggctggtc ttcaacgcct gacctcaggt

4141
gatccgcccg cctcggcatc ccaaagtgct gggattacag gcgtgagcca ctgcgcccgg

4201
cctctcatag tctcttagct ttctaaaatt tgaaaaatcc tgtaaagaca cacctgggtc

4261
aaagggctca gataacggac tgtggccctt aagtacttac gtcacaggtt attgagagga

4321
tcgatttagt taccagatgt aaaatgctgg gatcagtgcc tggcaaagga aaactttgta

4381
cagctgcagg ctttcaccat acacaacagc atcgctaacg aatgctatta caatattcat

4441
ttagcgttta ccaagtgcct actctataca aatcttgaga atacaacgtg aaggtgaact

4501
gctgactaaa gtttggtccc tttcgctccg tctccttgcg aaaatgctct aacggcagga

4561
ggtcacgcga gcgctggacg cgtttctccc cgcgagcccc tttccgaggc ctttcgggtc

4621
cccccggtta tccccgcccg ggcggtgcgc gcccccgctg ttcccgcttc cgctccagag

4681
aggcagggct ttccgagcct gctagccccg cggccgcaac taaccccggg tcggagtgtt

4741
ccggcccggc cagccccgcg gcgtgaggga aggggagctc agcagttccc cgcgcggggc

4801
ccaggcgtcg gcggcagggc gggcccctca ccgccagcgt gccagccccg cccctaccca

4861
ccagtgtgcc agccccgccc ttccccacgt cgccgcgcgc ccgggggcgg ggcctggcgc

4921
gcaccgcccg cgcacggcga ggcgcctgtt gattggccac tggggcccgg gttcctccgg

4981

cggagcgcgc ctccccccag atttcccgcc agcaggagcc gcgcggtaga tgcggtgctt

5041

ttaggagctc cgtccgacag aacggttggg ccttgccggc tgtcggtatg tcgcgacaga

5101

gcaccctgta cagcttcttc cccaagtctc cggcgctgag tgatgccaac aaggcctcgg

5161

ccagggcctc acgcgaaggc ggccgtgccg ccgctgcccc cggggcctct ccttccccag

5221

gcggggatgc ggcctggagc gaggctgggc ctgggcccag gcccttggcg cgctccgcgt

5281

caccgcccaa ggcgaagaac ctcaacggag ggctgcggag atcggtagcg cctgctgccc

5341

ccaccag
gta gcggggtggg ggtggggtcg aaggcggggg catagcggcg gggcgcttgg

5401
aacccggcga ggggaggctc gcacaggggg ttgggggggt gcacggcctg gccctgggct

5461
cggaggaggc ggggccgcag agttggcttg aatgagtgca ggggtcgagt ctggagcatt

5521
tgggggtgta gcttgtaaac agggtcggag gagagaggct gtgcaggaag agggctgcag

5581
gggagacgcg gagagttcgg gccttttgga gggaggagac gcgtcccgcc aggtgggggt

5641
gctgggctaa ggaaggggcg acgcgcgcag ctccgggtgg ggagggggcc tgggaggtgg

5701
gagcactggg ggtggggcga gaaggggaag gcgcccggcc cacttggtgg gcggggcggg

5761
gggcggggtg gcgggaagga ggaatgcctg cgggaggccg aacggggaga gtccggtggt

5821
gtggggtgcg aaaggaggtt cctcggccgg cgcggagata gtgagttggg gctccagtag

5881
tcgatcgagg tagacactta gaggtagtta agagccgcgg tcgccgagac gccttgggga

5941
cggtgggcct tcggcctagg tgaggggccg ccgagggggt gggccacgag ctgcgagcgc

6001
gggggggtgt gtcaccatgg ggaccgcggg gcctaattgg gcggggcggg gccgtgggga

6061
gccgaagtgc tgggatccgg ctgggtcctt cggtaggtag gctgcacgtg caccgagacg

6121
aagatagaat attttgacgt atgtggaaat tcgtgtcgag tggaaaatat tttattttat

6181
gaaatagtgt aatttttatg gggcaccact gggcttttag aggccttaat cgggcgctgg

6241
acaaagatgt gtggacgtga gtgactccgg ggaagcctgt cgggagttgt cctcacttta

6301
tgggcagtta agtgcttttt tttttttttc ctttttgaga gagagtttcg ctcaagtcca

6361
ggctggagtg caatggcgcg atctcagctc accgcaatct ccgcgtcccg gcttcaagcg

6421
attccccagc ttcagcctcc cgagtagtcg ggattacagg aatgcgcccc cacaccccgc

6481
caattttgta tttttagtag agacggggtt tctccatgtt ggtcaggcta gtctcggaat

6541
tcccgacctc aggtgatcca cccgcctcgg cctcaaagtg ctgggattac aggcgctagc

6601
caccgcgccc ggtctgttta gggcttttta tccgggcagc tggcgacatt ttgaaaagct

6661
tgcttttgct gtttgccaga tacatatata tgtattttga gacagagtct tgctcttttg

6721
tccaggctag agtgcagtgg cgcgttcttg gctcaccaca acctctgtct ctggatcaag

6781
agattatcct gcctcagcct cccaagtagc tgggactaca ggtgcgcccc accacgcctg

6841
gctaattttt gtatttttag tagagacggg tttcactatg ttggccaggc tggtatcgaa

6901
ctcctgacct cttgatcggc ccgcattggc ctaccaaagt gctgggatta caggcatgaa

6961
ccaccgagcc cggccgtttg tcagatacta aacacaaagt ttaatggtcg ctatttgaac

7021
aaacgaagaa ataaaggctc agaaaaaata actcattcaa gataagagcc agttcgtgtt

7081
ttttgtttgg ttttgttttg aaatggagtc tcgctctgtc gcccaggctg gagtgctgtg

7141
gcgctttctc ggctcactgc aacctctgcc cgccgggttc aagtgattct cctgcctcag

7201
cttcccgagt agctgggatt acgggtgtgc ccaccgcggt ccggctgatt tttcaccatg

7261
gagtttcacc atgttggcca ggctggtctt gaaactgctg acctcaagtg gtccacccac

7321
ttcagcctcc caaagtgctg ggattacagg tgtgagccac cgtgcccggc cgctagttag

7381
tggttttgag taatggattt caaatccatt taaatccagt ttaaagtgtc ctaaaggaat

7441
tctgagattt ttctaagtgt aattatagtg ttacccttgt ttaagcgacc ctttcccgca

7501
gtttaaatat atatagttgt gcattagtag aatatgcttg tggggaacag agccagcatc

7561
cgcaataaca aactcctggt tagaaaagca tgacgtattg tttacttgag catgaattga

7621
ttgttgaatc caaaccaaac gggtgtattt attgtaagga tgtactttac attcatattg

7681
aatagcgtat gttatttgtt tcttgaggtt gagtttaaga gacttgtaaa aataaaacgt

7741
atacatttca cctcccgtta tggagaggat tccagggtat tcaagaaaga tgggcatttg

7801
atactaggtt tctaaagaaa ctgcagtgtc tagatcactc tgccgagcac agcattaggc

7861
attatggatc ctggatacaa ccatgaacag gacaaagcaa agaggcaatt gtagactcca

7921
agtggaaagg ggacggagag gatgcgggtc aggctaggct ctcagctctg taaaccgaaa

7981
ccagaaggac aaataagctt agacagatta tagtgagagt gggaagctgg ttcaggaaga

8041
ggaaggtctg caaattgtgg gtaggatgaa aggaggagga gggagcattg gagaagttaa

8101
gcagagatcc aatcatgaac agtctgatga gctacagaga cattcggact tactccatga

8161
atcatttaag ccttaaaaca tgttgagcgt attttttttt tttttgagac ggaatttcac

8221
tcttgttgcc caagctggag tgcagtggtg tggtctcagc tcactgcaac ctccgcctcc

8281
tgggttccag cgattctcct gcctcatcct ctcaagtacc tggtattaca ggtgcctgcc

8341
accacgccca gctaattttt gtgtttatag tagagacggg tttcaccatg ttggtcaggc

8401
cagtcgtgaa ctcctgacct caggtgatcc acccacctca gcctcccaaa gtgttgggat

8461
tacaggcgtg aaccaccgca cctggccgtg agccaccgtg tctgtccgag catcttttaa

8521
tgtttgtcat ttagatttct tcttgtgctg aagtgtttgt cttttgctgt ttcttttttt

8581
tttcctagtt ctttgtcatt tgtgtgtgat ataaatgtct tctttcacaa tgagttcttt

8641
catttagttt atggctttgt tgttgttgtt gaataataga ggtctcactt tgttgcccag

8701
gctggtgttg aactcttgct ctcaagcgat cctcccactt cagcctccca acctgttggg

8761
attacaagtg tgagccacca cacccagcct tatggcatct ttcgatgaac aaattattga

8821
ttataatgtg gaatttgtcc ttttattttc tctgtggtta gtgtttctat aggttttatt

8881
taagaaatcc acagggaggc tgggtgcagt ggctcatgcc tgtaaaccca acactttggg

8941
aggccaaggc aggccaacat ggctagaccc tgtctctcca aaaaataaga aaattagcca

9001
ggcatggtgg cgtgtgcctg tagtcccagc ttcttgggag actgagatgg gaggatcgct

9061
tgagtccagg aggttgaggc tgcagtaagc caagagatca tgccatgcac tccagcctgg

9121
gtggcagagt cagaccctgt ctgccaaaaa ataaaataaa agttggtgaa aatgttgatt

9181
atatatttta ggaacaacta gtaattgaca tcaaaattat gggctaaaga gaaagcaaaa

9241
ataatgtgat tttaaaccag aattcaaaag atctgtttag cgtatgttta gacaaagcca

9301
ttacttatta tatcaaagtt ttaacattta ttttgtgagc tgtcagcttt tcctcttaac

9361
atttttcccc accgtcttaa aaaaccccaa gaataccgga catttaagac tcacttaaag

9421
ctttaaaagc acttgcaaaa tcctaaaatc ataatttaag gtgtttttgg agggcaggag

9481
caatggtggc aggcagtgtt ttgctttgtt gcccaggctg aagtacagtg gcagatctcg

9541
gttcactgca ccctcgacct attcggctca agtgatcctc ccacctcagc tttctgagta

9601
gctgggaccc caagtgcaca ccaccccatg cctggctaat ttttaaattt ttttgtagaa

9661
acaaggtctc actgtgtagc ccagatggtc tcgaattcct gggctcttaa gagatcctcc

9721
caaagtgctg ggatcatagg tgtgagccac cacacctggc ctattttggc attcttgaaa

9781
accgcaggat taccacggat aaaattttaa aattaccttt aaagaattca ggtttacaca

9841
caaaaaaaat ttggtttgtt agcagtgagt gaagaaaaat tttgagaaat gtttaaaatt

9901
tttagttttg ttacacaata cattttacta cctgtttaat tatctttttt gactcagaaa

9961
ccagtttcct gggtccagga tgtttagtgg tactcttttt cttcaagctt tttagcattg

10021
gaggaactgc atattagtaa aatttttagt cttagcattt tatagcttac tgctatttct

10081
tttctttcat tctttctttc tttttttttt tttttttttt ttttttgaga tggagtctcg

10141
ccctgtcacc caggctggag tgcagtggca cgatctcggc ttactgcaac ctctgccttc

10201
caggttcaaa tgattctcct gcttcagcct cccgagtagc tgggattaca gatgcccgcc

10261
accatgccca gctaattttt atttttttag tagagatggg gtttcaccat gttggccagg

10321
ccagtctcga actcctgacc tcgtgatcaa cccgccttgg cctttcaaag tgctgggatt

10381
acaggcgtga gccaccgtgc ccagcctttt tctttttctt tttctttttt tttttttttt

10441
tgagacggag tcttgctctg ttacccaggc tggagtgtag tggcatgatc tgggctcact

10501
gcaacctcca cctcccgggt tcaagggagt ctcctgcttc agcctcccga gtagctggga

10561
ttacaggcgc ctgccaccat gcccagctaa tttttgtatt tttttagtag agatggggtt

10621
tcgccatgtt ggccaggctg gtcttgaact cctgacctca ggtgatctgc ctgcctcgtc

10681
ctcccaaaat gctgggatta taggagtgag ccactgcgcc cggcccagca tactgctatt

10741
tctttctttc tttcttcttc cttttttttt tttttgtttt tttttttttt tttttttttt

10801
tgtgagacgg agtctgtcgt ccaggctgga atgcagtggc gttttcttgg ctcactgcaa

10861
cctctgctgc ccgggttcaa gtgattctcc tgcttcaggc tcccaagtag ctgggattat

10921
aggcctctgc cactgcactt ggctaatttt tgtatttttg gtagagacgg ggtttcacca

10981
tcttggccag gctggtcttg aactcctgac ctcgtgatcc acctgccttg gcctcccaaa

11041
gtgctgggat tacagacctg agccaccgca cccggcccat actgctattt cttaacagca

11101
gagaaattat gtgtcagatt ctgtaagtgt aatggtatat aaaggataaa atgatgttga

11161
aaaacaaaat tttttgttta aatgcttatg tttctaatat tttatttcag aaaggaattt

11221
atttcaaaac tgataatggt tggatccagc ttttcacaca aacttttttt tcctagtgag

11281
gatgcacatt tatcctgtaa acaaatggaa gacattattt ttttaattgc ttgcttagaa

11341
atgaaataat tcttttctaa tgatctttta aagcatgaga cctcatacat catttaaaac

11401
aatttatact gtattttaca catgacaaag ttctaaggta acagcccttt tctaagacta

11461
aagttacagt cctccctttg tatctgaggg ggattggttg caggaccccc ctgtgaatac

11521
ccaaatcctt ggatgtccaa gtcccttatg agatgtagta tttgcatata acctatacac

11581
atcttcccct gtactttatc tctagattac gtacaatacc taatagaatg taaatgcttt

11641
gaaattagtt gttcagctgt attttaaatt ttgtattttt tttccttttt ttttgagaca

11701
gagtcttgct ctgttgccca ggctggagta cagtacagtg atcacagctc actgcacctt

11761
taacctccca ggctcaagct gtcctgcctc ggcctcccca agtgttggga ttacaggtgt

11821
gagccatcat acctggtcac tgttttttat tggttttaaa tttttgattt aaaattttta

11881
atctaggttg gttgaatctg gactggaacc caaggatatg tttgttgagc atactgtatt

11941
tactttggaa tacaactaga atgcttaact tgtatgttaa aaatacttta tttggccagg

12001
cgcggtggct cacgcctgta atcccagcac tttgagaggc caaggcgggt gaatcatttg

12061
aggtcaggag tttaagacga gcctggccaa catggcaaaa ccctgactct acaaaaaaaa

12121
ggtaaaaata agccaggtgt gatggcgtgt gcctgtagtc ttggctattc aggaggctga

12181
gacacaagaa tcgcttgaac cggggaggca cgttacgccc tcagttgttg acttgagttt

12241
ttccgtagtt tgtaggggga gggtaataga gtattaggta gcttttggaa tacataggag

12301
tgtaactgga aaaagattcc aagcaagtct aatgaattag ataatttacc taattagtaa

12361
attatgtaat cagtatgctt tataataata ttgtgagtta gatcctgttt ctgatatgta

12421
cataccatat tgtataggtg ctactaattt ggagagcata tacagtgagt ccatgccttt

12481
ttcctgccat cagcattata ccaaaattct gccatggttt ttaaactttg attctgagaa

12541
agtttctcac cctaataaca taactatatt tgtgtttgtc ttcatagtta aatatgcatt

12601
atgatatcag cttgcataca ttttttaaat gacttgaata tctgacttta aaaattattc

12661
tagaatttct gtgcttcaat attaatgcca gaagacttgg aattgtttat ttgtaggtaa

12721
ctgcctttaa ggaaacttga ccaaatatta actaagttat gtatttcctt ttggcaacag

12781

ttgtgacttc tcaccaggag atttggtttg ggccaagatg gagggttacc cctggtggcc

12841

ttgtctggtt tacaaccacc cctttgatgg aacattcatc cgcgagaaag ggaaatcagt

12901

ccgtgttcat gtacagtttt ttgatgacag cccaacaagg ggctgggtta gcaaaaggct

12961

tttaaagcca tatacag
gta agagtcacta ctgccatgtg tgtgtgtttg tgtgtgcgtg

13021
tgtgtgtgtg agagaaacag acagacaggc agactttttt ctatatgatg aaattaagtg

13081
tattttaccc cagtaaattg caaggggtgg cagttgtgaa agcttctggc atgggaaagg

13141
gatgtaacat ggtctttagc tggtttgttt tgtggaatgg aatttttatt tctgtccttt

13201
gagtgactta cagcaatatt atacccttaa taagggtaaa ctaaactgtc cccccatctt

13261
gaagggtcca agagaaagtt aatgtcatca ggatacatag cctatagata gcgacattct

13321
ctagggaaag atggagatgc gcactacctg gccttcaaac tactcactaa tgaacacatc

13381
tgagttgagt ttcacaccaa actcctggaa ccataacttt cttttcccag atctagtctt

13441
gtttatcaca gacatcaaca gcctggcatg tttagcctca cttgggctag gtgcacccca

13501
tcgtctcttg tacaagttct ctttctttct tttttttttt tttttttctg gagacagagt

13561
ctcactctgt tgcctaggct ggagtgcagt ggcgcaatct cggcccactg caacctccgt

13621
ctcctgggtt caagagtttc ctacctcagc ctcccgagta gcttgggatt ataggcacac

13681
gccacgttgc ctggctatat atatatattt tttttttgag acggagtttt gctcttttgg

13741
cccaggctgg agtgcaatgg cgcaatctca gctcactgca accgccacct cccgggttca

13801
ggtgattctc cttcctcagc ctctaaagta gctgggatta caggtgcaca ccaccaagcc

13861
cagctaattt tttatttcta gtagagatgg ggtttcacca tgttggccaa gctggtcttg

13921
aactgctgac ctccagtaat ccacccacct ccccctacca aagtgctggg attataggcg

13981
tgagccactg tgcccagccg cccagctaat ttttgtattt ttagtagaga cggggtttca

14041
ccatgttggc caggctggtc tccaacttct gacctcaggt gatctgccca tttcggcctc

14101
ccaagagtct ccagtctagt acgttgtcgt actcggtgtt gtaaaatcca aacaagggtc

14161
agtttcccag gtaactggga aattcccaga atcacactct ttcgtcatag tgctcatcct

14221
acaaaaaagg attgggggca ttttgtctaa aattaaatgt aaatggtgat ctgacataca

14281
ggtggaaaga gaattgggaa gttttgttct ctcttctacc aacttgccac ataatcttgg

14341
ccaagcaaag taacttgttt tttcttttaa tctttttaaa agaaatagag acacagtttt

14401
gccatgttgc ccaagctggt ctcaaactcc tgcctgagct caagcagtct gcccacttcg

14461
gcctcccaaa gtgctgagac tacaggcata agccaccatg cccctgggct cggccaactt

14521
tttcgttttc ttttcaagag atgggggtct cactctgtca cccagcctgg agtatagtgt

14581
tgggatcata gctcactgga gccttgaact cctgggctca agtgattccc ccctgtttta

14641
gcctcctcag taaccgggac tagaggtgtc tgccaccaca cctggctaat ttttatatag

14701
tttttttttt tttttttttt ttttaaagag atgacggtct tgctatgttg cccccagggt

14761
ggtcttgaat tcttggcctc cagtgatcct tctgcatcag gctcccaagt agttgggtga

14821
tctggctaaa gtaacttatt ttctgatact gtttacttat atttagaatg aatctcattg

14881
gggttgcact ggggccgggc atggtggctc acacctgtaa tcccagcgct ttggaaggcc

14941
aaggcaggtg gatcacctga ggtcaggagt tccagactag cctggcaaac atggtgaaat

15001
cccgtctcta ctaaaaatac aaaaattagc tgggcatggt ggcacatgcc tgtaatccca

15061
gctacttggg aggctgaggc aagagaatcg cttgaatcta ggaggcggag gttgcagtga

15121
gtcaagatca tgccaccgca ctccaacctg ggtgacagag cgagactgtc tcaaaaaaaa

15181
aaaaaaaaaa aaaaaaaaaa aggctgggca cggtggctcg cgcctgtaat cccaacactt

15241
tgggaggccc aggcgggtgg atcacgaggt caggcgttcg agaccagcct gaccaagatg

15301
gtgaaacact gtctctacta aaaatacaaa aataagctga aatcccagct actcgtgaag

15361
ctgaggcaga gaattgctta aacctggtag gcggaggttg cagtgagccg agatcgcgcc

15421
actgcactcc agcctgggga acggagtgag acttcatctc aaaaataaat aaataaataa

15481
ataaataaaa taaaataata aataaagtaa aaagatctct cattgaacca gatgatatat

15541
gaagtctctt ttaggaccaa tttcgagatt taaaaaattt ggcagaatta cttttttttt

15601
ttgcagcgga gtccagcttt atcacccagg ctggagtgga atggcacaat ctcagctcac

15661
tgcaacctct gcctcctggg ttcaagcgat tctcctgcct ctgcctccca agtagctgtg

15721
attataggcg cccaccacca ggcccagctg atttttgtat ttttcagtag agttgaggtt

15781
tcaccacgtt gtccaggctg gtctcaaact cctgacctta agtgatccgt ccaccttggc

15841
ctcccaaagt gctgggatta ggtgtgagcc actgggctgg cccagaatga tttttaaaaa

15901
gagatcagta aggccaggca gtggtggctc acgcctgtaa tcccagcact ttgggagact

15961
aaggtaggtg gatcacctga ggtcaggagt tgcagacaag cctggccaac atggtgaaac

16021
cctgtctcta ctaaaaatac aaaaattagc caggcatggt gacacatgcc tgtaatctca

16081
gctactcagg agggtgaggc agaattgctt gaacccggga gtcagtttct tttttctttt

16141
tttgagatgg agacccactt tgtcacccag gctggagtgc aatggtgcag tcttggctca

16201
ctgcaatctc tgtctccggg gttcaagtga tcctcctgcc tcagtctcct tagtagctga

16261
gactacaggt gtgcaccacc acacctggct aatttttgta tttttaggag agatggatgt

16321
caccatgttg gccaggctga tctttaaact cgtgacctga agtgatccac ccgccttggc

16381
ctcccaaaat gctgggatta caggtgtgag ccaccacgcc cagccctaaa gttgtatttt

16441
gatggaacga actgttttga gaaataaatt ttaacgcgtt gagtctgaac tgggctgccc

16501
tttcaaaatg tgaaggcccc ttaaagtagc acattggttg gttattcttt tatttattta

16561
gatatatctg atctagttgt ctttgggaca aactcatatt taatatcata gctgcatgta

16621
actgacagtg tagtctttgt cttcctgaag tgtttgtttg ttttttgaga tggagtcttg

16681
ctctgtcgcc caggctgaag tgcagtggtg cgatcttggc tcactgcaac ctctgcctcc

16741
cgggttcaag tgattctcct tcctcagcct cccgagtagc taggactaca ggcatgtgcc

16801
accacaccca gctaattttt gtatttttag tagagatggg gtttcaccat attggtcagg

16861
ttggtcttga actcctgacc tcgtgatctg cctgcttctg cctcccagag tgctgggatt

16921
acaggtgcga gccattgtgc ccagctagta agtttttaag aaagattctc aaacctcttt

16981
taaatcgtct gcctcacttg aagaggtatg ccctacctgt ttagggctgt agacccaggt

17041
cattagaaga cagactaagt agtcctgggt gaacccatag ggcaccttca aggaggtaaa

17101
attggtgatt ttagtttcac cagtagtttt tccctgaata tttattcctt ttgtgcttta

17161
ttgatctatc tatatcaata aaaagtaatg gggcataaca aattatactt gtcattcttg

17221
ttcattaggg caaatgttgt aggttgagtc aagtgtccag ccaacaagtt attttatgtg

17281
tgtgtgtgtg tgtgtgtgtg tgtatacata tatacatttt tttttttttt tttcatcgag

17341
acagggtctt gcactgtcgc ccaggctgga gtgaagtggt gcaatctcgg ctcactgcaa

17401
cctctgcctc ccaggtttaa gtgatcttcc cacctcagcc tcccaagtag ctgggactac

17461
aggcgcacac caccaccctt ggctaatttt tgtatttttt ttttggtaga gatggggttt

17521
caccacattg cccaggctgg tcttgaattc ctgacctcaa gtagtccgcc cacctaagcc

17581
tcccaaaatg ctgggattac aggcgtgagc caccacacct ggcatatata tattttaaga

17641
tagagatggg gtttgctatg ttgcccaggc tggtcttgaa ctgctgggat tacaggcgtg

17701
agcctctgca cccggccctt attgtttata aatacatttc tttctaggtt caaaatcaaa

17761

ggaagcccag aagggaggtc atttttacag tgcaaagcct gaaatactga gagcaatgca

17821

acgtgcagat gaagccttaa ataaagacaa gattaagagg cttgaattgg cagtttgtga

17881

tgagccctca gagccagaag aggaagaaga gatgga
ggtg ggacacggca agcattcagt

17941
tgttatttat gttagggtga tgggggaaga aagggggagg gtgtattaac aagatacctt

18001
gttttatata tgtgtgtgta tatgtattat tttattatac atacatgcat acttctgtag

18061
ttccctggac tgtaggataa gttaggttac ttagaatctc aacagctagc atcgttttta

18121
cttaggtttt caagcctact ggcagggtaa gcaagaggta gtaccatttt ggtaagaagt

18181
agagagctag ggacagtaaa gatggagtaa tatatatatg agggtatagt caggccctag

18241
aaattaatta tccagtttta tgctttttat aaaaaaactg agatggggtc ttgctatgtt

18301
gcccaggctg gtctcaaact cctgagttca agggatctgc ccacctgggc ctcccaaagt

18361
gttgggatta caggcatgag ccacagcacc cagccccagc tttatgcttt taattctaaa

18421
actttttttg ttgtattttg cattcataag aatagatgtt aaataaacct tgaaatacaa

18481
ccttggctca aacgttaatg gtcatggata aagtgaatta aaacttgtta ggggccaggt

18541
gtggtggtta atgcctataa tcccagcact ttaggaagct gaggcagttg gatgtcctga

18601
ggacaggagt tcaagaccag cctggccaac acagtgaaac cctgtttcta taaaaaatac

18661
aaaaattagc tgggcgtggt ggcacacacc tgtagtccca accacttggg aggctgaggc

18721
atgagaattg cttgaacttg ggaggcagag ggacttggga ggcagagggt gtagtgagcc

18781
aagatcgcac cactgcattc cagccagggt gacagagcaa gaagactgtc aacaacaaca

18841
aaaaatgtta tagaagtgaa aaaaattgat taatttagaa caagcttgtc cagtctgtgg

18901
cccaggatgg cgtttaaatc agcccaacac aaatttgtaa actttcttaa aacattttgt

18961
gatttgttgt tgttgtttag ctcatcagct atcattagca ttagtgtatt ttatgtgtgg

19021
cctaagacaa ttcttccagt gtggcccagg gaagctgaaa gatcattatc ctctgatcta

19081
tcatattaat gagctgcatc ctaaaagaca ttcatctata actaagctca gtttcatgtt

19141
ttgttccttt ttcaatagat aagataggga atgagcaagt taataaagtg ggtattttaa

19201
ttttaaggtt gaaactaagg atcataacat tatcagaggt ctagaactgg atggcagcta

19261
cagagatcat ttagcctaat actggtttaa caaataatcc gggagatccg tgatatgtga

19321
atgtgctagg cctgagatga gacagccaat tgtggaagag caaacactag aaccagtata

19381
agttgcttac tgctttctta tgctattaat gagcatatcg cctcctgata tttatgatat

19441
atggtcatgc caacagcttt gtcataaata gaactcccat ggcagcaatc acttaatctt

19501
gtagttagag gtggggtctc accatgttgc cgagctggcc ttgaacttct gggctcagcg

19561
attttctcca caggcacctg ctactgtgct cggtgcagca ctttgtgttt ttgaacataa

19621
cctcaagatg ttattgtctt catagtaaaa caaaagatga ggcttagaac tggatcactt

19681
tgcctgtctc ttcttacctc ctcccagttc aaaatgcttg catctcttaa tagctagcat

19741
tcccttggat tttgcacatg agctcaaact caagcctcag cacaatcttt tttatagttt

19801
tagtctttta gccagagtcg acttaccccc catacccact ctgcttcctt cataatgctg

19861
ctttccctgg gcagagaatc cttgcccttc ttgtattatg tcactttgtg gggttggtgt

19921
ctgctacact tacagcaagt ccagagattt tttttccacc acgtttgcag gagaactatt

19981
ggcatggaaa atgacaattg ttttaatgtc aagtgaaact gaagttgatg ttcattgaga

20041
ggtttctaat ttctagaggt gggttctttt tttggcatat gaagttgcag catattaaga

20101
gaatttacag tagtacagat ggggttatcc catccacaac ttatgatggg gttacataaa

20161
ctaaaaacgt gtttaataca cctaccccac cgaatattgt agcttgggcg tagcctaacc

20221
tatctcagac gtgctcagaa cacttaaatg ttagcctaaa gttgggcaag atcatctaac

20281
acaaagccta ttttataata aggaattgcc tatctcatgt aattcatcga atactgtact

20341
aaaaatgaaa aacagtggct gcacgggtac cattataaag tcaaaaaatc ataagttgaa

20401
ctgtcttaca ttatggtttt ccaaattttg atttgttttt aaatactctt tccttgcctg

20461
gcaggtaggc acaacttacg taacagataa gagtgaagaa gataatgaaa ttgagagtga

20521

agaggaagta cagcctaaga cacaaggatc taggcgaagt agccgccaaa taaaaaaacg

20581

aagggtcata tcagattctg agagtgacat tggtggctct gatgtggaat ttaagccaga

20641

cactaaggag gaaggaagca gtgatgaaat aagcagtgga gtgggggata gtgagagtga

20701

aggcctgaac agccctgtca aagttgctcg aaagcggaag agaatggtga ctggaaatgg

20761

ctctcttaaa aggaaaagct ctaggaagga aacgccctca gccaccaaac aagcaactag

20821

catttcatca gaaaccaaga atactttgag agctttctct gcccctcaaa attctgaatc

20881

ccaagcccac gttagtggag gtggtgatga cagtagtcgc cctactgttt ggtatcatga

20941

aactttagaa tggcttaagg aggaaaagag aagagatgag cacaggagga ggcctgatca

21001

ccccgatttt gatgcatcta cactctatgt gcctgaggat ttcctcaatt cttgtactcc

21061

tgggatgagg aagtggtggc agattaagtc tcagaacttt gatcttgtca tctgttacaa

21121

ggtggggaaa ttttatgagc tgtaccacat ggatgctctt attggagtca gtgaactggg

21181

gctggtattc atgaaaggca actgggccca ttctggcttt cctgaaattg catttggccg

21241

ttattcagat tccctggtgc agaagggcta taaagtagca cgagtggaac agactgagac

21301

tccagaaatg atggaggcac gatgtagaaa gatggcacat atatccaagt atgatagagt

21361

ggtgaggagg gagatctgta ggatcattac caagggtaca cagacttaca gtgtgctgga

21421

aggtgatccc tctgagaact acagtaagta tcttcttagc ctcaaagaaa aagaggaaga

21481

ttcttctggc catactcgtg catatggtgt gtgctttgtt gatacttcac tgggaaagtt

21541

tttcataggt cagttttcag atgatcgcca ttgttcgaga tttaggactc tagtggcaca

21601

ctatccccca gtacaagttt tatttgaaaa aggaaatctc tcaaaggaaa ctaaaacaat

21661

tctaaagagt tcattgtcct gttctcttca ggaaggtctg atacccggct cccagttttg

21721

ggatgcatcc aaaactttga gaactctcct tgaggaagaa tattttaggg aaaagctaag

21781

tgatggcatt ggggtgatgt taccccaggt gcttaaaggt atgacttcag agtctgattc

21841

cattgggttg acaccaggag agaaaagtga attggccctc tctgctctag gtggttgtgt

21901

cttctacctc aaaaaatgcc ttattgatca ggagctttta tcaatggcta attttgaaga

21961

atatattccc ttggattctg acacagtcag cactacaaga tctggtgcta tcttcaccaa

22021

agcctatcaa cgaatggtgc tagatgcagt gacattaaac aacttggaga tttttctgaa

22081

tggaacaaat ggttctactg aaggaaccct actagagagg gttgatactt gccatactcc

22141

ttttggtaag cggctcctaa agcaatggct ttgtgcccca ctctgtaacc attatgctat

22201

taatgatcgt ctagatgcca tagaagacct catggttgtg cctgacaaaa tctccgaagt

22261

tgtagagctt ctaaagaagc ttccagatct tgagaggcta ctcagtaaaa ttcataatgt

22321

tgggtctccc ctgaagagtc agaaccaccc agacagcagg gctataatgt atgaagaaac

22381

tacatacagc aagaagaaga ttattgattt tctttctgct ctggaaggat tcaaagtaat

22441

gtgtaaaatt atagggatca tggaagaagt tgctgatggt tttaagtcta aaatccttaa

22501

gcaggtcatc tctctgcaga caaaaaatcc tgaaggtcgt tttcctgatt tgactgtaga

22561

attgaaccga tgggatacag cctttgacca tgaaaaggct cgaaagactg gacttattac

22621

tcccaaagca ggctttgact ctgattatga ccaagctctt gctgacataa gagaaaatga

22681

acagagcctc ctggaatacc tagagaaaca gcgcaacaga attggctgta ggaccatagt

22741

ctattggggg attggtagga accgttacca gctggaaatt cctgagaatt tcaccactcg

22801

caatttgcca gaagaatacg agttgaaatc taccaagaag ggctgtaaac gatactggac

22861

caaaactatt gaaaagaagt tggctaatct cataaatgct gaagaacgga gggatgtatc

22921

attgaaggac tgcatgcggc gactgttcta taactttgat aaaaattaca aggactggca

22981

gtctgctgta gagtgtatcg cagtgttgg
g taagactttg aacaagcttg ttctcaggct

23041
ttgataagta gtgctgtttg ccagctgtat attatcccta aaaataagta ataaggtata

23101
tatggtacat attttgacat gcatatacat atttgcatcc tgactaggct gcccacagca

23161
atttaagtta cttgaaactc gcttttatct tagtagccct ttggcctttc ttcagttttt

23221
tttttttttt tttttttttt gagacatggt cttgctctgt tgcccaggct agaatatggt

23281
gacacaacca tggctactgc agcctcgacc tcccaggctt aagtgatctt tccacctcag

23341
cctcccaagt agctgagatt acagatatgc accaccatgc atggctaata tttaaatgtt

23401
tgtagagaga tggggtctca ctgtgttgcc aggactggtc ttgaactcct gggctcaagt

23461
gatcctcctg cctcggcttc ccaaagtgct gaggttacag gcatgaccca ttgcgcctgg

23521
ccctttcttc agtctttaat aatcgaacaa aaggtttttg tttttagaca gtgtcttgct

23581
ctgttaccca ggacagacct ctcgtgtcag cctcttaggt agctaggatt tacaggtaag

23641
caccggcgtg ccctgcttta tttttttggt gggggaaggg ggaagggagt tgaagcttcc

23701
ctatgttgcc caggctggtc ttgaactcct ggcctcaagt gatcctccag tctcccaaaa

23761
gtgctgggat tacaggcatg agccaccgct cccggcccaa aagattttta aatgtgttat

23821
acttcatgag acaggcttta ttttagatcg aattttattt atcaataaaa agttgagctt

23881
tttattattt ggtgaatact gtttcaaggt gctttgttac actatctgtt gatccaacat

23941
ttaaaaattg ttttattaca acctttgcat ttcagtgaat ccatctgcat acaattttaa

24001
aagaatcatt ccttttttct gtagccaaat tgtcaaagat tctttcctac aattgatttt

24061
tcaaagccct gagttaggaa tttacaattt ggcaaccatc tcaacttcat aagcaatttt

24121
gttctttaaa tgtcacggcc aacattacct ggaaccattg ctgttttata gtttaggttt

24181
atgttgtata ttttttttaa ttttttagag acggggtctt gctgttttca gactggagta

24241
caatgggatg actagctcac tgcagcctca aactgctggg ttcaagtgat tctccttcct

24301
cagcctcctg agtagctggg actacaggtg gtcaccatca cacctggcta atttttgtat

24361
ttttggtaga gacagggttt ttcccgtgtt ggccaggctg ttcttgaatt cctgacctca

24421
aagcgatctg cccgccttga tctccgaaag agctgggatt acacgcatga gccactgcgc

24481
ccagccctgt tttttttttt tttttttttt taaataatgg tagtttactt gaatttgtaa

24541
cacagtaaca caaaactatt ttgatctgaa cgcaagtatc taatggaaca gaataatata

24601
cttcctttta gtgtgctgca tttggttact gggtaattta aaattcttcc tcagcacagg

24661
tgttcaaaaa ccagtcttca gagattgttt tcatatcagt gtgccaactt tggcacattc

24721
tgctaagtaa gaggcttaag tgtagcatgt ttctgctgtt ttgtgtttgt tttgttttgt

24781
tttttgagac agagtctctc tgtcgcccag gctggagtgc attggtgcga tcttggctca

24841
ttgcaacctc tgcctcccag gttcaagtga ttctcctgcc tcagcctcct gcgtagctgg

24901
gattacaggc atatgccacg tgtattaggc actgctaatt tctgtatttt tagtagagac

24961
gaggtttcac catgttggtc aggctggtcc tgaactgctg acctcgtgaa ctctgcccgc

25021
ctaggcctcc tgaagtgctg ggattacagg cgtgagccac cgtgcctggc ctctgctcta

25081
tcttttagct ttcccttggc acttctatgg tccagatgtt agagggtaag tattttgatg

25141
ggggagatcg ttggactgta attgaaagtt atgtcttata atgaaatgtg ttatataaag

25201
aagacctata aaacacttag gctgataaaa cccccaaacg atgaagcctc acttttaccc

25261
tctcttttaa cagatgtttt actgtgcctg gctaactata gtcgaggggg tgatggtcct

25321

atgtgtcgcc cagtaattct gttgccggaa gatacccccc ccttcttaga gcttaaagga

25381

tcacgccatc cttgcattac gaagactttt tttggagatg attttattcc taatgacatt

25441

ctaataggct gtgaggaaga ggagcaggaa aatggcaaag cctattgtgt gcttgttact

25501

ggaccaaata tggggggcaa gtctacgctt atgagacag
g taactgattc ttaaagtttt

25561
gttatcagaa agtcatttgt gacattagga ataacatact taggtgatca ttttccaaac

25621
acagttacat aaaagtcagc cagtgactta ataggaagca aagggaaatt actccctgtg

25681
ttataaaatt gagaattata tttagctgaa acatcgatgc ttaatgttaa ggggaatata

25741
tgttaaaaag gggaaggagg tcagtcattc aggtcatgag gccctttgac ttgaattcat

25801
ttcctcagaa ggtaggtata ttcatagtga acaaaaatac aaaggctgta tgaaaagatg

25861
aaaatgttac aggtttatcc ttaaattaga ctcatttgca gaaatgcaaa tgaggtaaga

25921
aagcaaatat agttcatgac ctctagcaac tgttgaaaac tgctctttag ggatgacatg

25981
ctggcccttt tttttttgtt gttgccaagg ctgaagtgca gtggcaccat cacagctcac

26041
tgcagcctcg aactcccagg ttcaaccctt cctcctgcct cagcctcccc agtagctggg

26101
actacagatg tacaccatca tgcctagctc atttttaaaa aaatttttta tggcattgta

26161
tttatcttct ctttataacc aggggttgac cagccacaga acttgtaaag ttttttatat

26221
ttttaaaagg ttgtaagaaa tagtagtagt tggctggtcc ccgctgctct cctgcattat

26281
agtatacttc tgttcaccta gtttgctaga gagaggcagt atagtgtgta tagtgatttc

26341
caaacttttt ctttaaatca gaatcacctg aaagaatgtg acaacgtgta aaaaaaaaaa

26401
aaaaggtgca gagattccat cctgacatgg attcacttga ttggaattga ttctaggcat

26461
ctcagtagtt ttaaagagct cctaggtgat tctattctgg ccagcgttga gaatcactag

26521
ggtagtgggt tggtaagcag gctctgatgt tttaaaggcc aggtgaggcc ctatgcctct

26581
tgtctctctt agcctcaact ttctccatgt tagcaaatgg atttcagaac agaaccaacg

26641
tacatgtgat tgtgaaagtt gttttagagt gcctagctct tacgtaaggg ttcataagaa

26701
agacaaaagt ttatgaaact gttactacca gtcataaaag accttttcct ccctcattca

26761
caggctggct tattagctgt aatggcccag atgggttgtt acgtccctgc tgaagtgtgc

26821

aggctcacac caattgatag agtgtttact agacttggtg cctcagacag aataatgtca

26881

g
gtgagtttt ttgtttccca cttaagttct cattcagtca tttagatgtg ataaaagata

26941
tttgcttctt gtatatgagc cttttaaatc taatatttga tttttctggt gttactttaa

27001
aaacatcact ttttaagaac tgcatagtct ctctctcttt tttttttttt tgagatggag

27061
tttccctctt gttgcccaag ctggagtgca atggcacgat cttggctcac tgcaacctct

27121
gcttccaggt tcaagtgatt ctcctgcctc agcctctcga gtagctggga ttacaggcgc

27181
atgccatcac gcccagctaa ttttttgtat ttttagtaga agcggggttt caccatgtta

27241
ggctggtctc ttaactcctg acctcaggtg atctgcttgc ctcggcctcc caaagtgctg

27301
ggattacagg cgtgagccac cgtgcccggc caataattgc atagtctctt aatgagattt

27361
aatcttttat accaatatgt gtagctcatg atagctatat aacctagaag atgaatttat

27421
gtaatatgat ttgcaaaatg agtattcatt tgtgattttt ttttttttaa ggtgaaagta

27481

cattttttgt tgaattaagt gaaactgcca gcatactcat gcatgcaaca gcacattctc

27541

tggtgcttgt ggatgaatta g
gtaagacat taaacttctc atttgaagac tatctatctt

27601
aaaaacattt gtacaaataa ctatttttat agaagattat ctgaagtaca tttaaacaat

27661
atgaatgttt ttagagcacg cactcaccat tgtggcacag accgatagtt ggagataaaa

27721
ggtgatattg tgaaaggttt ttgattaccc attaattatt aggccttaca ctgtttagtt

27781
gtaataaaac atttgttata ctacggggat gagaacacta ataggaggac tcaggaagtt

27841
tatgaccttg agcgatactg tattttcttt aaaagaaacc tcactcccca tgggctgcta

27901
agcagactcg tgtagctaaa caaggcctat ttatagaatg cttttagacg tggatgtact

27961
aaccgatgtt gcttttctgt cctagcattt ttgttttaat tccttttttg ttttaattcc

28021
tttgagttac ttccttatgc atattttact ttaacaggaa gaggtactgc aacatttgat

28081

gggacggcaa tagcaaatgc agttgttaaa gaacttgctg agactataaa atgtcgtaca

28141

ttattttcaa ctcactacca ttcattagta gaagattatt ctcaaaatgt tgctgtgcgc

28201

ctaggacata tg
gtatgtgc aaattgtttt tttccacaaa ttcggttttt tgagagggca

28261
cttctcttgc tagcacatgt atcgctaata tttttctttc ttaaggcatg catggtagaa

28321

aatgaatgtg aagaccccag ccaggagact attacgttcc tctataaatt cattaaggga

28381

gcttgtccta aaagctatgg ctttaatgca gcaaggcttg ctaatctccc agaggaagtt

28441

attcaaaagg gacatagaaa agcaagagaa tttgagaaga tgaatcagtc actacgatta

28501

tttcg
gtaac taactaacta taatggaatt ataactaact gaccttaagt ttcaaagaaa

28561
cagtaaaagg ggaagggatg atgcactatg aaaaaacaaa aaaacttttt tttttttttt

28621
tttaatttta agggaagttt gcctggctag tgaaaggtca actgtagatg ctgaagctgt

28681

ccataaattg ctgactttga ttaaggaatt atagactgac tacattggaa gctttgagtt

28741

gacttctgac aaaggtggta aattcagaca acattatgat ctaataaact ttatttttta

28801

aaaatga

The genomic sequence for human MLH1 (found on human chromosome 3) has GenBank Accession No. NG_007109.2. Sequence information related to human MLH1 (isoform 1) is accessible in public databases by GenBank Accession numbers NP_000240.1 (protein) and NM_000249.3 (nucleic acid). Sequence information related to human MLH1 (isoform 2) is accessible in public databases by GenBank Accession numbers NP_001161089.1 (protein) and NM_001167617.1 (nucleic acid). Sequence information related to human MLH1 (isoform 3) is accessible in public databases by GenBank Accession numbers NP_001161090.1 (protein) and NM_001167618.1 (nucleic acid). Sequence information related to human MLH1 (isoform 4) is accessible in public databases by GenBank Accession numbers NP_001245200.1 (protein) and NM_001258271.1 (nucleic acid). The genomic sequence for human MLH1 (GenBank Accession No. NG_007109.2; 79,540 bp in length), is found at nucleotide no. 5,001 and terminates at nucleotide no. 62,497, wherein exon 1 is located between nucleotides 5,001 and 5,314; exon 2 is located between nucleotides 8,270 and 8,360; exon 3 is located between nucleotides 12,606 and 12,704; exon 4 is located between nucleotides 16,052 and 16,125; exon 5 is located between nucleotides 18,642 and 18,714; exon 6 is located between nucleotides 20,465 and 20,556; exon 7 is located between nucleotides 23,471 and 23,513; exon 8 is located between nucleotides 23,662 and 23,750; exon 9 is located between nucleotides 26,083 and 26,195; exon 10 is located between nucleotides 29,157 and 29,250; exon 11 is located between nucleotides 31,961 and 32,114; exon 12 is located between nucleotides 37,288 and 37,658; exon 13 is located between nucleotides 40,435 and 40,583; exon 14 is located between nucleotides 51,837 and 51,945; exon 15 is located between nucleotides 53,919 and 53,982; exon 16 is located between nucleotides 59,170 and 59,334; exon 17 is located between nucleotides 60,168 and 60,260; exon 18 is located between nucleotides 60,555 and 60,668; and exon 19 is located between nucleotides 62,137 and 62,497. It is understood that intron sequence precedes and follows the denoted nucleotide regions comprising the exon sequences listed herein. Oligonucleotide compounds (e.g., exon skipping or intron retaining SSOs) can be directed to the nucleic acid sequence corresponding to the region of interest for each of the exons1-19 described herein, and intron-exon junctions, or exon-intron junctions listed with GenBank Accession No. NG_007109.2.

The genomic sequence for human PMS1 (found on human chromosome 2) has GenBank Accession No. NG_008648.1. Sequence information related to human PMS1 (isoform a) is accessible in public databases by GenBank Accession numbers NP_000525.1 (protein) and NM_000534.4 (nucleic acid). Sequence information related to human PMS1 (isoform b) is accessible in public databases by GenBank Accession numbers NP_001121615.1 (protein) and NM_001128143.1 (nucleic acid). Sequence information related to human PMS1 (isoform c) is accessible in public databases by GenBank Accession numbers NP_001121616.1 (protein) and NM_001128144.1 (nucleic acid). Sequence information related to human PMS1 (isoform d) is accessible in public databases by GenBank Accession numbers NP_001276337.1 (protein) and NM_001289408.1 (nucleic acid). The genomic sequence for human PMS1 (GenBank Accession No. NG_008648.1; 100,545 bp in length), is found at nucleotide no. 5,001 and terminates at nucleotide no. 98,545, wherein exon 1 is located between nucleotides 5,001 and 5,509; exon 2 is located between nucleotides 12,706 and 12,857; exon 3 is located between nucleotides 16,685 and 16,867; exon 4 is located between nucleotides 26,568 and 26,670; exon 5 is located between nucleotides 38,933 and 39,096; exon 6 is located between nucleotides 64,880 and 64,996; exon 7 is located between nucleotides 73,571 and 73,693; exon 8 is located between nucleotides 74,855 and 74,998; exon 9 is located between nucleotides 75,155 and 76,044; exon 10 is located between nucleotides 84,659 and 85,144; exon 11 is located between nucleotides 88,715 and 88,845; exon 12 is located between nucleotides 94,412 and 94,572; and exon 13 is located between nucleotides 98,188 and 98,545. It is understood that intron sequence precedes and follows the denoted nucleotide regions comprising the exon sequences listed herein. Oligonucleotide compounds (e.g., exon skipping or intron retaining SSOs) can be directed to the nucleic acid sequence corresponding to the region of interest for each of the exons 1-13 described herein, and intron-exon junctions, or exon-intron junctions listed with GenBank Accession No. NG_008648.1.

The genomic sequence for human PMS2 (found on human chromosome 7) has GenBank Accession No. NG_008466.1. Sequence information related to human PMS2 (isoform a) is accessible in public databases by GenBank Accession numbers NP_000526.1 (protein) and NM_000535.5 (nucleic acid). The genomic sequence for human PMS2 (GenBank Accession No. NG_008466.1; 42,868 bp in length), is found at nucleotide no. 5,001 and terminates at nucleotide no. 40,868, wherein exon 1 is located between nucleotides 5,001 and 5,110; exon 2 is located between nucleotides 8,076 and 8,215; exon 3 is located between nucleotides 10,049 and 10,135; exon 4 is located between nucleotides 10,315 and 10,417; exon 5 is located between nucleotides 11,471 and 11,654; exon 6 is located between nucleotides 14,832 and 14,999; exon 7 is located between nucleotides 16,684 and 16,781; exon 8 is located between nucleotides 18,474 and 18,573; exon 9 is located between nucleotides 22,050 and 22,134; exon 10 is located between nucleotides 24,152 and 24,307; exon 11 is located between nucleotides 26,487 and 27,348; exon 12 is located between nucleotides 31,116 and 31,283; exon 13 is located between nucleotides 35,411 and 35,511; exon 14 is located between nucleotides 36,350 and 36,519; and exon 15 is located between nucleotides 40,565 and 40,868. It is understood that intron sequence precedes and follows the denoted nucleotide regions comprising the exon sequences listed herein. Oligonucleotide compounds (e.g., exon skipping or intron retaining SSOs) can be directed to the nucleic acid sequence corresponding to the region of interest for each of the exons 1-15 described herein, and intron-exon junctions, or exon-intron junctions listed with GenBank Accession No. NG_008466.1.

Sequence information related to human MLH3 (isoform 1) is accessible in public databases by GenBank Accession numbers NP_001035197.1 (protein) and NM_001040108.1 (nucleic acid). Sequence information related to human MLH3 (isoform 2) is accessible in public databases by GenBank Accession numbers NP_055196.2 (protein) and NM_014381.2 (nucleic acid). The genomic sequence for human MLH3 (GenBank Accession No. NG_008649.1; 44,769 bp in length), is found at nucleotide no. 5,001 and terminates at nucleotide no. 42,769, wherein exon 1 is located between nucleotides 5,001 and 5,153; exon 2 is located between nucleotides 6,815 and 10,157; exon 3 is located between nucleotides 14,056 and 14,154; exon 4 is located between nucleotides 14,833 and 14,918; exon 5 is located between nucleotides 16,518 and 16,622; exon 6 is located between nucleotides 18,121 and 18,193; exon 7 is located between nucleotides 23,043 and 23,114; exon 8 is located between nucleotides 24,354 and 24,465; exon 9 is located between nucleotides 25,831 and 25,990; exon 10 is located between nucleotides 33,515 and 33,538; exon 11 is located between nucleotides 33,641 and 33,719; exon 12 is located between nucleotides 37,553 and 37,704; and exon 13 is located between nucleotides 39,332 and 42,769. It is understood that intron sequence precedes and follows the denoted nucleotide regions comprising the exon sequences listed herein. Oligonucleotide compounds (e.g., exon skipping or intron retaining SSOs) can be directed to the nucleic acid sequence corresponding to the region of interest for each of the exons 1-13 described herein, and intron-exon junctions, or exon-intron junctions listed with GenBank Accession No. NG_008649.1 and described in SEQ ID NO: 1. The human MLH3 gene sequence (GenBank Accession No. NG_008649.1; found on chromosome 14) is depicted in SEQ ID NO: 1 below, where the bolded italicized nucleotide bases correspond to EXON regions.

SEQ ID NO: 1

1 gatcatttga gcctgggagg ttaaggctgc aataagctgt gactgtgcca ccatccttca

61 gaaaaaaaaa agaaaaagga aaagaggtat tgacaattca cattcatgtt tcaaagattc

121 cttccaggtt agaatttgaa ttttaagtac cacagtccca ggaatgagac acttattttt

181 catttttatt ttttagcttt agttttagta tgaggataat gctggcttaa taaaatgtgt

241 tgggaagtgt ttcctcttct tttttttaac ccttaattct tggtttggat cttcttctat

301 tttttggaag agtttgtgaa gggttggtaa aggatttttt tttaaacatt tggtagaatt

361 tacccatgaa gccatctggt cctgggcttt ttatctgtgg gaagtttttg attacaaatt

421 ccatctcttg gtataggtct attcagactt tctatttctt cttgattcag ttttggttgt

481 ttgtctttct aggtattagg tttgttttca tccaaaaaaa tttaatctaa ttttttggca

541 tataattgtt catagaattc ccttttatcc tttttatctt tgtaaggttg gtggtaatgt

601 ctccttttat ttctgatttt agtaatttga ggctttcatc cgtttttctt tatcagtcta

661 gctaaaggtt gtcaattttg ttgatcctgt caaagaagca acttttgttt tattgatttt

721 ctctattctt ctgctttcca ttaatttctg ctctaatctt tatttccttc tttctgctca

781 ttttgaattc agtttgctct tctttttcta gtatcctaag gtggaaatct tgattattga

841 tttgaagaga gtttcttttt agattgcaac tagaggtggt gattacagtc cttgaaatgt

901 tgtcagatga ttgtttaagc aaaaatatgg ttatgaggta gaattcactt tttggaattt

961 tcttagggga gaaaaaccct gttggtgcaa tagatattca aaactgaaca tgagtcaata

1021 acattatgtc atcattaaaa aaataagaac agagttgagc acacaaatga aaccagtagg

1081 aaacgcccat tctcaacact ggtcaaattt tggagtgttc tgttcaactc tcaagagttc

1141 agataaggcc aggtgcagtg gctcatgcct gtaatcccag cactttggga ggccgaggca

1201 ggcggatcat gaggtcagga gatcgagacc atcctggcta acacagtgaa accccatctc

1261 tactaaaaat acaaaaaaat tagccgggag tggttgcggg cgcctgtggt cccagctact

1321 ggggaggctg aggcaggaga atggcataaa cctgggaggc ggagcttgca gtgagctgag

1381 atcacgccac tgcactccag tctgggcgac agagcgagac tccgtctcaa aaaaaaaaaa

1441 aaaaaaaaaa aaagagttca gataaatcac attgcaaatt tttaaagatt tatttgactg

1501 tgacaacctc ctgtttaaaa ctgcacattc ttacagctat ttaccatata agataactct

1561 taagaactgg agatagtcag ctcccctggg ttaatttgaa gcagaagagg gcagttgtta

1621 tactgccctg tcagttggat gcggagtctt actcaaaatt cattctcagc attcttcttt

1681 tatggtatct tctttggcac ttagcagcgc atcaggtagg catcttctat ttttcttcat

1741 tccttaattt cctttgtatc cctcaaatgg ttatttattt ggctggagtc tgttttgttc

1801 attaagcaaa catgtctttg ctttgaacat gtctttgatt tgatggatac ttaaattcct

1861 catcaaacat tttgttgcta tgcataacgt tttctttggc caactccagc aatttcccac

1921 attttgacat gcaatcatgt taactcccat tttcttttgt aatccaacat cttctattta

1981 gataattact ttaacaatca atgacttaat attctaatca taaatttata caaaaataaa

2041 attacctcca aaacattgct acctttccta aacattcagt cttgccacag tttaataaaa

2101 ggaaagaaca ttaaaaagga taagacactg taatgattag atgcttttta taagcctaaa

2161 ggcattgtga ttatttagac agaagagaag aaagtgaagt gaaaacctga tagttatgta

2221 gtctcatggt ttgctgttga gaggctgaac accagctgct ttccttttct aggaagataa

2281 taaagtgggc tttggctaca acataaagat gttgggttag acagtttcac tacagtaaga

2341 acaacgggat gagttgccca ggaaattgtg aaatactttc taatgatctt taaagatata

2401 atgaacacta attcatctgg atttgtttac gtgtggtcct ggttaaaggc aaagggaagg

2461 atcagataac ttcatgtttt ttccatttaa catacccaat agattcttga ttaggggaag

2521 ggaaaatgag caagatacag tccagtattc taaaaacaat cagccttagg ggatcatttc

2581 aaaagcatct gttttggact taagtctttg atacttaacc aaattgacta cacagtgaaa

2641 aattctagtg cctgggtttt atagggtaga agaaagacat gcagtcaagt ggccaatact

2701 tcatgtgaag ataagcaatg agatccttct tgctgtcttt cttttgactg ttctgggcaa

2761 tatcaaatta gtttcagtgg cttgattcta ggccaagatt ctggcaacag attgtagtct

2821 taccttgttt tcttcaatct cactggatct ctctctcttt ttacccccct taggctgagg

2881 gtaaaaagct gggattggta ggctgggtcc agaacactga ccggggcaca gtgcaaggac

2941 aattgcaagg tcccatctcc aaggtgcgtc atatgcagga atggcttgaa acaagaggaa

3001 gtcctaaatc acacatcgac aaagcaaact tcaacaatga aaaagtcatc ttgaagttgg

3061 attactcaga cttccaaatt gtaaaataat ggcctgaatt taagttttct aagataaact

3121 cagtggtttg gtttttatta ttaatagaga tagaactatt gtgtgttaat attagcatta

3181 gtcaataagt tattttaatg tcagattttt gaatgttatt atatattacc tgtatgatgg

3241 aaggattacc actgtacaca aatctaatca ataaaaacgt tagaaccttc tgcttagagt

3301 acttttaaaa aatcttcagt gaacttcctt ttgggcgaaa tgagaggtct ttattcagta

3361 aacatttgta ggaagaggat tttgaggtaa tttaaagagg tctgaaagaa aaaaagtctg

3421 agtcatttct ttaaatggtt tctatgaaat gttcttcaag aaattccatg cctaataaga

3481 acaaatacca caagttcaat ttgttagctc tgttcacctt atgtttggat gaattacttc

3541 tgctgttgtt tcttttctct gggtaaggaa ttcacataaa gttatgttat gggctgaact

3601 gtgtcccacc aaattcatat gttaaagtct cagtccccag tacctcagaa agtgactgta

3661 tttggacata gggcctttaa agaggtgatt aagattaaat gaggctgtga gggtgggccc

3721 taatccaatc tgactggtgt tcttataaga gaacatattg gctatagaca cgtgtgcaaa

3781 aatcaaagac cctgtgaaaa tggccatcta caagccaagg agagaggcct caggagaaat

3841 cgttgctgcc aacaccttga tctcagactt ccagtctcta gaactgagag gaaatagact

3901 tctgctgctt aagctactca gtctgaggta ttttgttatg gcagccctag gtatagtaat

3961 aatcataaac agttatcagg attttgctta atcagcccta gaagactggt tggttgggtt

4021 tggttagtca ctaaactagc atatatcaaa tgcttaccag gtctgacaaa ttcgttataa

4081 attccacttt aaattctcaa tgaaaatgag atagaaagca aaaactaagg actggttaac

4141 aattccaaaa cactttattc tcacagcatt ctcagagttc tgctctcctt tagttatttt

4201 tataactaaa agctgtggtg gcactgggga gattcaagtc agtgaagaga gtcttggtgt

4261 tgtcatctgt aaattaagag ttgagcaaag gccgggtgcg gtggctcacg cctgtaatcc

4321 caacactttg ggaggccgag gcgggcagat cacctgaggt ccggagttcg agaccagcct

4381 gaccaacatg gagaaacccc cgtctctact aaaaatacaa aattagccga gtgtggtggc

4441 gcatgcctgt aatcctagct actcgggagg ctgaggcagg agaatcactt gaaccctgga

4501 ggcggaggtt gcagtaagct gagatcgcac cactgaattc cagcctgggc aacaagagct

4561 aaactccgtc tcaaaaaaaa aaaaaaaaaa aaaaagagtt gagttagaca gtttctcagc

4621 cttttccagc tccaaatgcc ataattctaa gatggcaggc tctggaatta ttcattcatt

4681 cggtgcctac cataggccag gcactgttct tggtaactgg gatacagcag taaacaaaat

4741 atataaactc cttaccttca catagcttac attctaggga gagaagacaa taagtaaaca

4801 cataaaatat atattgaatt aaacggtagt taagagcaaa ggtgaaaatg agaaaaaaaa

4861 ggaaagggaa aacacgggaa aaaaaaataa aaacaaaagt aaaagctacg acatagtctt

4921 taacaccatg ccaaaaggga ataaggattg agactgtagg taccggttca ctgaaccctg

4981 ggatgcggat ccttggctgg aacaactggt gcgcatgcgc actggtgtct cgcggcctgg

5041 cgcgccccct ccgaagcgca tgctcgtggg cacgcacgag cctcaagatc caaggtgcgc

5101 gcgtcggcgt ccgaggcggt tggtgtcgga gaatttgtta agcgggactc cag
gtgactc

5161 tgggggaagc acgcgacgaa aagatgatgc cggggtctct tctaacacca gaagggccct

5221 gatgatggct gcgcgcagct ttcggagccg gatgcgcgag ggccccggag gcccggcggc

5281 ctggcggccg ggcgggccca gtttggggac aaggacgggg ctggccaggg aggggctggg

5341 cctggcggga aggcagcgct gccccggact cggcccgcgc ggccctccca ggcccccgtg

5401 ccctggatcc aggcccgtgc gtcccgtcag tcccaggcgc tgggaggcgt catcaggaaa

5461 tcattgggtt tacattaatc ggaatacttg ttaaacgttt acccgtcagt tgctgccggc

5521 tgcttagtgc attagcttag aaagcagcag aaattctgca gttaagagcc ctgatttgtc

5581 ccgagtttgg aaaaccggcc ccaccgcctg caggggccca cccacgtggc tctcactgat

5641 ggagaagaag gggagacctt taatggcact ggaatcttag ggtttggttt tttttgtttt

5701 gttttttgtt tttgtgtttt cacttgcagg aaaacttaaa atcaagttca ggcagcccca

5761 tgccatcatt attattacca aggtagttta tgcccatctg taaagaccaa aagaatatta

5821 ataatgacct tctggccggg cgcggtggtt cacacctgta atcccagcac tttgggaggc

5881 caaggcggga ggatcacttg agttcaggag ttcgagacca ggctggttaa catggtgaaa

5941 ccccatctct acataagaga caaaaattag ccaggcatgg tggcgagcgc ctgtaatccc

6001 agctactcgg gaggctaagg ctggaggatt gcttgagccc gggaggtgca ggctgcagtg

6061 agccgagatt gtgtcattgc actccagcct gggtgacaga gtgagactgt ttcaaaaaag

6121 aataataacg actttctaaa aacatcagag taatacatga acaggattta acaatcaaat

6181 ggcaaagaaa ggtttatatg aaaaggaatt ttcctgcttt atcttttccc attcccagcc

6241 tctttccctg aggtgactat acttaaccat aataataata acctcttaat gcccccaatc

6301 tctctttgat acaatttgat tcttaaaatt tcttcctatt gcttcacctt attacaatgt

6361 cttagcaatt cattataagt atttttctaa aagtcatggt tattgtgtaa tgtataaatg

6421 ttatgaaact agcttggaca tttttgtcat gaataatttc tagctaatgt tccttagctg

6481 tatttaattt aggcatctgt ttttggtgaa gtggttagaa tttcgaatac tgtgtttact

6541 ggtcaacttc aagtgtaatt atgatttcac tttaggacat gtgggattta gaaaggagca

6601 ttgaaaatta tgaaattatg aatttttttt ggatgttaat ccattgcacc aagcatgagc

6661 tgtgcctaga gatcagcggt ataactttgt tttgctttgt ttcacaattt ggtttaataa

6721 gagtgatttc atttacctca agtgctattt cttcataatg ctgtgtaatg ctaaagcttt

6781 gattatgtgc gtgtgtggtt tttttctcca ataggcaatt atttccagtc agagaaggaa

6841 accagtgcct ggcattctca ccatctttct acctaccatg atcaagtgct tgtcagttga

6901 agtacaagcc aaattgcgtt ctggtttggc cataagctcc ttgggccaat gtgttgagga

6961 acttgccctc aacagtattg atgctgaagc aaaatgtgtg gctgtcaggg tgaatatgga

7021 aaccttccaa gttcaagtga tagacaatgg atttgggatg gggagtgatg atgtagagaa

7081 agtgggaaat cgttatttca ccagtaaatg ccactcggta caggacttgg agaatccaag

7141 gttttatggt ttccgaggag aggccttggc aaatattgct gacatggcca gtgctgtgga

7201 aatttcgtcc aagaaaaaca ggacaatgaa aacttttgtg aaactgtttc agagtggaaa

7261 agccctgaaa gcttgtgaag ctgatgtgac tagagcaagc gctgggacta ctgtaacagt

7321 gtataaccta ttttaccagc ttcctgtaag gaggaaatgc atggacccta gactggagtt

7381 tgagaaggtt aggcagagaa tagaagctct ctcactcatg cacccttcca tttctttctc

7441 tttgagaaat gatgtttctg gttccatggt tcttcagctc cctaaaacca aagacgtatg

7501 ttcccgattt tgtcaaattt atggattggg aaagtcccaa aagctaagag aaataagttt

7561 taaatataaa gagtttgagc ttagtggcta tatcagctct gaagcacatt acaacaagaa

7621 tatgcagttt ttgtttgtga acaaaagact agttttaagg acaaagctac ataaactcat

7681 tgacttttta ttaaggaaag aaagtattat atgcaagcca aagaatggtc ccaccagtag

7741 gcaaatgaat tcaagtcttc ggcaccggtc taccccagaa ctctatggca tatatgtaat

7801 taatgtgcag tgccaattct gtgagtatga tgtgtgcatg gagccagcca aaactctgat

7861 tgaatttcag aactgggaca ctctcttgtt ttgcattcag gaaggagtga aaatgttttt

7921 aaagcaagaa aaattatttg tggaattatc aggtgaggat attaaggaat ttagtgaaga

7981 taatggtttt agtttatttg atgctactct tcagaagcgt gtgacttccg atgagaggag

8041 caatttccag gaagcatgta ataatatttt agattcctat gagatgttta atttgcagtc

8101 aaaagctgtg aaaagaaaaa ctactgcaga aaacgtaaac acacagagtt ctagggattc

8161 agaagctacc agaaaaaata caaatgatgc atttttgtac atttatgaat caggtggtcc

8221 aggccatagc aaaatgacag agccatcttt acaaaacaaa gacagctctt gctcagaatc

8281 aaagatgtta gaacaagaga caattgtagc atcagaagct ggagaaaatg agaaacataa

8341 aaaatctttc ctggaacata gctctttaga aaatccgtgt ggaaccagtt tagaaatgtt

8401 tttaagccct tttcagacac catgtcactt tgaggagagt gggcaggatc tagaaatatg

8461 gaaagaaagt actactgtta atggcatggc tgccaacatc ttgaaaaata atagaattca

8521 gaatcaacca aagagattta aagatgctac tgaagtggga tgccagcctc tgccttttgc

8581 aacaacatta tggggagtac atagtgctca gacagagaaa gagaaaaaaa aagaatctag

8641 caattgtgga agaagaaatg tttttagtta tgggcgagtt aaattatgtt ccactggctt

8701 tataactcat gtagtacaaa atgaaaaaac taaatcaact gaaacagaac attcatttaa

8761 aaattatgtt agacctggtc ccacacgtgc ccaagaaaca tttggaaata gaacacgtca

8821 ttcagttgaa actccagaca tcaaagattt agccagcact ttaagtaaag aatctggtca

8881 attgcccaac aaaaaaaatt gcagaacgaa tataagttat gggctagaga atgaacctac

8941 agcaacttat acaatgtttt ctgcttttca ggaaggtagc aaaaaatcac aaacagattg

9001 catattatct gatacatccc cctctttccc ctggtataga cacgtttcca atgatagtag

9061 gaaaacagat aaattaattg gtttctccaa accaatcgtc cgtaagaagc taagcttgag

9121 ttcacagcta ggatctttag agaagtttaa gaggcaatat gggaaggttg aaaatcctct

9181 ggatacagaa gtagaggaaa gtaatggagt cactaccaat ctcagtcttc aagttgaacc

9241 tgacattctg ctgaaggaca agaaccgctt agagaactct gatgtttgta aaatcactac

9301 tatggagcat agtgattcag atagtagttg tcaaccagca agccacatcc ttaactcaga

9361 gaagtttcca ttctccaagg atgaagattg tttagaacaa cagatgccta gtttgagaga

9421 aagtcctatg accctgaagg agttatctct ctttaataga aaacctttgg accttgagaa

9481 gtcatctgaa tcactagcct ctaaattatc cagactgaag ggttccgaaa gagaaactca

9541 aacaatgggg atgatgagtc gttttaatga acttccaaat tcagattcca gtaggaaaga

9601 cagcaagttg tgcagtgtgt taacacaaga tttttgtatg ttatttaaca acaagcatga

9661 aaaaacagag aatggtgtca tcccaacatc agattctgcc acacaggata attcctttaa

9721 taaaaatagt aaaacacatt ctaacagcaa tacaacagag aactgtgtga tatcagaaac

9781 tcctttggta ttgccctata ataattctaa agttaccggt aaagattcag atgttcttat

9841 cagagcctca gaacaacaga taggaagtct tgactctccc agtggaatgt taatgaatcc

9901 ggtagaagat gccacaggtg accaaaatgg aatttgtttt cagagtgagg aatctaaagc

9961 aagagcttgt tctgaaactg aagagtcaaa cacgtgttgt tcagattggc agcggcattt

10021 cgatgtagcc ctgggaagaa tggtttatgt caacaaaatg actggactca gcacattcat

10081 tgccccaact gaggacattc aggctgcttg tactaaagac ctgacaactg tggctgtgga

10141 tgttgtactt gagaatg
gta agtacgtagt attcatgtgc atgagatgct tttgaagatg

10201 ggaatgctgg acaaggagta agatcctcat tatccaaaga gattatctca acagatagaa

10261 cattttgaag accacttata aaacatatgt tgtattttca tgctatatga agatttgcta

10321 cgtccaacac tacttttttt tttttttttt tttgagacgg agtcttgctc tgtcacccag

10381 gctggagtgc agtggtgcga tctcagctca ctgcaacctc tgcccccctg gttcaagcga

10441 ttctcctgcc tcagcctcca gtgtagcttg gattacaaga gcatgccacc atgcccagct

10501 aaattttttt attttcagta gagacaggat ttcactatgt tggccaggct ggtctcaaac

10561 tcctagcctc aagtgatcca cctgcctcgg cctcccagag tgttgggatt ataggtgtga

10621 gcccgcacgc agcctctagg tccaacacta tttaaatgga gaagtacagt gaaaggatgt

10681 cagtgtgtgt atatgtgtct atgggtgtgt gtatgtgtaa gaaagggggt gatgggtata

10741 tacctagagc aacaacaaaa ggggtgagac tgtccaattt taataaaaat gatactatta

10801 atagttggga agatccacag tactttccta ccttgttttc ccttcctgtt aaatgcccag

10861 ctttcctaga aatgtgtctt taaaggcacc tgcagtgtca gcatggtatt ttactggaag

10921 tttctctgcg tggcttccag gttaagggcc agaatgattc tgtaatcatc ctaggtttcc

10981 cagtaaactc tatggcttag tacagtacta catttatgag ctttttcttt ctagaaagta

11041 ggacttgtcc atgaattttc aaaatatagg tcaagtatat actttcatga ctatactttt

11101 caaatgtact ttattataat gcatatggta aaatctcctt gatgtgttta tggtacaagt

11161 agctgttatt caggaagaca ataatatggc atttatttat ttgagacaga gtttcactct

11221 tgttgcccag gctggagtgt agtggcgtga tctcggctca ctataacctc cgcctcccag

11281 gttcaaggga tcctcctgcc tcagcctccc aagtagctgg gattacaggc acccgctacc

11341 acacccggct aattttttgt gtttttgtag agacagggtt ttaccgtgtt ggccaggctt

11401 gtctcgatct cctgacctca ggtgatccac ctgcctcggc ctcccaaagt ggtgggatta

11461 taggcgtgag ccactgcacc caaccaatat agcttttaga tcgtaccaca gggctacatt

11521 agagcctgca ctaggactct gggttctcgg tccacattta aagtgatatt gctttggcag

11581 tgtgttgaga gccagatgtt gggaggtgat aaagctgatt tccatctggc tataatcttg

11641 gctttgcact aggaggataa acattacatc tgtatccgta tttaaaattt tgaatgctgc

11701 agaagaatgg aacatgtata aatgtagtta taagtcaatt tctaatttct cctgacgatg

11761 aatatctcta gcagaaagct gtggtttctt taaggttaaa tgaaaccttt acttttcagt

11821 gtttttctgt ttttccttgt gaaaaaaaaa tcactgtagt aacttcagta gtatttcata

11881 gtattttgta gtattcatta ggagaaaact catttctttc actgttgttc ctgagactat

11941 caagttgctc ttaaagccac tttatgtgct tttcaatcta tgtggtttta tgtcctgact

12001 tacactgaag gcttttcaga ggtaaatcta cagcactgac acctcatctt cctgaaatgc

12061 aactgcttct gatatggtgc caaaaaataa ctggattaca ttatactgtg agatggctgt

12121 tgctaaaaga aagggggaaa tcttagctgc catattcaac gtaaatattt cttccttttg

12181 aatatagacg tttaaaacac tttaacctac ttaacattct ggattagcat acttttttat

12241 gaacttttga gggtcatgtt aactgatagt ttcctaatga aaaatatttt tggtatagaa

12301 agaagttcca tgaacagatg gctcctcagt accctgctac ctttctggaa atggctgata

12361 tacatcatga catcaggaca ttgtcaaact ttacataaaa gtgttacatg aaataaattg

12421 ttaaaagaca attttttttt tttgagacgg agtttcgctc ttgttgccca ggctaaagtg

12481 aaatggtgcg atctcggctc actgcaacct ccgccttctg agttcaagcg attctcctgc

12541 ctcagcctcc tgagtagctg gaattacagg catgcgccac catgcctggc taattttgta

12601 tttttagtag agacgggatt tctccatgtt ggtcaggctg gtctcaaact cccaacctca

12661 ggtgatctgc ccgcctcggc ctcccaaagt gctgggatta caggcgtgag ccaccagcct

12721 aaaagataca ttttaatgta cagaaggagg atctataaag gtgattttga ggagtagtgt

12781 caagtcccca agccttggta attttttctt taaaatatat tttatgcagt tctttctatt

12841 gtagttataa accttatttc acttcattat cttaatttta tcattcttaa cctctccagg

12901 tttagtccaa cagatttatc tctcatgttt tcctcctgtt ctaaagctgt caaattcagc

12961 attgtttcta aaactcaaat ctgattatcc ttcatggaga agaacagcat ttgggccaga

13021 tcttactaga ctctggagtt tgaattgaga acctttcatt tgtctacctg ttcacttttt

13081 catgcagcat ttgttgatca tccacactgt gctaggtgct gtcacgttgc aggttaactg

13141 actttgcttt taggagttca cagtactatt agggaagata gctatctaaa tataattgta

13201 ttataatgag acaaatgtta ggatagagac atacattgga gataagagac ataagttgga

13261 gatttgtgaa ggttgcacca agttatggct gacaagtact gctgtatgtc taacacaggg

13321 cttctctggg taattttttg ttgtaaggtt tgtcctatgc attgtagaat atttagcagc

13381 atccgtggcc tttatctgct agatgctagt agaacgtaga accctttact caagttgtga

13441 caatgaaaag tgtctcaagc cgggcacagt ggctcacgcc tgtaatccca acactttggg

13501 aggccgaggc gggtggatca caaggtcaag agtttgagac cagcccggcc aatatggtga

13561 aaccccatgt ctactaaaaa tacaaaaaaa ttagccgggt gtggtgacgc atgcctgtaa

13621 tcccagctac tcgggaggct gaggcaggag aattgcttga acccgggagg cggaagttgc

13681 agtaagccga gatcacgcca ctgtactcca gcccgggcga cagagcgaga cgccgtctca

13741 aaaaaaaaaa aaagggtctc aagacattgc caatgtcccc tcgtgcaaaa ttgcccccca

13801 gtgagaatca cagtgaccta ttctaggtct tcattctgtc tggtatgaat agataatgga

13861 actaagcatt aatatagagt cctatatgga cagcataatt tcattaaagg atttctgatt

13921 ctcaatctag tgtttagtag tctgctttat gtcttgactc agtttgtgca gaaagaggtt

13981 ttatgtgatt aaatttttaa agagtttact tgtattttaa gttcacattt ctaggttttt

14041 ctttcttata tttagggtct cagtacaggt gtcaaccttt tagaagcgac cttgttcttc

14101 ctttccttcc gagagctcga gcagagagga ctgtgatgag acaggataac agag
gtaagg

14161 gtggcagaga gtgctggggc acacagtaca catcgtacca cctaactcaa aaaacaaagc

14221 aggcttgcat taagcttgat cagtgccagc tgtgctgtac tggaatcagg aattccccac

14281 ggccctgttt aacagcggaa ctaattaatt cagcgcatgc atttaggtca gaaattactt

14341 ttccagactt gaggtcaata tttgctgatt atgcatagtc tgtggtgtct tgtgattagg

14401 ggacatgttt cttattttta aaaaggctgg acttgcactc caagttgctg tagttttatg

14461 taagcaaaac tcttcaaaaa aagctacaga catcaatttt gtgtgtatgt ttaaaaatag

14521 ataaggtata ttttgcttaa aggcagtaag gctataaatt tgagccatag cttctctgtc

14581 agtattccac aatatctctt ttaacagttt ggagaaaagc tgtaatgcca gaagatacag

14641 tcaccaactc tagtttaaaa agtaacagat caaaataatt tagagataca gcaaacactg

14701 aacatttgta gaacatagaa acaatgtacg tgacctttgg tcttgaggct atggctcacc

14761 aaacaggaaa tgccattccc tcctttccct gtggttctgg atgccaactt tacctgttcc

14821 cttttgccct agatactgtg gatgatactg ttagtagcga atcgcttcag tctttgttct

14881 cagaatggga caatccagta tttgcccgtt atccagag
gt gggtctgtag cagtttttct

14941 ttgccttttt tttttcttct tcttcttctt attttttaaa ttttgagatg gagttttgct

15001 cctgttgccc agactgaaat gcaatggcat gatctcggct cacgtaacct tcgcttccca

15061 ggttcaagca attctcctgc ctcagtctct cgagcagctg ggattacagg cacctgccac

15121 cacgcccagc gaattttgta tttttagtag agatggggtt tctccatatt ggtcaggctg

15181 gtctctaact cccaacctca ggtgatctgc ccgcctcagc ctcccaaagt cctgggatta

15241 caggtgtgag ccatcacgcc cagcctttct ttgcctgtta ttttaaactt tgagtttcaa

15301 gatgggtaca gtggcatgca cttatagtct cagctactca ggaggctgag gtgggaggat

15361 cacttgaggc cagaagttca aggctatata gtgtgctgat tgcacctgtg actagccact

15421 gcacactata gcctgggcaa cacagtgaga ctccatctct taaaaaataa gtaaattagt

15481 taaaaataaa atttgaattt tatttaagct gggtatgggc atgcaagact atattaattt

15541 gtaattatga ttggaaactg ggcatatttc caaattatgt atttggaaat tactgtttaa

15601 tgtagagggg agaaataaaa tttatagcat ttgggaactc aggcattttg gattaaacat

15661 gtataatcaa tgtgagaaca gtgaatgtgt ttaccactct cagaaaccct gacttgtagc

15721 tttggcacag tattcagggt ggtgataatg aaattgttat cattagataa agccacacat

15781 cttctactgg aggaacagtt cctcattggc cccgcatgga tgttctctcc tcagtgtatt

15841 ttcacagaat gtactgtgca tgttataaac agggtgtata atagatgagc atttgtttac

15901 cagttctttc ctcaagatat catttgagga cttgatcatg gaacatagac aaattgttgt

15961 tgtgaaacaa ttttttttgt tttgtcagta attttgttta taagcaaaaa tttttatgta

16021 agactcatta ttcataaatt cttttgaatt tttttttttt tttttttttt tttttttttt

16081 tttgtgacag agtcttgctg tgtcgcccag gctgaagtgc agtggcgtga tctcagctca

16141 ctgcaagctc cgcctcccgg gttcacacca ttctcctgcc tcagtctccc aagtagctgg

16201 gactaaaggc gcctgccgcc acgcccagct aattttttgt attttttagt agagatgggg

16261 tttcaccatg ttagccagga tggtctcgat ctcctgacct cgtgatctgc ccgcctcagc

16321 ctcccaaagt gctgagatta caggtgtgag ccaccacgcc cggccgattc ttttgaattt

16381 ctataaattc cctgaattaa acccacctct agtaggctaa ataaaaatga atgcttttaa

16441 agcactctga tatctttggt aaaacaattc tgacacaaaa attaaaattt caattatatt

16501 ttgctgagtc tttctaggtt gctgttgatg taagcagtgg ccaggctgag agcttagcag

16561 ttaaaattca caacatcttg tatccctatc gtttcaccaa aggaatgatt cattcaatgc

16621 ag
gtaaaaga ttaacttcaa aatctcataa atatatatat atatatatat atatatatat

16681 atatatggcc taactcttct aaaatttgat ttatttaatg atatagacca ccttggtcaa

16741 ggcacgtgtg tgtgtgtgtg tgtgtgtgtg tgttcttact agaactttta tcatcagaag

16801 aaaaagcaag ggattaatta taattacaaa gaaagaaata tctttagtag tgtaagtagg

16861 tttggaggga atgaattatg gattcaggct gacagtttca aggattctca aggttctcct

16921 gcagaagatt atatattgac tttatatttg gcaataattt ataaatcaaa gtcctttagt

16981 gaccttgtcc ttaagtgtgc aacagtgccc ttttagaaat taccttctat tgtgtgagcc

17041 tacacaggtt tgggaaagcc catattgaga agtcatttca caaaagggac cctttgtctg

17101 atctgctttt ctattttgcc ttgtgtttac caccatggct gtcttattcc ctttgttttc

17161 agtacggaaa caaattagga ggctaacaaa tttgggtaaa ctcctaacct ggcattgacc

17221 tggtaccaga gcaggcaagc agttgatcca gaaatagacc ctggggcagt ggtataggct

17281 cgggggtatt ggagagcctc aagtacctag acagagagga accaccaagg tctaaagcca

17341 ccagtggctt ttaacttact tctttaatag actagcagcc gggcgcgctg gctcacgcct

17401 gtaatcccag cactttggga ggccgaggtg ggtggatcat gaggtcagga gttcgagacc

17461 atcctggcta acacggtgaa accccgtctc tactaaaaat acaaaaaatt agctgggcgt

17521 ggtggcgggc acctgtactc ccagctactc gggaggctga ggcaggagaa tggcatggag

17581 gcaggagaat ggcatgaacc caggaggcag agcttgcagt gagccgagat cacaccattg

17641 cactccagcc tgggtgacaa agtgagactc cgtctcaaaa aaaaaaaaat taaaaataaa

17701 taaaaataaa aatagactag tttctccata atcgttttcc ttgctttcat ataaatttta

17761 aaaatttatt tcagcctgta tgttagcaat ctctggtcac attacctatt ctcaagagtc

17821 actctgttgg gaacatctca ggccttctct aaagtattgc tagcacacca ctctgagacg

17881 tgggagtctg tcttcccctg tcgcctccct ccccttacat cttctgatca actccacctg

17941 cccattcttt catccttcct agtgactttt tctggttcat cagaacctta cccagtctca

18001 aagaaaggag tgtgataagg caaacaactc taaataaaat acagttctgt atctttctat

18061 gcaaataagc ctttgttaat ttttatttag tgttgtacca cttatttgaa tgtctgacag

18121 gttctccagc aagtagataa caagtttatt gcctgtttga tgagcactaa gactgaagag

18181 aatggcgagg cag
gtaagaa tggagtttag cctgaatgat taaccagctg gtcttcttgt

18241 aacctttttt taatgattat ttttggtata taatatagtt tcaatgattt ggatggatta

18301 gggaaagcca atctaaaact aagaatatta tgaaggctgg gtgcggtggc tcacacctgt

18361 aatcccaaca ctttgggagg tgaggcaggt ggatggcttg aggccaggag ttcaggacca

18421 gcctgagcaa catggcaaaa ccccgtctct actaaaaata caaaaattag ctgggcatgg

18481 tggtgtgcac ctgtagtccc agctactcag gaggctaagg cacaagaatc gcttgaactt

18541 gggaggcaga ggatgtagtg agccgagatt gcgctattac actccagcct gggcgacaga

18601 catttagttt tatgttcata cacatacata tactcaggat gcacattaaa ctatcaagag

18661 tatttacttt gggagtgaaa atgagggtgt gcaaaggaga tatttcaact tttactcttt

18721 ttgtcttttt tttttccttt ttgtggagaa cggggtctcg ctatattgcc caggtaggtc

18781 tcgaactcct gggcttaagc tatccttcca cctctgcctc cctaacagct gggattacag

18841 gtgtaagcca ccacgcccag cccatctttt attcttttaa atattttcta ttgtttgact

18901 ttttaataat attttcatgt tttcccttta taataataga aagtgtatat aatgaactct

18961 gctaggtttg tcaatattta cacagaatct cttggtgacc tgctctaatg aaacaataat

19021 agtttgtaaa tatattaact cattcaacaa gtattaattg agtgcccatt gtatgccagg

19081 gactgtacca ggccctaggg atacaataat gagaaaagat atatatgcaa gagaaaaaaa

19141 taattacaca aacataaaat tttagttcta gcccctgtaa tggagaaaga gatacaagat

19201 gctctaagaa cctgtattgg gagatttgat ttcatcaggg agttcaggga agtgcttctt

19261 ggtaaaatga ggtctgaata tgaatatgag ttaactaagt aaagaaagga gggaacaacc

19321 atgcaatttg tgatttattc tcctcagtga gtggaaacag ggcaagggga actggagatg

19381 aggctagaga agtaggtagg agtcagatct taaatgccag ctacaagctt tgctttgtcc

19441 caatactgtt ggaaaccatt gaaagctttt ttttttcatt ttttatcatt tgtattttga

19501 tgttgttttt ggggtggtac atgtcacatc acatttgcat tttaaaaaga ttgctctggt

19561 tgtagtgtgg agaatggagt aggagtaggg gtggagtccc agttaactgt agattgactg

19621 ttaaaagaat attgcaatat ctgagacatg gttagatggg accagggtgg actagagagc

19681 agtgaatgta ttggaaagat atttaggaga tagagtcagc agggcttggt gatggattgg

19741 atatggggaa agtatgtaaa agatgacttc tggtgttctg gcctccatag ctgagttgat

19801 gatgttgcag gacccgtgtt gcaggcagag cagagaggat gaaaatcatg agttcagtct

19861 ttgaaatgct ccagaagaga tgtcaaggtg gccaggcacg gtggctcacg cctgtaatcc

19921 cagcactttg ggaggccgag gtgggtggat cacctgaggt caggagttga gaccagcctg

19981 gccaacatgc tgaaacgctg tctctactaa aaatacaaaa attagctgga catggtggca

20041 gatgcctgta atcccagcta ctcgggaggc tgaggcagag aattgcttta acccgggagg

20101 cggaggttgc agtgcgccaa gatcgcacca ctgcactcca gcctgggtga cagagcgaaa

20161 ctctgtcaaa aaaataaaat aaaaagatgt caagaatgtg gttggatatt acaagtctgg

20221 cctagacaag tgttctgaca tggagataca aatttgagcc atcagatgca gaaaagtgat

20281 cactgaaacc ctggatataa atgttatcac ctagaaaaaa gaagagtgag gagaggtctt

20341 gaatctagaa taattctagc acttaagagc tcggtagaag aggatgaacc agcaaaagag

20401 actgtgaaag aacagctaga gaaataggaa gaaaacagaa gaatgttatg ttggaaaacc

20461 aggatgtcat gccaaacgct gccaagacat taagtaagat gaggatcatc tgattttgtg

20521 acagtagaga ttagggattt tagcaagtga gagctgtttc agagaataga caggacgaga

20581 tgctgatggt gctaatggag actgcaagga gatactcaga aatgtgccca tgacacggaa

20641 gagggactac agagggtgag ggtgacagag gacctttttt ttttaatggg agaaacttga

20701 gcacatttgc aagggatgcc tgatgggata aagtccctga aaaggcctga gtggctgagc

20761 cctgagcagc agtggagggc tggctccagc ttgatagtac ctgcaggcaa gtgctaaagt

20821 tttagagcat ttattctcta tgtatttcaa aatattccct ttaccttgtt tagtaaatgt

20881 catagcagat caaaggcaaa ttaacctaaa gttctgtcag aattatcatg aatatagaat

20941 ttgtgaagat caaattccta tagttttact atttctagaa aagtaatagt tttttgttgt

21001 tgttgttttt gagacgaagt ttcactcttg ttgcccaggc tggagtgcaa tggtgcaatc

21061 tcagctcact gcaacctccg cctcctgggt tcaagcggtt caccagcctc agccttccaa

21121 gtagctggaa ttacaggcac ccgccaccca cgcccagcta attttgtatt tttagtagag

21181 acggggtttc tccatgttgg ccaggctggt ctcgaactcc tgatctcagg tgatccgccc

21241 gccttggcct cccaaagtgc tgggattaca ggcatgagcc aacccgcctg gccaattttt

21301 tttttaagtt ttcattatct ggaattcttt gttgtttttg agatcagaat ctctactaat

21361 gatactcaag atgggtgtaa ctacaaaatc tgcagatgtt gaaagaagca aaatggatct

21421 gttgaaattt ctcttttttt ctttactttt tttttttttt tttttttttt gagatggagt

21481 cttgctctgt cgcccaggtg ggattgcagt ggtgcaatct cggctcactg cagtctcccc

21541 ttcccgagtt caaggaattc ttctgcctca gcctcccaag tagctgggac tacaggcacc

21601 tgccaccacg cccggctaat tcttgtattt ttagtagaga cagggtttca ccatgttggt

21661 cagctgatct tgaattcctg acctcaagtg atctgccctc ctcagcctcc caaagtgttg

21721 ggattacagg catgagccac aggcccagcc agaatctgtg ttttaataag atccccaggg

21781 ggtatgtgat atgaacgttt gagaagccca agtctaaggc aggataaaca tggcccgagg

21841 ggaatctggg catcacctct ttctgtacag ccagggagat gggaatggtt tttacatttt

21901 ttaatggttg ggaaaaaaaa agaagactat ttcttgacac attaaaatta tatgaaatta

21961 gtatttcagt gtccataaat aaagttttat tggaacacag ctacgatatt cacatattgt

22021 ctatggctgc tttggctcaa aaagaattgt tgagtaggca tgacagaaat cgtgtggccc

22081 aaaaagccta aaatatttat gatctggccc ttcagaaaac gttctccggc ttgtgatcta

22141 aagtgcactg gaagtacatt ctttgtgggt ttccaagtag cttgtacacc aaggttagct

22201 gattataaca atgtgttgtt taacgatcct ttatcatatt aaagttgaat taataaggca

22261 aaatcagtat ttaaacataa gaattatata ttcaagcttc aaacttgagg aaatgccact

22321 ttgacttttt tttttttttt taagatggag tctcactctg tcaccaggct ggagtgcagt

22381 ggtgcaatct cggctcactg caacctacac ctcctgggtt caagcgattc tcctgcctca

22441 gcctcccaag tagctggaac tacaggcgcg tgccaccaca cccagctaat ttttgtattt

22501 ttagtagaga cagggtttca ccatgttggc caggatgatc tcgatttctt gacctcatga

22561 tccgcccgcc tcagcctccc agtgtgctgg gattacaggt gtgagacact gcacccagcc

22621 ctgactatgt tttctaaatg agatgctggt gaactaaaga gtttcttagt ttaccttttg

22681 tttcctaaaa aatgaaatta tggggaaaat aataaaactt ataggataaa aattagtaat

22741 actggtaatc tctggagaga gaaactagaa ggggtggggg caagagtata aaggagactt

22801 tattaaattc ccttttgtgc ctttcagaat tggatctggg attcaaacat atgggataag

22861 tttgctatca ttttccccat aatttcatgt tttaggaaat aaaagataca attgttactc

22921 ctcagaatgt tttgtcttat gctgttgtta gatagtttga attaagtctt ctctgtcctc

22981 aacacacatg atggttgtcg tcttgctctg agaatcaaag ttcacaatcc ttgctcatct

23041 aggtgggaac ctgctcgtgc tggtggatca gcacgctgcc catgagcgta tacgtctgga

23101 gcagcttatc attg
gtaagg atctgtttgc agccagaaaa aatttgagac tcccagcaga

23161 atcagtacac ctcaaagagc ttttctaact gcaactgttg tgagaaagca cactttgtac

23221 tgtatgttca aaaattaaac taaagtcctg cagtttggcc tgtgttataa ttgaaagaat

23281 gtggtagttc actaattgaa agatgtgaac ttatttatgt ggtcagaggc atatttgcca

23341 gatgttatta acaacccaga agtacgcaag gataggctgc ttactctgac atccagcaca

23401 aagctctgcc cctccatatg cccggtgatc ttggcatgcc ttcccacact ccatcaggca

23461 gagaccagtg gcagctcaga aggcccagta tgctgttttg attttaaaat ttagtttgct

23521 tttgaatatg cagtataatc aaatagatcc aattctgaaa ggcacaaaag ggaatataat

23581 aaagtctcct ttctatcctt gtccccaccc ctcctagttt ctctccccag agacaaccaa

23641 tatctctggt ttttaaatct atctttccaa agcaatatat gctttttaaa tgtttttttt

23701 tgctaccctt ttcctcatca tgaggcagtg tgttcaccaa gtggtaaaat accaggaaag

23761 tctttactcc tgtggccaat gagtaatgtc aaagagttag gcaaggacct aaggagtgct

23821 tcatggacta gaatggcaat gatattacta ctgttttcac ttttccttat aaaatgacgg

23881 cctaagagtt gggaaagata tagtaaaaac agtgttagaa aaacttacct ctacaggccc

23941 tgcaccattt gagaatagta ctaattctct ccagagactg ggagaccctg ctcaaggaaa

24001 gggaagcctt ctggagccat aagactgtgt ggtcaaccca catatgttcc tggtaatcca

24061 cattattcac ctctgtcatc aaaacaacat ttattaacta caggaacatt ggcttgttca

24121 tctgaatgta agatgcaagt cttatatttc ttattttgaa aaatactacc aaaattgtta

24181 ataggctgaa tatttattag tttgacttta aaaatggaaa tccaaaatct aaaactgaac

24241 attaaacatt ctgctttgca ttagaatgat tatctcaagg ccgtgcttta tttttatctg

24301 aatctagaag acttcccttc ctactcttaa cccattcatc tttctgccaa tagattccta

24361 cgagaagcaa caggcacaag gctctggtcg gaaaaaatta ctgtcttcta ctctaattcc

24421 tccgctagag ataacagtga cagaggaaca aaggagactc ttatg
gtcag taccaccatg

24481 agaatgtgat gttgatggta tcaattctca caataattct gtgaggaata agcataacta

24541 ttgttccatt tctcagatga gaccaagaga ggttacaaga cttgtctaag gtcatagcag

24601 ttattaagta gtggagcagg cactcaaagg caggtcgtct gactccacag gctgtttgtt

24661 tcctttttta aaatatattt ttttctgcat ccaatcatcc attaaaatat ttattttgtg

24721 agacatggtc tcactctgtt gtccaggctg gagtgcagtg gcacaatctc tgctcactgc

24781 aacctccacc ttccaggctc aagcagtcct cccacttcag cctcccaagt acctggaact

24841 acaggtgagc accaccatgc tggactaatt tttgtaattt tttggtagag atgggttttc

24901 gccatgttac ccaggctggt ctgaactcct aggctcaagc gatcctccca ctttggcctc

24961 ccaaagtgct gggattacag gcgtgagcca ctggacccag cctattcttt ccactgtatc

25021 ccagctgaag agtataggat agagtttccc tgataatttt ttccagcagt tggcttagaa

25081 cactcttctc atttcttgga acagtctgtg tttatgccat gctttgtgaa agcaaagttt

25141 ctgcagtaat tggtcccatt ctgctttgct ttggtgcatt ctagactctt agttttcatt

25201 atcgtaatca taatgggaag aaagtaaaac attgggatgt aaggaagggg cgaatcatat

25261 agtgagtcac aagataggat atttttcctc ttctaggcag cacaaattat tgaaaatgaa

25321 aaagaaattt ctcccaatat gtttcaggtt tgggaatatg atactgccag taaatctttt

25381 tttccctatg acataattta tattagaggc agatctattt acttacatcc cgttagtcag

25441 gacttctcaa ttccttgggc tgttaattac ctccctgctt taatgctgat tcaaatcact

25501 ctcagacctt ttcttcatgc cagactccct taagtgccag cagccagaag ttagcagttc

25561 ctcaatgaag aaaacacact tagaaacaag acttagacac agattgattc ccaatggaaa

25621 agagaaaaac aataaaggtt actgtaattc aaaaaggaaa tttgataata aaaaatccca

25681 aacacccaca cataagtgtg agaaaaacaa tacggttgtt aagccctgtg gcagcttttg

25741 aaaaactatt aagtagtatt tggaaccagt agtgaagtgc aaatgaagta gcactctttt

25801 tttttttaac atttgaggtt atctctttag gtgttaccac aaaaatctgg aagatctggg

25861 ccttgaattt gtatttccag acactagtga ttctctggtc cttgtgggaa aagtaccact

25921 atgttttgtg gaaagagaag ccaatgaact tcggagagga agatctactg tgaccaagag

25981 tattgtggag
gtaagacaca gctgcagatg ttaaggaaat tgctgagtga tggtaacctc

26041 atattttgtc tgtagattct ctgaattcat cttttcttca acaagtattc atggtattta

26101 cctgcggtgt gccaggagct agaaattcag agatgagtta gctcaaaata aggaagaagg

26161 gcatttaaag aattatagtc agtgaacttt ggctagtaga ggtatgtgtt ttgctttctt

26221 cccagtgata aaggtaatga aaactaagag tctagaatgc accaagatag tttttcagaa

26281 gtgttatgga aacacagaat gacttgtgtg cctggggagg tgaagtcaat gaaaatgtca

26341 cagaaaagct gttttcttaa gaaaagtttt tacttttgag agaccaggtc tcactctgtt

26401 gcccaagctg gagtgcagtg gtgcaatcat agctcactgt agccttgatc tcctgggcac

26461 aagcaatctt cctgcctcag cctcccaagt aactcggact acagacacgc accaccatgc

26521 ctggctaatt ttttcctttt ttttttttgt agagatgggg gcctcactat gttgcccagg

26581 ctggtcctga acttctggcc tcaagcaatc cccttgcatt ggcttcccaa acccccggca

26641 ctacaggcat gagccactgc acacagccag aaaagtctta cagaatagtg ggaaactagg

26701 ctgggcaagg tggctcacac ctgtaatgcc agcactttgg gaggccgtgg tgggtggttc

26761 acctgaggtc gggagttcga gaccagcctg acaagcatgg aaaaacccca tctctactaa

26821 aaatagaaaa ttagccgggc gtggtggtgc atgcctataa tcccagctac tcaggaggct

26881 gaggcaggag aatggcttga acctgggagg cggaggttgc attgagccaa gattgcacca

26941 ttgtactcca gcctgggcaa caagagtgaa actctgtctc aaaaaaaaaa agaaaaaaag

27001 aatagtggga aagtgctgag tacagaagag gggagaaggg cattctagac agaggaatac

27061 atatcagaga cttgagatac atcctatgtt tagggaacta tcaatagata tggctcaaat

27121 taagatctac agttgatcct tggataactt gggtctgaac tgcgcaggta cacttaaatg

27181 cagattttct ttcacctctg ccattcctga gacagcaaga ccaacctttc ctcttcttcc

27241 tcctccttag cctattcaat gtgaaaatga tgagggtgaa gacctttatg atgattcatt

27301 tccacttaat gaataataaa tatattttct gtttcttatg attttcttaa cattttcttt

27361 ttctttcttt ctttctttct ttcttttttt tttttttttg cgatgcccag gctggagtgc

27421 aatggcacaa tctcagctca ctgcaacctc cacctcccgg gttaaagcga ttctcctacc

27481 tcagcctccc aagtagctgg gattacaggc gcacaccacc atgccccggc taatttttgt

27541 atttttagta gagatggggt ctcaccatgt tggccaagct ggtattgaac tcctgacctc

27601 aaatgatcca cctgccttgg cctcccaaag tgctgggatt acaggcatga gccactacgc

27661 ctggccaaca ttttcttttc tctagcttac ttaattgtaa gaatacagtg tgtaatatat

27721 acaacacaca aaatatgtgt taatcaactg tttatattat tggtaatgct tctgatcaac

27781 gttaggctat tggtagttaa gatttgggga aatcaggcca ggtgcagtgg ctcacgcctg

27841 taatcccagc attttgggag gccaaggcgg gcggatcatt tgaggtctgg agttcaagac

27901 cagcctggcc aacatgatga gaccccgtct ctactaaaaa ttaaaaaatt agccaggcat

27961 gatggtatgt gcctgtaatc ccagctactt gggaggctga ggcatgagaa ttgcttgaac

28021 ccggaaggtg gaggttgcag tgatccaaga tcactgcact gcactccagc ctgagtgaca

28081 cagtaagact ccgtctcaaa aaaaaaaaaa aaaaaattgg ggggaaatca gaagttatac

28141 ttggatcttt gactggatgg gagttggcta gatgggttgt caaggtcaaa tcataaaata

28201 atttagcata ttctactaag tagtttgcat tttatcctat aggcagccag tgaaaacttt

28261 taagcaggag agtaaggtcc attttagatt gcattccaaa tatgattact ttacatatat

28321 gttttggtaa aggtttggaa agctactgct gggctgtggc attggaggta ttagagagaa

28381 ctattacttt ttattttgat tacttatatg tttctttttt tatagcaaat atcatttatt

28441 ttgtctttat tgtctttttc ctctcctttc ctgttttcta ttatagtttt ctgtatttct

28501 cttttttttt ttgctctact cgtttagaag ttatatattc catttatatc cttttagaga

28561 ttagccttaa cttttagcat gcatacttat caaagtttaa aagtaattca cttctctatc

28621 ctcttcctaa ctgtaccaaa atcttacaaa ctgttcatct cccatcaccc tcctcccatc

28681 ttccatgttg ctgtctagta ctttaattcc accttgtttt taattccctg gtggtcattt

28741 attttatagt caatctttat ttagattaaa aaaagtgttc accagagtct ttgttcacca

28801 ttgcctcttg catcctactt attctgtgtt cttttgggtt tttttttttt tttttttttt

28861 ttttttttac tgaagtaaat ctcttacaag atcttttatc cacaaacaga tcttgagaca

28921 aaaaatacaa aaatataaaa ataaatagaa gttattttag taagagtccg tgggccaggc

28981 atggtggctc atgcctgtaa tcccagcact ttgggaggcc aaggcgggca gatcacctga

29041 ggtcaggagt tttgagacca gcctggccaa cgtggtgaaa ccctgtctct accaaaaata

29101 caaaaattag ctgggcatgg tggcaggtac ttgtaattcc agctactcgg gaggctgagg

29161 caggagaatc acttgaaccc gggaggcgga agttgcaatg agctgagatc acgccattgc

29221 accccagcct gggtgacaag agtgaaactc tgtctcaaaa aaaaaaaagt ccgtgaatga

29281 taagctgtct tttaaagtat ctttattttg tcttcatact tgcatgatta tctagctaaa

29341 aacagaattt cacattgcca gttatccccc ccgcattttg aaaatatttc actacttttg

29401 actttctatt ttattttatt ttatttcatt ttttgagaca gagtctcact ctgttaccca

29461 ggctggagtg caatgggatg atctcagctc actgcaacct ccgcctccca ggttcaagcg

29521 attctcctgc ctaagcctac aaagtagctg ggattacagg tgcccaccac cacacccagc

29581 taatttttgt atctttagca gagatggggt ttcaccatgt tggccaggct ggtctcaaac

29641 tcctgacctc aggtgatctg cctgccgtgg cctcccaaag tgctgggatt acaggtgtga

29701 gccactgcac ccagcctgac tttctgtttt agccattgaa gtccttagtc aatccaatta

29761 ttactctgta ggtaatctga ttttccctct ggttgctttt acgatggttc actttgtctt

29821 tggtgttctg caatttccct atgatgtatc tttgtatgga tttattttca tttccttctg

29881 gagattcact atgcttcttc agtttggaaa gtatgcagtc attatcacct caaacgtttc

29941 tttcccattc tctctcttgt ctcttggaaa ttttgtcaga tatctactgg gccttatctt

30001 tctactctgc atgcctcttc aactctcttc catatttccc attgctttct ctctgtgata

30061 cattctgtga aatctcctca cttatcttcc agtgaactaa agtatctctt catcttctgt

30121 ttaattatgt gtttagctta tccactgagt tttttatttc agtgactaaa ttttttattt

30181 caatggctat attttttatt tccagaagtt gtgttttttt ccccaaatct cactcttcct

30241 tttttatggt gtcctgcttt tttcatgatt gttccttctc ctttatcagt agctttcttg

30301 atttggagca caggcaaatc cacacccacc tcatctagaa gtctgaagtg ctttagtaat

30361 atctaaaata gtaaaatcta aaattagagg atagatgtag tagataaatt gggggatgga

30421 tttatgtggg aagatttgag tcagggagag cagctgacgg gttattgcag tggttaaggt

30481 aagagaagat gaggattgga ctgagtcagt agcagaggga gtagagagga gtgacatatc

30541 tgagacagaa tttgggaggc agaagcaaga ggacttggtg agcaatcaaa cgtgaaagga

30601 ataaaggaga aaacaaaagc aacagtgatt ctgagggttg tagcatggat ggctggaagg

30661 atggtggtgc cattgacaga gaagggacac agatgaggag gaacaaacct gcctaggagt

30721 tccatcgtga aggtgggttt agacacagtg aatgtgcctg caggtggaat gcccagtgga

30781 taggcacact taaaagatct ggtacttgga aaataaatct gggctgtaca tatctatctg

30841 agagttaaca gtatatatat attgatagta tcagaagctg gagaagggcc ttgaggaagt

30901 gaggttgtcc agggagaata tggagagtga aaaggaagag acaaaagagt ggagcctgtg

30961 gaacatcagc atccagggtg cgtatgcttt ggaaaaggtc tggaaagctc tgctgaaaag

31021 aggaaattct aaggatggct gaaaatactt ggttagagag ggaggaggaa aaccaggaaa

31081 aagcaatatt gccaaaacta aggaaagttt taagcaagga gggaatggtc aataacggct

31141 atgaagtaga gcctgaaacg ctggacttga ctgtcatgag gacattagtt gtcttgtgga

31201 atcatttcct ggggatgagg tggggcagaa gataagagtg cagtgagtca ggagtgcgtg

31261 aagcaggagg aagtgaagct aagtagacaa ctctttcagg aactttggct acacacagaa

31321 gaagagagat ggggtgagag ctaaaggtta gaagtggttg ggaagtgtgg ggtgggattc

31381 aaggctgagg aaaggtgttt gtttgcttta agacaaaaaa gacgtaagca ggtttgcagg

31441 ccaaggggga aacattcaag aggaagagca gctgtcagtc agctcttggg tcatcttggc

31501 tttgcccttt ccagaatcgc ctttggatca gacagatcca aagatttgct ctgcttccag

31561 caaagctctt tcctccccac agacatgctt tctctaagac ccctgctcca aattgtccta

31621 aatccaaaag ataggatctg ttagagtctt ttattgtaat tcaatcccta atatgtaacc

31681 agaaaattat aagaaaaact tttcctgaag gaacttaaaa gactactgtt aaggagtttg

31741 cttcaaatgg aaacaaacct ctggtttcat aaaatcttga ttatcctatg tcaggctgat

31801 caggagagac tttgtgtgat tctggaagaa gctggaacag ttttggtgtt ttgaggaggt

31861 agctatttga atcctagcag ttgaaagatc attaatacat ccttctataa agctctatct

31921 tctagcctgg tgaggaggtg gaggctgcag tgagctgaga tcgcgccatg gcactccagc

31981 ctgggcaaca acgagactac gtctcaaaaa attaaaaata ataaataaat acggccaggt

32041 gcagtggctc acaactgtaa tcccagcact ttgggaggcc gaggcaggcg gatcatctga

32101 ggtcgggagt tcgagaccag cctgaccaac atggagaaac cctgtctcta ctaaagatac

32161 aaaattagcc aggcgtggtg gtgcatgatt gtaatcccag ctactcagga ggctgaggca

32221 ggagaatcgc ttgaacccag aaggcagagg ttgcggtgag ccaagatcgt gccattgcac

32281 tccagcctgg gcaacaagag tgaaactcca tctcaaaata agtaactaac taactaaata

32341 aataaataaa aaataaaata aaaatattaa attatccagc attaaacaga ccattggaat

32401 ataaaatgga ctgagatgag acccatcacc ccttcactaa gcttacaaac agatgtttta

32461 ccatcccaga tgtacaaaca caagaatgtt tgctctccat ccacactaag ctttgtcagt

32521 ggattgagtc ttatgagacc atctttctgt ttaggtttgt ttgggaaaac atgtgatttt

32581 taaattatat gtaatatagt atctactacc tagctcttga taactgtgcc catttatagg

32641 catggcagac tagagtatag atgaaggagg gaagagaatt tcctgagtct ggttttaaaa

32701 gaaaaggtac catgttgtaa tcttaggagc agatttacac atagcctttt ccaggagtaa

32761 agaaatgatt ttcttataaa gaaatcatta aatgtgcaac aggttttttt ctttttacct

32821 ccagaggtta aacaaattgg tagcatgaaa tagtgtctgc tttaaatttc tttaagcttc

32881 ttttgtaaat attaatatcc acagggatat aactattgaa aaggttaccc cagggacaga

32941 gacatgcaag ggaggagaaa tttctcaaca aagggcctca gaaaataaac tttctgacct

33001 gaagggaatt gtccccaaaa agcccgtggg aactaaagtg actgtttact tatgtttatg

33061 atatttaagc aagtgttata ctgagcagtg gttgtgttga tggttgttgt gtatttattc

33121 tcagtttgtg tgtaatatta cgcttgttct ggctaagatt acacacacaa acagacagcc

33181 ttttagtgag aagccttcct gatgttttga attggttgaa tcatcaaggc tactctcacc

33241 agctacataa cctcattagt tgagaagcta tctctggagg aagtctcagt agctaaatat

33301 attctgtgtt caataagagt tctattcagt tgcttggttt tttcatattc aaagtgcctt

33361 acaacaaata aatttgtacc caagtgctgt attgctggaa ctctttcttt caggaggcat

33421 gatttaaaga agaaaattga gatattttgt aaactatcaa agggcaagta aaacataacc

33481 gtagattaaa gccgattttc cttttgcttt gcaggaattt atccgagaac aactggaggt

33541 aagcttttcc tttattttgg ggtttcacac agaagcacca cacataaaag gcttcgttgt

33601 tccgttttct tttcctgtta ataacgtgtt tatgcattag ctactccaga ccaccggagg

33661 catccaaggg acattgccac tgactgtcca gaaggtgttg gcatcccaag cctgccatg
g

33721 taagcccttc aacatagcag tgatcaaaca cctctgcatg cagagccctg gcagggctgc

33781 cgggattaca aaagaggcag agggtacatt tactacttga ctcttggaga atacaattta

33841 gatcaagaaa ctgaatatac ccttttaaaa atcactttta caaaaacaaa atcccaacac

33901 aaaaatttga ctatagtgct gcctgaccta tatgagtgga ggaataactc ctagaggaga

33961 tgcattttga gatgaatttt gaatgagctg gggaacatgg gttcatagca tggaaagggg

34021 aattcactat agggagagta aacagcctgt gcagagaggc agaacagaaa caagttagtt

34081 atatgagaag cagcaaagag tatcaagaaa ctaggcaggt aagagaacag aaacctagaa

34141 ggtcacagca aaacctttgg atttgataca gaaggcatta gaattagcaa gcgttctttt

34201 tttatctttc caactttcat tttaggttca gggggtacat gtgcactttg taacatgggt

34261 gagttgcatg tcgcaggggt ttggtgtaca gattattttg tcacccaggt aatgagcata

34321 gtacccaata ggtagttctg atcctcaccc acctcccacc ctccaccctc aagtaggccc

34381 aggtgtctgt taccccttct ttgtttctat gtgtattcaa tgtttggctc ccacttttaa

34441 gtaagaacat gcagtatttg gttttctaat tcacttagga tgatggcctc tagctctatg

34501 tttttgcaaa ggatgtgatc tcgttctttt ttatggcgac atagtattcc atgatgtata

34561 tgtaccacat tttctttatc caatccacca ttgatgagca ttgaggttga ttccatgtct

34621 ttgctattgt aaatagtgct gcagtgagca taacacatgc atgtgtcttt atggtagaat

34681 gatttctgtt tctttgggca tattcccagt aataggatta ctgggttgaa tggtagtttt

34741 gctttaagtt ctttaagaaa tctctaaact gttttccaca gtggctgaac taatttatat

34801 ttcccaccag cagtgtataa gtgttctgtt ttctttgcag cctcaccagc atctgttatt

34861 ttttaacttt ttaataccag ccattctgac tggtgtgaga tggtatgtca ctgtgatttt

34921 gatttgcatt tctctaatga ttagtgacat tgaacatttt ttatatgctt gttggccatg

34981 tgtatgtctt cttttgagaa gtgtgtgttc atgtcctttg cccatttttt aatagggttt

35041 tttggttttt gcttgttaat ttatttaagt tccttataga ttctggatat tagttcctta

35101 tatagattct ggatattaga cctctgttgg atgcagtttg caaatatttt ctcccattct

35161 gtaagttgtc cgtctattct gttgatagtt tcttttgctg tggagaagct ctttagttta

35221 attaagtctc acttgtcaat tttcgtttta ttgcagttgc ttttagagtc tttaccagga

35281 aatctttgcc aaggcctacg tccagaatgg tatttctgag gttttcttct agggtttttg

35341 taaaaagatg gaatgcgccc aggcgcggtt gcttacgcct gtaatcccag cactttggga

35401 gaccaaggcg gatggatcac ctgaggtcag gagttcgaga ccagcctggc caacatggtg

35461 aaaccccatc tctactaaaa acataaaaat tagccaggcg tggtggcggg cacctgtaat

35521 cccagctact caggaggctg aggcaggaga atcacttgaa accaggaggt agaggttgca

35581 gtgagccaat atcacgccac tgcactccag cctgggtgac agagtgagac tccatctcaa

35641 aataataata ataataataa taataatgaa gatggaatgc ttcatgaatt tgcatgtcat

35701 ccttgtgcac ggaccatgct aatcttctcc gtatcactcc aattttatta tatgtgctgc

35761 caaagcgaac accattgagg gttcttgagc agggtaatat gatacaatat agaaaatggg

35821 gagctgggtg aggccctgtg aggaaggtgc ctgcttctcc ttcaccttcc gccataattg

35881 taagttttct gaggcctccc cagccatgca gaactgtgag tcatttaaat ggtctttcct

35941 ttataaatga ccgagtcttg gtcatttctt catggcagtg tgagaacaga ctcatactcc

36001 tctatcacca aatgtcatca ttgccacctt caaaatgtat cttcggtccg tctgattcag

36061 tctgtctggc tcctccctca tttttgccac tgttgagctc tcttacttag ttcttcacat

36121 ccactctggc tccctttcaa gctgctcttc tctcagcacc cagaggaatc tttttgaaac

36181 atccctccaa gcatgttgtt cttctaacta aaatgtgtca atgatgaccc attgctcttg

36241 ggatagaaat gtgatttttt actgtggccc acaagggtct tcgtgatttg gcccctgcct

36301 gcctctccag tgtcacgtca tatcactctc caccttgttc tttacatccc agcctgcttt

36361 tttggtcttc tgcttcctgt gtgctaaaca tttgtcagcg tcggggcctc tgcactggcc

36421 ttctcctcta cctgaaacac ttttctctcc ttcttcaatt agccacgtct ttcttaacct

36481 aaagtctcag cttgaatact actttgccag gaaagccttc cttaagctcc taaactgggt

36541 ttggtcttcc tattatatat tcatatcctg tattttatag cacctatatt tatcatttaa

36601 attattgtat aattatctgc ttcatgtttg tctcccctat tagaccataa gcgccatgag

36661 gatggaaatt atctgtctca ctaactgcct gacaaagctc ccaatacaaa gtatatactt

36721 aataaatatt tgttgactgg gtaaattgtg cataaaggca ggttataaca atatgagagt

36781 ttagattagg aagattatta tggaaattag aaaaaaagaa gcaaataaaa tgaacattgt

36841 aaggaaacaa tcagagattg ctattggacg atccatggag aaagataacc ccaacttcac

36901 aaacccataa agagtaggca aaaatactgc tcttgcctct tggagaaaaa ctctgaaagc

36961 cagatcctaa caagttaagg aatgcctcct acagagatta gaaatacaaa tactccgtaa

37021 gaaaaagctg agagacagaa ggtcagtcct gaagccttag tttcatccat ggtcataagc

37081 actaatttat ttccctcttt tttttttttt tttttttttt ttgagaacgg agtctcgctt

37141 tgttgcccag gctggagtgc agtggcgtga tctcggctca ctgcaacccc cgcctcctgg

37201 gttcaagtga ttcttctgcc tcagcctccc aagtagctgg gactataggc gcgtgccacc

37261 acacccggct aatttttgta tttttagtag aggcgggatt tcaccatatt ggccaggctg

37321 gtcttgaact cctgacctca tgatctgccc gcctcagcct cccaaagtgc tgggattaca

37381 ggtgtgagac accatgcccg gcctataagc accaatttca agcaggatta aagcttcatt

37441 aaagaagcct aagaaacatg ttactttttc cagatatttg gcagtgaccc aaggtcagca

37501 ttggtttccc actatataca ctataacctt cttgtttctg tctttcattt aggggccatt

37561 aagtttaatg atggcctgag cttacaggaa agttgccgcc ttattgaagc tctgtcctca

37621 tgccagctgc cattccagtg tgctcacggg agaccttcta tgctgccgtt agctgacata

37681 gaccacttgg aacaggaaaa acag
gtacat taatgactaa caggagcagg agggagcaaa

37741 gtttattttc tctttctttt ctggctttta ctatgcaact gtaatctgtc ttaacctttc

37801 caaaatagga gagttttgca aaatctgcac tggaagtttc acataaagcc tcacccattt

37861 cattttccag ctcaaatcca aaatggaact tagactgaac tgtcaggtca cctacataat

37921 ggctttgtat tttaaaactt aacacatgta cagacttatg taccctgaat atctattgcc

37981 tgctggctct aggtttcata atatgcagtt ctttctgaag ctactgaaag ttggtagaag

38041 cacagcaaat tcactgttgg caatatagag cctcaacttc cagtttcttg atagtacctg

38101 ctggaaacgg tgatctgtga ctgtagctgg ccatccccaa ggatcttgag ttatggtttg

38161 aggatcatca gacagggacc caagcttggg gatctctaca ttatgctttg gccctaatta

38221 cccagcaacc tgcaaactac aatgtggata atgcagaaaa ggcacagaat ggcttaaatg

38281 aagaacttct gtccttgtta taatcttcag tgaaggagaa catggagaac ataacaggaa

38341 tgtccctgtt gctgctttcc aaaatctttc cttttttttt gttttttgag acggagtttt

38401 gctgttgttg cgcaggctgg agtgcaatgg tgcgatcttg gctcaccgca acctctgcct

38461 cccgggttca agcaattctc ctgcctcagc ctcccgagta gctgggatta caggcatgcg

38521 ccaccacgcc cggctaattc tgtattttta gtagagacag cgtttctcca tgttggtcag

38581 gctggtctcg aactcccgac ctcaggtgaa ctgtccacct tggcctccca aagtgctggg

38641 attataggca tgagccactg cgccctgccc aaaatctttt cttttgctaa cctttccctg

38701 ctaagggata taaaatcttt tcttgcatgg agttttcagg tactaatgtt gtctgactca

38761 actagctgat tttacttcat taaaacctct ttgtttcaca aagaaaaata tgaagaaagt

38821 ttttctccat tgctgagagc tgggaatcta gacttaaagt tctgagtccc cttggatcct

38881 taaaacaatc tgtaatctct attgaatcca gaagttagaa atctaaacag attataacag

38941 ccagactttg gaaatatttg tatgctacct acattttcaa tttttttttt ttgagacgga

39001 gtctcactct gtcgcccagg ctggagtgca gtggcatgat cttggctcac cacaacctcc

39061 gcctcccagg ttcaatcaat tctcctgcct cagcctcctg agtagctggg attataggca

39121 tgcgccacca cacccggcta attttttttt gtatttttag tagagacagg gtttcaccat

39181 attggccagg ctggtctcaa actcctgacc ttgtgatccg cctgccttgg cctcccagag

39241 tgctgggatt acaggtgtga gtcacctcgc ccagcctgta tgctacctac cttttcatat

39301 tgctaattga agttattctt tcttcccata gattaaaccc aacctcacta aacttcgcaa

39361 aatggcccag gcctggcgtc tctttggaaa agcagagtgt gatacaaggc agagcctgca

39421 gcaatccatg cctccctgtg agccaccatg agaacagaat cactggtcta aaaggaacaa

39481 agggatgttc actgtatgcc tctgagcaga gagcagcagc agcaggtacc agcacggccc

39541 tgactgaatc agcccagtgt ccctgagcag cttagacagc agggctctct gtatcagtct

39601 ttcttgagca gatgattccc ctagttgagt agccagatga aattcaagcc taaagacaat

39661 tcattcattt gcatccatgg gcacagaagg ttgctatata gtatctacct tttgctactt

39721 atttaatgat aaaatttaat gacagtttga ttggttgctt ggtttgttat ttgaagggtg

39781 tgatttttgt ttttgtacag ttttttttca agcttcacat ttgcgtgtat ctaattcagc

39841 tgatgctcaa gtccaagggg tagtctgcct tcccaggctg cccccagggt ttctgcactg

39901 gtcccctctt ttcccttcag tcttcttcac ttccctatgc tgctgcttca tgtgctacat

39961 ctcagactta aagagtttct ctactacagt gaaaacattc tctagggtct ttcatcaggc

40021 ctttagttat tttagggata aaaactattg ataaaaagga caaggataga acagagaaaa

40081 tttaaagtcc tgttccgggt tttttgttat gttttcttta aaaactcaga gactgatgtt

40141 caatatccca aaccagtaaa atggtgaaaa tactatgagc ttgtttttta aaatatgatt

40201 ttttttggta ctttataaag tatctcttta tgtgaaagca attgtcatat caaaacacag

40261 catacatacg ttcaacctaa ccaaatatct ttacactttt tctttcagga gacaagggtt

40321 ctttgggtcc ctttcaaacg gtatcttggt gttattacat tatgcctatc tattgccctt

40381 ataatatcac ttgggaccag gactgatcgt tctgcaaatg cttgttatgc cattctcaat

40441 ctatttttcc cgcacctttt cacatgattt gtggttaata ggactcaaca gactaaaatt

40501 gcatagtaga aaaaaaatgc aaaaagccag ctggtaatgt ttattgcaac tggggtgcta

40561 tacaattagt aagatgatgc aatgagaatt tctacttttg tatttcctga ccagcctgct

40621 caaagtggct tttatatcaa ttgaatgatt ttcctcattt tttaatacag gaaaccaatt

40681 cgtgctcatg gaagaaaagt tcctttgcca gcagccttga agtgaatctt acaggagcaa

40741 tgaaagtatt gcattcatta gcgtctgccc cagagaaggt tcagagaaaa ccttcacttg

40801 ttttcaaggg gatccttgta gatttacgta attggaatcc tgaagaacag gccctactgt

40861 ctaaaaaatg gcttttattc ttctaaatac atataaacgg atgttttata gatgggaaga

40921 catgacctta gaaaggagag agttttcaga ggatttgcca ggctgtcagg ggctctgcct

40981 ccaggcccag tgtggcagtg tggcctcagg gcctccgcct ccctgcttga gggctgcatg

41041 gaggccaact gtcctgggag ttgtaaaaat cttttaaggc cagaccaatt tgagggattt

41101 taaaaagtgt ctcagtgcct cttatgattt cagaaggttt tgctatatgt aatcccaact

41161 actgttttct tgagagtagc agaggattag aaaaagtcct ccataaatta tgtaaccggc

41221 cttcctgact agcctgactc aagcaatgta agagataatt attctgtttt cataatttat

41281 aagtgtgggg gcatgcctca gcataaaaac aacctattag ggaaaaatat ctaatagatt

41341 acctttatcg cctgttaggg ttttatgttg tttttaactc agatgccata agaacaaaga

41401 tacatgtaat ttataatagt aatcattaat acctatattg tgctttaagg tttacaaaat

41461 aatttttctc atactttatc ttagtttagt ttcttgacag tccatgaggt aaggtggtag

41521 ctttatcacc attttacaaa gtgggaaacg aaggttcctc ttaggaacct agttgtcacc

41581 tttgtataat aaaacttcga agctcggagc tgttaactgg tttgctgaag gcttagctgt

41641 aagagccaga attcagaccc aggtctgagt gacttcaaac tgcacagtcc ttcccattat

41701 tacccatatg ctatccctta tatttttaat ttattaggaa ttcattcatt tataaacttg

41761 gtgattcacc tttattagat tctggtcgct gaaggcttta gtaacttcag agtaaaactt

41821 gagagatgag atgtaaaatg cagccattct tgagagttcc tttttctgta acattcatca

41881 acacttcatt gagaagtgaa ggttcctatg gctgtctcta ccttcaagag gcttagcttt

41941 agtcactgag aaagacaagg aaactaatga tagaatatag tagcttcttc tggcgttagg

42001 tatcacagag tcacagctag ttacagctag ccctttatta ttgaaagaag aggagctagc

42061 agtcccacta tcagaattaa gactagagat ggtaatagga gctagtatca gaaaagctta

42121 aggcaaagca taaagtgtag gctagaatga agctggagaa tggggagggg gcttgggtaa

42181 catccagaac ctggctgggg acctggaact acatgagatg taagaatgga gaggttctag

42241 cagtcagagg tcaggtacaa atgaacagct gggatctgcg catggcagac agtgaaaaaa

42301 cccaggcaag caaaatggtc agagcagaaa ggggcccaag gccacgttct tgagatgtgg

42361 agggggctga ggaagccacg ccaagtaagg acagatgcag ctcagcagtt cctagcgagc

42421 cctgacaagc cagctcagct gaagcttcgg gtgggagcca gtcatggcac agtggagtga

42481 aggaagagca gtttcaggca cccaaaacct gacccccacg acctgttttc cacctgaaga

42541 gccacccatt ccatccaaac ccttggcaaa agtctgctaa cagagagaac cggccagtat

42601 gctggccagt cgcgatcatg cctgtcttta ccctctaagc tgaagctgct catcaacggt

42661 gagatggcaa aaaggtgggt ccagaagagg ggaaaagaag ggagtctgtg aaaacaaaat

42721 gctgaagaat ctgcatcaaa taaacccttc cttccttcct ttttccttc
c atccctctct

42781 tcattcagca aatcttcagt gagttccttt ctaaatgtat tgtatcagaa cttgtggggg

42841 atataaagaa aaaaaagagg ttatcacatg gcttaggatg tttccaaccc ttggatcatt

42901 ttcaccccag gcccattagt tacagagcac tcagtcttac ccagaagcat cggatatagt

42961 ccaggcaacc ggatccaagc gtgtgggaat cgcagtgtgc tgcatgtggg aatctatcag

43021 gccaggatga ttcaagcagc agcctgcaga agtgggattt ggccagcctt gcctgactct

43081 ggccttccaa cttgctctta tctacttcca aagtaaggaa tgccccatga tgggccgagc

43141 agtccaaaaa aagtggcttg gagttagcta ccgcaattag cagtttctca ttattcaaca

43201 ctgcattacc tctttttcat attaactgca gaattttatt tttatgtatt tattatcctt

43261 ccaaacccag tgttgtagga agaatattac atataaatga taacttagga atcttgtcaa

43321 gtttttgttt cttcagcact aagtagctta ttttctaggc aggtcttaat tctaaatgta

43381 acatgcttga aaaaaacact attgaaagga tccgtctctt cagcatagta tttaatatac

43441 atatcaggca atcaccatct caagcatgat tagactcaag tgctgccctc catcgtgtga

43501 gggatgctgg gagactggaa gcttagactt tagctcttta agttggtgca aaagcaattg

43561 cagtttttac tattaataat aataattgcc tagccattgt gcagtacctt gcaagagctc

43621 tttggcaaat gaaagaataa ttcaaagcaa agtaggccag atataaaaca cttccaaatc

43681 acatttcaaa attgttcttt catgtcaact aggtgcatgt ctgctaaggg agtttttgca

43741 aggacaggtg gatagtgtga ggcctttaca ataatcacat caccatctgg tactatctgt

43801 atggcagagc agttctgatg tagttgtagt attatagatc attacagagt atgccagtca

43861 ttccatgatg gaactgtgac tggcagcctt cattagccca aagactgatg caacagattg

43921 ctttgagaaa tagttttgaa gcacatccat gaaacatatg atcctatggg ttccagcatt

43981 gatttctttg aggtatcatt ttacacttca gtttttcctg gtagagtatt tttcagagat

44041 gtctcttctg aggctaaatt aggaatttct gttccttatt caagttccaa gaattgtgcc

44101 tttcctgccc tctgcccaaa ctggaagata gaaaccgtgg gaaaaaagtg tctttacaaa

44161 ctgcactgtt atctaacttg gtttatttag catattgagg aagactttca catccatgga

44221 atcctagttc tattaattct ctccaagcta caacagttgt ttttttgttt gtttttttcc

44281 gagtttctcg gttccgccag caaggcaggg gtgggaggtg ggcacccctg ataccaaggc

44341 tgacaggtag tgagttgatg tggaacttct gtttcctcct gttcagttca ggttctctct

44401 ttctgatact tacccctcct caaataggga aacggagaga ggggaaataa gaagatgagc

44461 cttaataggg tttacttaat tggggtcata aagattctaa aaagtatgca ttcctgcagt

44521 tctgttctag gcactgtaag agctgaccag aaagagagct tctaccctta taaccttcat

44581 ccaactcagt catgcctggg aaggatgttg gtgctaattt tagacacaga gactaagatg

44641 gacaaaggac aagtatcata aagcattttt ggagccagga acagatttaa gttccttgcc

44701 tcactgctgc tcttactcca aaccacacaa ttgtacccaa gtgaattgct gttagaattg

44761 gtgcctttg

SEQ ID NO: 2 below corresponds to the reverse complement of chr14:75500097-75500218 that shows the sequence in the direction of transcription, where Exon 7 of MLH3 is underlined:

5′-aagttcacaa tccttgctca tctagGTGGG AACCTGCTCG

TGCTGGTGGA TCAGCACGCT GCCCATGAGC GTATACGTCT

GGAGCAGCTT ATCATTGgta aggatctgtt tgcagccaga

aa-3′

The protein MLH3 (as half of MutLgamma) works downstream of MSH3 (half of MutSbeta) to cleave DNA near bound MutSbeta. MSH3 has variable expression, and is often absent in tissues that do not exhibit somatic repeat expansion. When ectopic expression of MSH3 is introduced, the repeat expands (A5). In one embodiment, MSH3 expansion can be inhibited by inhibiting the next step in the process, DNA nicking by MutLgamma via MLH3 isoform switching. While MLH3 has not been identified as a major contributor to cancer in humans (A6,A7) and MLH3 knockout mice were healthy and showed no susceptibility to morbid cancer in the first 9 months of life (A8), longer term studies have shown that complete loss of MLH3 throughout life increased the propensity for late onset tumors (A9) which was modified by loss of other tumor suppressor genes (A10).

Oligonucleotides

“Oligonucleotide compounds” of the invention can include oligonucleotides, e.g., Antisense oligonucleotides (ASOs), splice switching oligonucleotides (SSOs), siRNA, shRNA, and the like as well as modified nucleotides discussed herein that are incorporated into the same. ASOs are single stranded nucleotide molecules that are complementary to a target nucleic acid sequence. For example, “target nucleic acid” and “nucleic acid encoding a subunit of the MMR system” encompass DNA encoding a subunit of the MMR system, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA, as well as DNA or RNA sequence described herein that further encompasses noncoding sequence. In one embodiment, the target sequence comprises a nucleic acid sequence encoding a subunit of the MMR system (e.g., MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2), where the nucleic acid sequence includes but is not limited to sense and/or antisense non-coding and/or coding sequences associated with a nucleic acid sequence encoding a subunit of the MMR system.

Hybridization involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleotides which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances. Complementary, as is understood by the skilled artisan, refers to the capacity for precise pairing between two nucleotides. For example, an oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. It is understood in the art that the sequence of an oligonucleotide does not need to be 100% complementary to that of its target nucleic acid to be specifically hybridizable. For example, an oligonucleotide can hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (such as, e.g., a loop structure, mismatch or hairpin structure). Thus, “specifically hybridizable” and “complementary” are used to indicate a sufficient degree of complementarity or precise pairing where stable and specific binding occurs between the oligonucleotide and its target nucleic acid (e.g., the DNA or RNA target).

In one embodiment, the specific hybridization of an oligonucleotide compound with its target nucleic acid interferes with the normal function of the nucleic acid. In one embodiment, an oligonucleotide compound is specifically hybridizable when binding of the oligonucleotide to the target nucleic acid interferes with the normal function of DNA, the normal function of RNA, or the normal function and/or expression of the product encoded by the target nucleic acid, causing a modulation of function and/or activity. In one embodiment, an oligonucleotide compound (e.g., an SSO) can cause an intron to be retained. When an intron is retained, for example, the mRNA is de-stabilized and subsequently degraded. Thus, intron retention mediated by an oligonucleotide compound, such as an SSO, can lower expression of the target gene just like shRNA or siRNA.

The DNA functions to be interfered include, for example, replication and transcription. The RNA functions to be interfered include, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and/or catalytic activity, which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of an encoded product or oligonucleotides. In one embodiment, the modulation is a decrease or loss of the activity of the encoded product. In one embodiment, the modulation is a decrease or loss of expression of the encoded product.

The oligonucleotide compounds described herein comprise about 70%, or about 75%, or about 80%, or about 85%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence complementarity to a target region within the target nucleic acid sequence to which the oligonucleotide compound is targeted. For example, an oligonucleotide in which 18 of 20 nucleotides of the oligonucleotide compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity.

In one embodiment, the oligonucleotides are specific for polynucleotides of a subunit of the MMR system (e.g., MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2), which includes, without limitation, non-coding regions. In one embodiment, the oligonucleotide is an antisense RNA molecule. In one embodiment, the oligonucleotide is an antisense DNA molecule. In one embodiment, an oligonucleotide targets a natural antisense sequence (natural antisense to the coding and non-coding regions) of a subunit of the MMR system (e.g., MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2). In one embodiment, the oligonucleotide is an antisense RNA molecule. In one embodiment, the oligonucleotide is an antisense DNA molecule.

The oligonucleotide compounds discussed herein can also include variants in which a different base is present at one or more of the nucleotide positions in the oligonucleotide compound. For example, if the first nucleotide is an adenine, variants may be produced which contain thymidine, guanosine, cytidine or other natural or non-natural nucleotides at that position. The base substitution can be done at any of the positions of the oligonucleotide. The oligonucleotide compounds can then be tested using methods described herein to determine the oligonucleotide compound's ability to inhibit expression and/or function of a target nucleic acid, such as a subunit of the MMR system (e.g., MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2).

In one embodiment, homology between an oligonucleotide and its target nucleic acid sequence (e.g., the nucleic acid sequence of a subunit of the MMR system such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2) is from about 50% to about 60%. In some embodiments, the homology is from about 60% to about 70%. In some embodiments, the homology is from about 70% to about 80%. In some embodiments, the homology is from about 80% to about 85%. In some embodiments, the homology is from about 85% to about 90%. In some embodiments, the homology is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%. In one embodiment, sequence identity between an oligonucleotide and its target nucleic acid sequence (e.g., the nucleic acid sequence of a subunit of the MMR system such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2) is from about 50% to about 60%. In further embodiments, the homology is from about 60% to about 70%. In further embodiments, the homology is from about 70% to about 80%. In further embodiments, the homology is from about 80% to about 85%. In further embodiments, the homology is from about 85% to about 90%. In further embodiments, the homology is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%. In one embodiment, complementarity between an oligonucleotide and its target nucleic acid sequence (e.g., the nucleic acid sequence of a subunit of the MMR system such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2) is from about 50% to about 60%. In another embodiment, the homology is from about 60% to about 70%. In another embodiment, the homology is from about 70% to about 80%. In another embodiment, the homology is from about 80% to about 85%. In another embodiment, the homology is from about 85% to about 90%. In another embodiment, the homology is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%.

Modifications

According to the invention, oligonucleotide compounds can comprise at least one region where the oligonucleotide is modified in order to exhibit one or more desired properties. The desired properties of the oligonucleotide include, but are not limited, for example, to increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. Modified oligonucleotides can include, for example, synthetic nucleotides having modified base moieties and/or modified sugar moieties (see e.g., described generally by Schcit, Nucleotide Analogs, John Wiley, New York, 1980; Freier & Altmann, (1997) Nucl. Acid. Res., 25(22), 4429-4443, Toulme, J. J., (2001) Nature Biotechnology 19:17-18; Manoharan M., (1999) Biochemica et Biophysica Acta 1489:117-139; Freier S. M., (1997) Nucleic Acid Research, 25:4429-4443, Uhlman, E., (2000) Drug Discovery & Development, 3: 203-213, Herdewin P., (2000) Antisense & Nucleic Acid Drug Dev., 10:297-310); or 2′-O, 3′-C-linked [3.2.0]bicycloarabinonucleosides. Such modified nucleotides include synthetic nucleotides designed to enhance binding properties, e.g., duplex or triplex stability, specificity, or the like.

An oligonucleotide compound, whether DNA, RNA, DNA or RNA with modified nucleotides, DNA or RNA with substituted nucleotides, and the like, can specifically hybridize when binding of the oligonucleotide compound to the target nucleic acid (e.g., a DNA or RNA molecule) interferes with the normal function of the target DNA or RNA. Further modifications can include conjugate groups attached to one of the termini of an oligonucleotide compound or to selected nucleotide positions of an oligonucleotide compound, conjugate group(s) added to various positions on the sugar ring, or conjugate group(s) added to one of the internucleoside linkages. In one embodiment, the interference can cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide compound to non-target nucleic acid sequences under conditions in which specific binding is desired. Conditions in which specific binding are desired include, but are not limited to, physiological conditions in in vivo assays or in therapeutic treatment, or conditions in which the in vitro assays are performed.

ASOs comprise a more general grouping of antisense compounds, which include but are not limited to siRNA, ribozymes, external guide sequence (EGS) oligonucleotides, single- or double-stranded RNA interference (RNAi), and other oligonucleotides that hybridize to at least a portion of the target nucleic acid sequences and modulate its function. The antisense compounds can be single-stranded, double-stranded, circular or hairpin and can comprise structural elements such as mismatches or loops. Antisense compounds are routinely prepared linearly but one of ordinary skill in the art can prepare antisense compounds to be joined or otherwise prepared to be circular and/or branched.

In one embodiment, oligonucleotide compounds directed to a nucleic acid sequence of a subunit of the MMR system (such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2) can comprise one or more modified nucleotides. In one embodiment, oligonucleotide compounds directed to a nucleic acid sequence of a subunit of the MMR system (such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2) can comprise shorter or longer fragment lengths (e.g., 15-, 16-, 17-, 18-, 19-20-, 21-, 22-, 23-, 24-, 25-, 26-, 27-, 28-, 29-, 30-, 31-, 32-, 33-, 34-, 35-, 36-, 37-, 38-, 39-, 40-, 41-, 42-, 43-, 44-, 45-, 46-, 47-, 48-, 49-, or 50-mers). In one embodiment, oligonucleotide compounds directed to a nucleic acid sequence of a subunit of the MMR system (such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2) can comprise modified bonds or internucleotide linkages. Non-limiting examples of modified bonds or internucleotide linkages include phosphorothioate, phosphorodithioate, and the like. In one embodiment, the oligonucleotide compounds can comprise a phosphorus derivative. In one embodiment, the phosphorus derivative (or modified phosphate group) can be attached to the sugar or sugar analog moiety in the modified oligonucleotides of the present invention. Non-limiting examples of a phosphorus derivative (or a modified phosphate group) include a monophosphate, diphosphate, triphosphate, alkylphosphate, alkanephosphate, phosphorothioate and the like. The preparation of the exemplary phosphorus derivatives (or modified phosphate groups), and their incorporation into nucleotides (e.g., those comprising an oligonucleotide compound of the invention), is well-known in the art.

A number of nucleotide modifications have been shown to make the oligonucleotide into which they are incorporated more resistant to nuclease digestion than the native oligodeoxynucleotide. Oligonucleotides that have been modified to enhance their nuclease resistance survive intact for a longer time than unmodified oligonucleotides. As discussed herein, embodiments of the present invention encompass modified oligonucleotides, such as modified ASOs directed to a nucleic acid sequence of a subunit of the MMR system (such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2). Modified oligonucleotides can comprise 2′-O-methyl modified oligoribonucleotides, which render the antisense oligonucleotide resistant to RNase H degradation. In one embodiment, modified oligonucleotides comprise a phosphorothioate backbone. For example, the phosphorothioate backbone increases the stability of an oligonucleotide compound against nucleases and enhances cellular uptake. In some embodiments, oligonucleotide compounds can comprise a full length phosphorodiamidate DNA. In some embodiments, oligonucleotide compounds can comprise one or nucleotides having a 2′O-methyl modification. In some embodiments, oligonucleotide compounds comprise one or more modifications discussed herein. Non-limiting examples of modified backbones include phosphorothioates, phosphinates, phosphorodithioates, phosphoramidates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates (e.g., phosphonates comprising 3′ alkylene phosphonates), short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. In one embodiment, oligonucleotide compounds directed to a nucleic acid sequence of a subunit of the MMR system (such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2) comprise phosphorothioate backbones.

In one embodiment, the region of a modified oligonucleotide compound directed to a nucleic acid sequence of a subunit of the MMR system (such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2) comprises at least one nucleotide modified at the 2′ position of the sugar. In some embodiments, the nucleotide having a modification at the 2′ position of the sugar comprises a 2′-O-alkyl, 2′-O-alkyl-O-alkyl, or 2′-fluoro-modified nucleotide. In some embodiments, RNA modifications include 2′-fluoro, 2′-amino and 2′-O-methyl modifications on the ribose of pyrimidines.

As discussed herein, oligonucleotide compounds can comprise additional modifications such as morpholino phosphorodiamidate DNA, locked nucleic acids (LNA), and ethylene bridged nucleic acids. These modifications can render the oligonucleotide compounds RNase H and nuclease resistant as well as can increase the affinity for the target RNA. In one embodiment, oligonucleotide compositions of the invention have morpholino backbone structures (e.g., as disclosed by Summerton and Weller, in U.S. Pat. No. 5,034,506, which is hereby incorporated by reference in its entirety). Morpholinos, for example, are commercially available through Gene Tools, LLC, Philomath OR; http://www.gene-tools.com/).

For example, the morpholino backbone of oligonucleotide analogues makes them resistant to nucleases and proteases so that they are long-lived in the cell. Some morpholino oligomers can be diluted by cell division and gradually become ineffective after a single dose in rapidly dividing tissues or in growing organisms. In contrast, morpholino splice switching oligonucleotides (SSOs) remain active in post-mitotic tissues such as brain and spinal cord for several months (A2). In one embodiment, the region of a modified oligonucleotide compound directed to a nucleic acid sequence of a subunit of the MMR system (such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2) comprises at least one nucleotide modified with a morpholino subunit.

A number of nucleotide modifications incorporated into an oligonucleotide (e.g., resulting in an oligonucleotide analog), makes the oligonucleotide useful for steric blocking applications, such as exon skipping. For example, negatively charged oligonucleotide analogues, such as oligodeoxynucleotide phosphorothioate (DNA-PS), 2′-O-methylphosphorothioate (OMe-PS), 2′-O-methoxyethyl (MOE), 2′-deoxy-2′-fluoronucleotides (2′-F), locked nucleic acids (LNA; also referred to as bridged nucleic acids (BNA)), ethylene-bridged nucleic acids (ENA), tricycloDNA analogue (TcDNA), and 2′-O-[2-(N-methylcarbamoyl)ethyl]uridine (MCE), as disclosed in Jarver et al. (2014) Nuc. Acid Therap., 24(1):37-47 (incorporated by reference in its entirety), can be used to induce exon skipping:

embedded image

where R can be O or S in the above-listed negatively charged oligonucleotide analogues. In one embodiment, the oligonucleotide compounds disclosed herein, such as for example, nucleic acid molecules set forth in SEQ ID NOS: 3 and 4, comprise one or more substitutions or modifications. In one embodiment, the oligonucleotide compounds disclosed herein, such as for example, nucleic acid molecules set forth in Table 4, comprise one or more substitutions or modifications. In one embodiment, the oligonucleotide compounds are substituted with at least one locked nucleic acid (LNA). In one embodiment, the oligonucleotide compounds are substituted with at least one phosphorothioate (PS). In one embodiment, the oligonucleotide compounds are substituted with at least one 2′-O-methylphosphorothioate (OMe-PS). In one embodiment, the oligonucleotide compounds are substituted with at least one 2′-O-methoxyethyl (MOE). In one embodiment, the oligonucleotide compounds are substituted with at least one 2′-deoxy-2′-fluoronucleotide (2′-F). In one embodiment, the oligonucleotide compounds are substituted with at least one ethylene-bridged nucleic acid (ENA). In one embodiment, the oligonucleotide compounds are substituted with at least one tricycloDNA analogue (TcDNA). In one embodiment, the oligonucleotide compounds are substituted with at least one 2′-O-[2-(N-methylcarbamoyl)ethyl]uridine (MCE). In one embodiment, the oligonucleotide compounds are substituted with at least one oligodeoxynucleotide phosphorothioate (DNA-PS), 2′-O-methylphosphorothioate (OMe-PS), 2′-O-methoxyethyl (MOE), 2′-deoxy-2′-fluoronucleotide (2′-F), locked nucleic acid (LNA), ethylene-bridged nucleic acid (ENA), tricycloDNA analogue (TcDNA), 2′-O-[2-(N-methylcarbamoyl)ethyl]uridine (MCE), or a combination thereof.

Charge-neutral peptide nucleic acids (PNA) and phosphorodiamidate morpholino oligonucleotides (PMO) are further examples of oligonucleotide analogues, as disclosed in Jarver et al. (2014) Nuc. Acid Therap., 24(1):37-47 (incorporated by reference in its entirety), that can be used to induce exon skipping:

embedded image

In one embodiment, the oligonucleotide compounds disclosed herein, such as for example, nucleic acid molecules set forth in SEQ ID NOS: 3 and 4, comprise one or more substitutions or modifications. In one embodiment, the oligonucleotide compounds disclosed herein, such as for example, nucleic acid molecules set forth in Table 4, comprise one or more substitutions or modifications. In one embodiment, the oligonucleotide compounds are substituted with at least one peptide nucleic acid (PNA). In one embodiment, the oligonucleotide compounds are substituted with at least one phosphorothioate (PS). In one embodiment, the oligonucleotide compounds are substituted with at least one peptide nucleic acid (PNA), phosphorothioate (PS), or a combination thereof.

Due to the uncharged backbone of the morpholino subunit, these oligonucleotide analogues can bind their complementary target RNA very tightly (A1). Morpholinos work simply by binding their complementary sequence and excluding binding by proteins or nucleic acids. In one embodiment, binding to a splice donor or acceptor sequence can interfere with recognition of those sequences by the splicing machinery and cause exon skipping. Morpholinos have most often been used for protein knockdown experiments. A morpholino designed to bind the initiating AUG in an mRNA will block translation initiation by ribosomes. An advantage of morpholinos is the predictable way that they work in different species and different tissues since they are not dependent on accessory protein expression such as RISC, dicer, or RNaseH for activity.

In one embodiment, oligonucleotide compounds disclosed herein can bind to a selected target nucleic acid sequence to induce exon skipping. In some embodiments, masking a donor splice site can induce exon skipping. In some embodiments, masking an acceptor splice site can induce exon skipping. In one embodiment, an oligonucleotide compound (e.g., an SSO) can cause an intron to be retained; thus, when an intron is retained, for example, the mRNA is de-stabilized and subsequently degraded, wherein intron retention mediated by an oligonucleotide compound, such as an SSO, can lower expression of the target gene. In one embodiment, the oligonucleotide compound is a modified oligonucleotide directed to a target nucleic acid sequence of a subunit of the MMR system (such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2). In another embodiment, the modified oligonucleotide compound directed to a target nucleic acid sequence of a subunit of the MMR system comprises at least one morpholino subunit. For example, to induce exon skipping in exons of the MLH3 gene transcript, the antisense molecules are selected from the group of SSOs shown in Table 4.

TABLE 4

SSOs for human MLH3 exons 2 through 12.

SEQ ID

NO:
SSO
SEQUENCE

7
hMLH3X2ac
TCTCTGACTGGAAATAATTGCctat

8
hMLH3X2dn
ATTCTCAAGTACAACATCCACAGCC

9
hMLH3X3ac
TGACACCTGTACTGAGACCctaaat

10
hMLH3X3dn
tctctgccacccttacCTCTGTTAT

11
hMLH3X4ac
CATCCACAGTATctagggcaaaagg

12
hMLH3X4dn
ccacCTCTGGATAACGGGCAAATAC

13
hMLH3X5ac
CAGCAACctagaaagactcagcaaa

14
hMLH3X5dn
gttaatcttttacCTGCATTGAATG

15
hMLH3X6ac
TGCTGGAGAACctgtcagacattca

16
hMLH3X6dn
actccattcttacCTGCCTCGCCAT

17
hMLH3X7ac
GTTCCCACctagatgagcaaggatt

18
hMLH3X7dn
tgcaaacagatccttacCAATGATA

19
hMLH3X8ac
GAATctattggcagaaagatgaatg

20
hMLH3X8dn
acattctcatggtggtactgacCAT

21
hMLH3X9ac
GTAACACctaaagagataacctcaa

22
hMLH3X9dn
taacatctgcagctgtgtcttacCT

23
hMLH3X10ac
Cctgcaaagcaaaaggaaaatcggc

24
hMLH3X10dn
tacCTCCAGTTGTTCTCGGATAAAT

25
hMLH3X11ac
CGGTGGTCTGGAGTAGctaatgcat

26
hMLH3X11dn
ctatgttgaagggcttacCATGGCA

27
hMLH3X12ac
AATGGCCCctaaatgaaagacagaa

28
hMLH3X12dn
tgctcctgttagtcattaatgtacC

In one embodiment, an oligonucleotide compound directed to a nucleic acid sequence of a subunit of the MMR system comprises SEQ ID NO: 3. In one embodiment, an oligonucleotide compound directed to a nucleic acid sequence of a subunit of the MMR system comprises SEQ ID NO: 4. In one embodiment, an oligonucleotide compound directed to a nucleic acid sequence of a subunit of the MMR system comprises a nucleic acid sequence depicted in Table 4. In one embodiment, an oligonucleotide compound is directed to a nucleic acid sequence (or target complementary nucleic acid sequence) corresponding to a region of interest for any one of the exons 1-16 described herein for GenBank Accession No. NG_007110.2, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007110.2. In one embodiment, an oligonucleotide compound is directed to a nucleic acid sequence (or target complementary nucleic acid sequence) corresponding to a region of interest for any one of the exons 1-24 described herein for GenBank Accession No. NG_016607.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_016607.1. In one embodiment, an oligonucleotide compound is directed to a nucleic acid sequence (or target complementary nucleic acid sequence) corresponding to a region of interest for any one of the exons 1-10 described herein for GenBank Accession No. NG_007111.1 or SEQ ID NO: 33, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007111.1 or SEQ ID NO: 33. In one embodiment, an oligonucleotide compound is directed to a nucleic acid sequence (or target complementary nucleic acid sequence) corresponding to a region of interest for any one of the exons 1-19 described herein for GenBank Accession No. NG_007109.2, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007109.2. In one embodiment, an oligonucleotide compound is directed to a nucleic acid sequence (or target complementary nucleic acid sequence) corresponding to a region of interest for any one of the exons 1-13 described herein for GenBank Accession No. NG_008648.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008648.1. In one embodiment, an oligonucleotide compound is directed to a nucleic acid sequence (or target complementary nucleic acid sequence) corresponding to a region of interest for any one of the exons 1-13 described herein for GenBank Accession No. NG_008649.1 or SEQ ID NO: 1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008649.1 or SEQ ID NO: 1. In one embodiment, an oligonucleotide compound is directed to a nucleic acid sequence (or target complementary nucleic acid sequence) corresponding to a region of interest for any one of the exons 1-15 described herein for GenBank Accession No. NG_008466.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008466.1.

In some embodiments, an oligonucleotide compound directed to a nucleic acid sequence of a subunit of the MMR system is a modified oligonucleotide. According to the invention, a combination or “cocktail” of two or more oligonucleotide compounds can be provided that bind to a selected target nucleic acid (such as a subunit of the MMR system) in order to induce exon skipping. For example, to induce exon skipping in exons of a subunit of the MMR system gene transcript (such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2), the oligonucleotide compounds in the cocktail are selected from the group of SSOs shown in Table 4. In one embodiment, the cocktail comprises at least 2 SSOs selected from Table 4. In one embodiment, the cocktail comprises SSOs comprising SEQ ID NO: 3 and SEQ ID NO: 4. In one embodiment, the cocktail comprises SSOs comprising SEQ ID NO: 3 and an SSO selected from the group of SSOs shown in Table 4. In one embodiment, the cocktail comprises SSOs comprising SEQ ID NO: 4 and an SSO selected from the group of SSOs shown in Table 4. In one embodiment, the cocktail comprises SSOs directed to nucleic acid sequences (or target complementary nucleic acid sequences) corresponding to a region of interest for any one of the exons 1-16 described herein for GenBank Accession No. NG_007110.2, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007110.2. In one embodiment, the cocktail comprises SSOs directed to nucleic acid sequences (or target complementary nucleic acid sequences) corresponding to a region of interest for any one of the exons 1-24 described herein for GenBank Accession No. NG_016607.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_016607.1. In one embodiment, the cocktail comprises SSOs directed to nucleic acid sequences (or target complementary nucleic acid sequences) corresponding to a region of interest for any one of the exons 1-10 described herein for GenBank Accession No. NG_007111.1 or SEQ ID NO: 33, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007111.1 or SEQ ID NO: 33. In one embodiment, the cocktail comprises SSOs directed to nucleic acid sequences (or target complementary nucleic acid sequences) corresponding to a region of interest for any one of the exons 1-19 described herein for GenBank Accession No. NG_007109.2, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007109.2. In one embodiment, the cocktail comprises SSOs directed to nucleic acid sequences (or target complementary nucleic acid sequences) corresponding to a region of interest for any one of the exons 1-13 described herein for GenBank Accession No. NG_008648.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008648.1. In one embodiment, the cocktail comprises SSOs directed to nucleic acid sequences (or target complementary nucleic acid sequences) corresponding to a region of interest for any one of the exons 1-13 described herein for GenBank Accession No. NG_008649.1 or SEQ ID NO: 1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008649.1 or SEQ ID NO: 1. In one embodiment, the cocktail comprises SSOs directed to nucleic acid sequences (or target complementary nucleic acid sequences) corresponding to a region of interest for any one of the exons 1-15 described herein for GenBank Accession No. NG_008466.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008466.1

Target site(s) useful in the practice of the invention are those involved in mRNA splicing (such as splice donor sites, splice acceptor sites or exonic splicing enhancer elements). Splicing branch points and exon recognition sequences or splice enhancers are also potential target sites for modulation of mRNA splicing. In one embodiment, oligonucleotide compounds disclosed herein can bind to a selected target nucleic acid sequence to induce exon skipping. In some embodiments, masking a donor splice site can induce exon skipping. In some embodiments, masking an acceptor splice site can induce exon skipping. For example, owing to the nature of morpholino oligomers, one of ordinary skill in the art can identify sequences that will reliably bind splice junctions. As described in the examples herein, the efficacy of targeted morpholino SSOs can be quickly ascertained in tissue culture.

Another modification of the oligonucleotide compounds disclosed herein involves chemically linking one or more moieties or conjugates to the oligonucleotide, which enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Non-limiting examples of moieties and conjugates include lipid moieties (such as a cholesterol moiety, a cholesteryl moiety, a thiocholesterol moeity), intercalators, reporter molecules, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety, a phospholipid, aliphatic chains (such as dodecandiol or undecyl residues), polyamine chains, polyamide chains, polyethylene glycol chains, polyether chains, cholic acid, and adamantane acetic acid. Examples of conjugate groups include, but are not limited to, cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.

Oligonucleotide compounds comprising lipophilic moieties, and methods for preparing such are known in the art, for example, as described in U.S. Pat. Nos. 5,138,045, 5,218,105 and 5,459,255, each of which is incorporated by reference in its entirety.

Representative United States patents that teach the preparation of oligonucleotide compound conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241; 5,391,723; 5,416,203; 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is hereby incorporated by reference in its entirety. Representative conjugate groups are disclosed in U.S. Pat. Nos. 5,578,718; 6,153,737; 6,287,860; and 6,783,931, each of which are incorporated by reference in its entirety.

The oligonucleotide compounds can be conveniently and routinely made through the established technique of solid phase synthesis. Equipment useful for such syntheses can be obtained through several commercial vendors, including Applied Biosystems (Foster City, Calif.). Synthesis of the oligonucleotide compounds is well understood by one of ordinary skill in the art. It is also well known in the art to use similar techniques to prepare other oligonucleotides such as the phosphorothioates and alkylated derivatives. It is also well known to use similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products such as biotin, fluorescein, acridine or psoralen-modified amidites and/or CPG (available from Glen Research, Sterling Va.) to synthesize fluorescently labeled, biotinylated or other modified oligonucleotides such as cholesterol-modified oligonucleotides. Morpholinos, for example, are commercially available through Gene Tools, LLC, Philomath OR; http://www.gene-tools.com/). For example, the oligonucleotide compounds of the invention (such as ASOs and SSOs) are synthesized in vitro and do not include antisense compositions of biological origin, or genetic vector constructs designed to direct the in vivo synthesis of oligonucleotide compounds.

In one embodiment, the oligonucleotide compounds (e.g., modified oligonucleotide compounds) bind to coding and/or non-coding regions of a target nucleic acid sequence of a subunit of the MMR system (such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2), and modulate the expression and/or function of the target molecule. In one embodiment, the oligonucleotide compounds (e.g., modified oligonucleotide compounds) bind to a natural antisense target nucleic acid sequence of a subunit of the MMR system (such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2), and modulate the expression and/or function of the target molecule. In one embodiment, the oligonucleotide compounds bind to a sense target nucleic acid sequence of a subunit of the MMR system (such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2), and modulate the expression and/or function of the target molecule.

Embodiments of the present invention are directed to oligonucleotide compounds that hybridize with a complementary sequence of the human MSH2 gene and/or mRNA. For example, mRNA may be pre-mRNA. The human MSH2 gene comprises SEQ ID NO: 35. Embodiments of the present invention can be directed to modifying MSH2 gene expression. For example, embodiments of the present invention may be directed at skipping MSH2 exons. Embodiments of the present invention encompass splice switching oligonucleotides (SSOs). In one embodiment, the oligonucleotide compound comprises at least one modification described herein. Embodiments of the present invention encompass morpholino oligonucleotides complementary to the target nucleic acid sequence of MSH2.

Embodiments of the present invention are directed to oligonucleotide compounds that hybridize with a complementary sequence of the human MSH3 gene and/or mRNA. For example, mRNA may be pre-mRNA. The human MSH3 gene comprises SEQ ID NO: 36. Embodiments of the present invention can be directed to modifying MSH3 gene expression. For example, embodiments of the present invention may be directed at skipping MSH3 exons. Embodiments of the present invention encompass splice switching oligonucleotides (SSOs). In one embodiment, the oligonucleotide compound comprises at least one modification described herein. Embodiments of the present invention encompass morpholino oligonucleotides complementary to the target nucleic acid sequence of MSH3.

Embodiments of the present invention are directed to oligonucleotide compounds that hybridize with a complementary sequence of the human MSH6 gene and/or mRNA. For example, mRNA may be pre-mRNA. The human MSH6 gene comprises SEQ ID NO: 37. Embodiments of the present invention can be directed to modifying MSH6 gene expression. For example, embodiments of the present invention may be directed at skipping MSH6 exons. Embodiments of the present invention encompass splice switching oligonucleotides (SSOs). In one embodiment, the oligonucleotide compound comprises at least one modification described herein. Embodiments of the present invention encompass morpholino oligonucleotides complementary to the target nucleic acid sequence of MSH6.

Embodiments of the present invention are directed to oligonucleotide compounds that hybridize with a complementary sequence of the human MLH1 gene and/or mRNA. For example, mRNA may be pre-mRNA. The human MLH1 gene comprises SEQ ID NO: 38. Embodiments of the present invention can be directed to modifying MLH1 gene expression. For example, embodiments of the present invention may be directed at skipping MLH1 exons. Embodiments of the present invention encompass splice switching oligonucleotides (SSOs). In one embodiment, the oligonucleotide compound comprises at least one modification described herein. Embodiments of the present invention encompass morpholino oligonucleotides complementary to the target nucleic acid sequence of MLH1.

Embodiments of the present invention are directed to oligonucleotide compounds that hybridize with a complementary sequence of the human PMS1 gene and/or mRNA. For example, mRNA may be pre-mRNA. The human PMS1 gene comprises SEQ ID NO: 39. Embodiments of the present invention can be directed to modifying PMS1 gene expression. For example, embodiments of the present invention may be directed at skipping PMS1 exons. Embodiments of the present invention encompass splice switching oligonucleotides (SSOs). In one embodiment, the oligonucleotide compound comprises at least one modification described herein. Embodiments of the present invention encompass morpholino oligonucleotides complementary to the target nucleic acid sequence of PMS1.

Embodiments of the present invention are directed to oligonucleotide compounds that hybridize with a complementary sequence of the human PMS2 gene and/or mRNA. For example, mRNA may be pre-mRNA. The human PMS2 gene comprises SEQ ID NO: 40. Embodiments of the present invention can be directed to modifying PMS2 gene expression. For example, embodiments of the present invention may be directed at skipping PMS2 exons. Embodiments of the present invention encompass splice switching oligonucleotides (SSOs). In one embodiment, the oligonucleotide compound comprises at least one modification described herein. Embodiments of the present invention encompass morpholino oligonucleotides complementary to the target nucleic acid sequence of PMS2.

Embodiments of the present invention encompass oligonucleotide compounds that hybridize with a complementary sequence of the human MLH3 gene and/or mRNA. For example, mRNA may be pre-mRNA. The human MLH3 gene comprises SEQ ID NO: 1. Embodiments of the present invention are directed to modifying MLH3 gene expression. For example, embodiments of the present invention may be directed at skipping of MLH3 exon 7 (SEQ ID NO: 2). Embodiments of the present invention encompass splice switching oligonucleotides (SSOs). Embodiments of the present invention include oligonucleotide compounds comprising SEQ ID NO: 3 or SEQ ID NO: 4. In one embodiment, the oligonucleotide compound comprises at least one modification described herein. Embodiments of the present invention encompass morpholino oligonucleotides complementary to the target nucleic acid sequence of MLH3.

Target nucleic acid sequences of about 5-100 nucleotides in length, comprising a stretch of at least five (5) consecutive are suitable for targeting. Target nucleic acid sequences can include DNA or RNA sequences that comprise at least 5 consecutive nucleotides from the 5′-terminus of the gene encoding a subunit of the MMR system (such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2). Target nucleic acid sequences can include DNA or RNA sequences that comprise at least 5 consecutive nucleotides from the 3′-terminus of the gene encoding a subunit of the MMR system (such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2).

In one embodiment, the oligonucleotide compound binds to an antisense strand of a particular target nucleic acid sequence (for example, a subunit of the MMR system (such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2)). The target nucleic acid sequences include coding as well as non-coding regions. Generally, the oligonucleotide compound can be from about 10 nucleotides in length up to about 50 nucleotides in length. In one embodiment, the oligonucleotide compounds of the invention are 10 to 50 nucleotides in length. In one embodiment, the oligonucleotide compounds are at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the oligonucleotides are 15 nucleotides in length. In some embodiments, the oligonucleotides are 20 nucleotides in length. In some embodiments, the oligonucleotides are 25 nucleotides in length. In some embodiments, the oligonucleotides are 20 nucleotides in length. In some embodiments, the oligonucleotides are 30 nucleotides in length.

Kits, Diagnostics, and Therapeutics.

The oligonucleotide compounds of the present invention can be utilized for diagnostics, therapeutics, and prophylaxis, and as components of kits. For example, the specificity and sensitivity of antisense oligonucleotides can be harnessed for therapeutic uses. Oligonucleotide compounds (such as antisense oligonucleotides disclosed herein) can be employed as therapeutic moieties in the treatment of disease states in subjects, such as human subjects. For example, oligonucleotide compounds can be useful therapeutics utilized in treatment regimens for treatment of cells, tissues and animals, especially humans.

Transfer of an exogenous nucleic acid into a host cell or organism, such as an oligonucleotide compound of the invention, can be assessed by directly detecting the presence of the nucleic acid in the cell or organism. Detection can be achieved using several methods well known and practiced in the art. For example, the presence of the exogenous nucleic acid can be detected by Southern blot or by a polymerase chain reaction (PCR) technique using primers that specifically amplify nucleotide sequences associated with the nucleic acid. Expression of the exogenous nucleic acids can also be measured using conventional methods including gene expression analysis. For instance, mRNA produced from an exogenous nucleic acid (or its absence thereof) can be detected and quantified using a Northern blot and reverse transcription PCR (RT-PCR).

Kits. For use in kits and diagnostics and in various biological systems, the oligonucleotide compounds of the present invention, either alone or in combination with other compounds or therapeutics, are useful as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues, such as a subunit of the MMR system (e.g., MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2).

The invention also provides kits for treatment of a subject with a genetic disease, wherein the kit comprises at least an oligonucleotide compound, packaged in a suitable container, together with instructions for its use. In one embodiment, the invention provides for a kit for the treatment of a DNA Repeat Expansion Disease (DRED), the kit comprising an oligonucleotide compound discussed herein. In one embodiment, the invention provides for a kit for the treatment of a DNA Repeat Expansion Disease (DRED), the kit comprising at least two oligonucleotide compounds discussed herein. In one embodiment, the DRED is any one listed in Table 1. In one embodiment, the oligonucleotide compound comprises SEQ ID NO: 3 or SEQ ID NO: 4. In one embodiment, the oligonucleotide compound comprises a nucleic acid sequence depicted in Table 4. In one embodiment, the oligonucleotide compound comprises at least one modification described herein. In some embodiments, the kits will contain at least one oligonucleotide compound (e.g., an ASO or SSO), such as shown in Table 4, SEQ ID NO: 3 or SEQ ID NO: 4, or a cocktail of antisense molecules comprising a combination of SEQ ID NO: 3, SEQ ID NO:4, or a nucleic acid sequence depicted in Table 4. The kits can also comprise any one, or a combination thereof, of the following: an oligonucleotide compound that is directed to a nucleic acid sequence (or target complementary nucleic acid sequence) corresponding to a region of interest for any one of the exons 1-16 described herein for GenBank Accession No. NG_007110.2, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007110.2; an oligonucleotide compound that is directed to a nucleic acid sequence (or target complementary nucleic acid sequence) corresponding to a region of interest for any one of the exons 1-24 described herein for GenBank Accession No. NG_016607.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_016607.1; an oligonucleotide compound that is directed to a nucleic acid sequence (or target complementary nucleic acid sequence) corresponding to a region of interest for any one of the exons 1-10 described herein for GenBank Accession No. NG_007111.1 or SEQ ID NO: 33, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007111.1 or SEQ ID NO: 33; an oligonucleotide compound that is directed to a nucleic acid sequence (or target complementary nucleic acid sequence) corresponding to a region of interest for any one of the exons 1-19 described herein for GenBank Accession No. NG_007109.2, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007109.2; an oligonucleotide compound that is directed to a nucleic acid sequence (or target complementary nucleic acid sequence) corresponding to a region of interest for any one of the exons 1-13 described herein for GenBank Accession No. NG_008648.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008648.1; an oligonucleotide compound that is directed to a nucleic acid sequence (or target complementary nucleic acid sequence) corresponding to a region of interest for any one of the exons 1-13 described herein for GenBank Accession No. NG_008649.1 or SEQ ID NO: 1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008649.1 or SEQ ID NO: 1; or an oligonucleotide compound that is directed to a nucleic acid sequence (or target complementary nucleic acid sequence) corresponding to a region of interest for any one of the exons 1-15 described herein for GenBank Accession No. NG_008466.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008466.1. The kits may also contain peripheral reagents such as buffers, stabilizers, and the like.

The invention provides kits for monitoring the efficacy of treatment in a subject with a genetic disease, wherein the kit comprises at least one primer, packaged in a suitable container, together with instructions for its use. In one embodiment, the kit comprises at least two primers. In one embodiment, the genetic disease is a DNA repeat expansion disease (DRED) listed in Table 1. In one embodiment, the status of a subunit of the MMR system (e.g., MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2) can be monitored. In one embodiment, the primer comprises MLH3 L3324 (TCCTTTCCTTCCGAGAGCTC, SEQ ID NO: 5). In one embodiment, the primer comprises MLH3X7L3138 (GCATTTCGATGTAGCCCTGG, SEQ ID NO: 29). In one embodiment, the primer comprises MLH3X7L3449 (TTGCCCGTTATCCAGAGGTT, SEQ ID NO: 30). In one embodiment, the primer comprises MLH3 R3757 (TTTTCCGACCAGAGCCTTGT, SEQ ID NO: 6). In one embodiment, the primer comprises MLH3X7R3862 (CAAGGCCCAGATCTTCCAGA, SEQ ID NO: 31). In one embodiment, the primer comprises MLH3X7R4013 (AGCTCCAGTTGTTCTCGGAT, SEQ ID NO: 32). The kits may also contain peripheral reagents such as buffers, stabilizers, and the like.

The invention provides kits for monitoring the progression of a DNA repeat expansion disease (DRED), wherein the kit comprises at least one primer, packaged in a suitable container, together with instructions for its use. In one embodiment, the kit comprises at least two primers. In one embodiment, the DRED is selected from the list in Table 1. In one embodiment, the DRED is Fredreich Ataxia. In one embodiment, the status of a subunit of the MMR system (e.g., MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2) can be monitored, indicative of the progression of a repeat expansion. In one embodiment, the MMR subunit is MLH3. In one embodiment, the primer comprises MLH3 L3324 (TCCTTTCCTTCCGAGAGCTC, SEQ ID NO: 5). In one embodiment, the primer comprises MLH3X7L3138 (GCATTTCGATGTAGCCCTGG, SEQ ID NO: 29). In one embodiment, the primer comprises MLH3X7L3449 (TTGCCCGTTATCCAGAGGTT, SEQ ID NO: 30). In one embodiment, the primer comprises MLH3 R3757 (TTTTCCGACCAGAGCCTTGT, SEQ ID NO: 6). In one embodiment, the primer comprises MLH3X7R3862 (CAAGGCCCAGATCTTCCAGA, SEQ ID NO: 31). In one embodiment, the primer comprises MLH3X7R4013 (AGCTCCAGTTGTTCTCGGAT, SEQ ID NO: 32). The kits may also contain peripheral reagents such as buffers, stabilizers, and the like.

Treatments and Therapy for Diseases. As used herein and as is well understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminution of extent of disease, a stabilized (i.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also refer to prolonging survival as compared to expected survival if not receiving treatment.

The term “in need thereof” refers to the need for symptomatic or asymptomatic relief from a condition such as, for example, a DRED, a cancer, a neurodegenerative disease, or a combination thereof. The subject in need thereof may or may not be undergoing treatment for conditions related to, for example, a DRED, a cancer, a neurodegenerative disease, or a combination thereof.

European patent application 13170245.8 claims an antisense oligonucleotide directed against exon 43 of the dystrophin pre-mRNA, which facilitates the exclusion of exon 43 from the final mRNA. U.S. Pat. No. 8,361,979 claims antisense oligonucleotides that may be used for the treatment of Duchenne Muscular Dystrophy. U.S. Pat. No. 8,455,634 claims an antisense molecule capable of binding to a selected target site to induce exon skipping in the dystrophin gene. U.S. Application Publication No. US 2014/0039037 discloses claims directed to antisense oligonucleotides that induce skipping of exonic sequences that comprises the trinucleotide repeat expansion. These disclosures use antisense oligonucleotides to target the diseased gene itself, rather than disease modifying genes, such as a subunit of the MMR system (e.g., MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2). A drawback of targeting the diseased gene itself is that the proposed therapeutic has limited effectiveness for only a single disorder. For many of the expansion disorders, such as FXS, DM1, DM2, and FRDA the repeat is in an intron, or in the first or last exon, which cannot be skipped. Presently, there is no cure for any of the DNA repeat expansion disorders. Furthermore, the treatment options for these devastating disorders are generally inadequate. In particular, there remains an urgent unmet need for new therapeutic compositions that prevent the unwanted, progressive trinucleotide repeat expansion associated with such disorders. The object of this present invention is to address this unmet need.

The present disclosure relates generally to small molecule therapeutics (e.g., oligonucleotide compounds, naked or modified) useful for the treatment of DNA repeat expansion diseases (DREDs). In one embodiment, an oligonucleotide compound (e.g., an antisense oligonucleotide) is administered to a subject to prevent or treat diseases or disorders associated with DNA repeat expansion. In one embodiment, an oligonucleotide compound is directed to a target nucleic acid sequence of a subunit of the MMR system (e.g., MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2). In one embodiment, an effective amount of the oligonucleotide compound is administered to the subject. In some embodiments, the oligonucleotide compound is a modified oligonucleotide that is nuclease-resistant. In some embodiments, the oligonucleotide compound comprises a pharmaceutical composition administered to a subject in a pharmaceutically acceptable carrier. In some embodiments, the oligonucleotide compound (e.g., an antisense oligonucleotide that directs exon skipping) can serve as a therapeutic method for the treatment of various DREDs.

Embodiments of the invention may be used to treat human DRED. In particular, embodiments of the present invention may be used to treat diseases associated with expanded DNA repeats. For example, expanded DNA repeat disorders may include trinucleotide repeat disorders. Examples of diseases which may benefit from embodiments of the present invention may include Friedreich ataxia, ALS, Huntington's disease, Fragile X syndrome, Myotonic dystrophy Types I and II, Spino Cerebellar Ataxias (SCAs). SCAs may include SCA1, SCA2, SCA3, SCA6, SCA7, SCA8, SCA10 and SCA17. Additional examples of diseases, which may benefit from embodiments of the present invention may include those disorders listed in Table 1. In one embodiment, the DRED is Duchenne Muscular Dystrophy, Fredreich Ataxia, or Huntington's disease. In some embodiments, the DRED is a disease selected from Table 1. Embodiments of the invention may slow the rate of or inhibit repeat expansion. Embodiments of the present invention may slow the rate of or inhibit the progression of repeat expansion disorders. Embodiments of the invention may slow the rate so as to inhibit the progression from an asymptomatic size to a disease causing size, thus preventing onset of an expansion disease.

For therapeutics, a subject, for example, a human, suspected of having a disease or disorder (such as a DRED), which can be treated by modulating the expression of a nucleic acid sequence of a subunit of the MMR system (e.g., MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2) is treated by administering an oligonucleotide compound (such as an ASO) in accordance with this invention. In one embodiment, a pharmaceutical composition comprising a an oligonucleotide compound disclosed herein, such as a nuclease-resistant oligonucleotide 15 to 30 nucleotide bases in length targeted to a complementary nucleic acid sequence of a gene or gene product encoding a MutS or MutL subunit, is administered to a subject. In one embodiment, the oligonucleotide hybridizes with and decreases the expression of the human MutS or MutL subunit (such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2) by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or 100%, as compared to a normal control. In one embodiment, the oligonucleotide compound comprises at least one modification. In one embodiment, the oligonucleotide is 17 to 28 nucleotide bases in length. In one embodiment, the oligonucleotide is 18 to 25 nucleotide bases in length. In one embodiment, the oligonucleotide is 19 to 23 nucleotide bases in length.

In one embodiment, a pharmaceutical composition that is an oligonucleotide compound comprising an oligonucleotide complex can be administered. In one embodiment, the complex comprises a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide comprises a sequence complementary to an acceptor region of an exon of a gene encoding a MutS or MutL subunit, and wherein the nucleic acid sequence of the first oligonucleotide comprises a nuclease-resistant modification, and wherein the second oligonucleotide comprises a sequence complementary to a donor region of an exon of a gene encoding a MutS or MutL subunit, and wherein the nucleic acid sequence of the second oligonucleotide comprises a nuclease-resistant modification. In another embodiment, the complex comprises a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide comprises a sequence complementary to an acceptor region of an exon of a gene encoding a MutS or MutL subunit, and wherein the nucleic acid sequence of the first oligonucleotide comprises a nuclease-resistant modification, and wherein the second oligonucleotide comprises a sequence complementary to a donor region of an exon of a gene encoding a MutS or MutL subunit. In a further embodiment, the complex comprises a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide comprises a sequence complementary to an acceptor region of an exon of a gene encoding a MutS or MutL subunit, and wherein the second oligonucleotide comprises a sequence complementary to a donor region of an exon of a gene encoding a MutS or MutL subunit, and wherein the nucleic acid sequence of the second oligonucleotide comprises a nuclease-resistant modification. In one embodiment, the human MutS or MutL subunit comprises MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2. In one embodiment, the methods comprise the step of administering to the subject in need of treatment, a therapeutically effective amount of the oligonucleotide complex. The oligonucleotide complex of the present invention effectively modulates the activity of a MutS or MutL subunit, or modulates the expression of a MutS or MutL subunit. In one embodiment, the activity or expression of a MutS or MutL subunit in an subject is decreased by about 10% as compared to a control. In other embodiments, the activity or expression of a MutS or MutL subunit in a subject is decreased by about 20%. In yet other embodiments, the activity or expression of a MutS or MutL subunit in a subject is decreased by about 30%. In some embodiments, the activity or expression of a MutS or MutL subunit in a subject is decreased by about 50%. In some embodiments, the activity or expression of a MutS or MutL subunit in a subject is decreased by about 60%. In some embodiments, the activity or expression of a MutS or MutL subunit in a subject is decreased by about 70%. The oligonucleotide compounds disclosed herein (e.g., ASOs, SSOs, or oligonucleotide complexes) can modulate mRNA expression of a MutS or MutL subunit (such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2) by at least 10%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 98%, by at least 99%, or by 100% as compared to a control. In one embodiment, the oligonucleotide comprises SEQ ID NO: 3. In one embodiment, the oligonucleotide comprises SEQ ID NO: 4. In one embodiment, the oligonucleotide comprises a nucleic acid sequence depicted in Table 4. In one embodiment, the oligonucleotide comprises an oligonucleotide compound that is directed to a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-16 described herein for GenBank Accession No. NG_007110.2, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007110.2. In one embodiment, the oligonucleotide comprises an oligonucleotide compound that is directed to a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-24 described herein for GenBank Accession No. NG_016607.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_016607.1. In one embodiment, the oligonucleotide comprises an oligonucleotide compound that is directed to a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-10 described herein for GenBank Accession No. NG_007111.1 or SEQ ID NO: 33, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007111.1 or SEQ ID NO: 33. In one embodiment, the oligonucleotide comprises an oligonucleotide compound that is directed to a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-19 described herein for GenBank Accession No. NG_007109.2, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_007109.2. In one embodiment, the oligonucleotide comprises an oligonucleotide compound that is directed to a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-13 described herein for GenBank Accession No. NG_008648.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008648.1. In one embodiment, the oligonucleotide comprises an oligonucleotide compound that is directed to a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-13 described herein for GenBank Accession No. NG_008649.1 or SEQ ID NO: 1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008649.1 or SEQ ID NO: 1. In one embodiment, the oligonucleotide comprises an oligonucleotide compound that is directed to a nucleic acid sequence corresponding to a region of interest for any one of the exons 1-15 described herein for GenBank Accession No. NG_008466.1, or to an intron-exon junction, or to an exon-intron junction listed with GenBank Accession No. NG_008466.1.

For example, the decrease or reduction of the expression of a MutS or MutL subunit (such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2) can be measured in serum, blood, adipose tissue, cerebral spinal fluid, liver, or any other body fluid, tissue or organ of the subject. In one embodiment, the cells contained within the above-listed fluids, tissues or organs that are being analyzed contain a nucleic acid molecule encoding a MutS or MutL subunit (such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2).

Formulations and Administration

The oligonucleotide compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the oligonucleotide compounds and methods of the invention may also be useful prophylactically.

An “effective amount”, “sufficient amount” or “therapeutically effective amount” as used herein is an amount of a composition that is sufficient to effect beneficial or desired results, including clinical results. As such, the effective amount may be sufficient, for example, to reduce or ameliorate the severity and/or duration of an affliction or condition, or one or more symptoms thereof, prevent the advancement of conditions related to an affliction or condition, prevent the recurrence, development, or onset of one or more symptoms associated with an affliction or condition, or enhance or otherwise improve the prophylactic or therapeutic effect(s) of another therapy. An effective amount also includes the amount of the composition (e.g., the oligonucleotide compounds discussed herein) that avoids or substantially attenuates undesirable side effects.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Non-limiting examples of such pharmaceutical carriers include liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical carriers may also be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents may be used. Other examples of suitable pharmaceutical carriers are described in Remington: The Science and Practice of Pharmacy, 21^stEdition (University of the Sciences in Philadelphia, ed., Lippincott Williams & Wilkins 2005); and Handbook of Pharmaceutical Excipients, 7^thEdition (Raymond Rowe et al., ed., Pharmaceutical Press 2012); each hereby incorporated by reference in its entirety.

The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. A pharmaceutically acceptable carrier can comprise any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent that is compatible with the active compound can be used. Supplementary active compounds can also be incorporated into the compositions. For oligonucleotide compounds, examples of pharmaceutically acceptable salts and their uses are further described in U.S. Pat. No. 6,287,860, which is hereby incorporated by reference in its entirety.

In one embodiment, modulation of a subunit of the MMR system (e.g., MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2) can be effected by administering one or more oligonucleotide compounds (e.g., ASOs or SSOs, naked or modified) to a subject in need thereof. In one embodiment, the prevention, amelioration, or treatment of a DRED that is related to abnormal expression, function, activity of a subunit of the MMR system (e.g., MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2) as compared to a normal control can also be effected by administering one or more oligonucleotide compounds (e.g., ASOs or SSOs, naked or modified) to a subject in need thereof. Embodiments of the present invention can be administered alone, or can be administered in a therapeutic cocktail or as a pharmaceutical composition. For example, a pharmaceutical composition can comprise embodiments of the present invention, and a saline solution that includes a phosphate buffer. Embodiments of the present invention can be administered using the means and doses described herein. Embodiments of the present invention can be administered in combination with a suitable carrier. In one embodiment, the oligonucleotide compounds of the invention (e.g., ASOs and SSOs) encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to a subject, provides (directly or indirectly) the biologically active metabolite or residue thereof.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EM™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it can be useful to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, examples of useful preparation methods are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The oligonucleotide compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Non-limiting examples of United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,165; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.

For treating tissues in the central nervous system, administration can be made by, e.g., injection or infusion into the cerebrospinal fluid. Administration of antisense RNA into cerebrospinal fluid is described, e.g., in U.S. Pat. No. 7,622,455, which is incorporated by reference in its entirety. When it is intended that an oligonucleotide compound (e.g., an ASO or SSO) will be administered to cells of the central nervous system, administration can be with one or more agents capable of promoting penetration of the oligonucleotide compound across the blood-brain barrier. Injection can be made, e.g., in the entorhinal cortex or hippocampus. See also U.S. Pat. Nos. 6,632,427 and 6,756,523 for additional disclosures relating to direct delivery to the brain, each patent which is incorporated by reference in its entirety. For treating cardiac tissues, administration can be made by, e.g., injection or infusion into the bloodstream. The injection can be administered by the following routes: intraperitoneal injection, subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, intra-arterial injection, or a combination thereof. In one embodiment, administration into the bloodstream is useful to treat the heart, which is a major affected target in Friedreich ataxia.

Formulations useful for topical administration include those in which the oligonucleotide compounds of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Exemplary lipids and liposomes include neutral (e.g. diolcoyl-phosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol (DMPG)) and cationic (e.g. diolcoyltetramethyl-aminopropyl (DOTAP), and diolcoyl-phosphatidyl ethanolamine (DOTMA)). For topical or other administration, oligonucleotide compounds of the invention can be encapsulated within liposomes or can form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotide compounds can be complexed to lipids, in particular to cationic lipids. Exemplary fatty acids and esters, pharmaceutically acceptable salts thereof, and their uses are further described in U.S. Pat. No. 6,287,860, which is hereby incorporated by reference in its entirety.

The formulation of therapeutic compositions and their subsequent administration (dosing) is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC₅₀s found to be effective in in vitro and in vivo animal models. In some embodiments, the therapeutically effective amount is at least about 0.1 mg/kg body weight, at least about 0.25 mg/kg body weight, at least about 0.5 mg/kg body weight, at least about 0.75 mg/kg body weight, at least about 1 mg/kg body weight, at least about 2 mg/kg body weight, at least about 3 mg/kg body weight, at least about 4 mg/kg body weight, at least about 5 mg/kg body weight, at least about 6 mg/kg body weight, at least about 7 mg/kg body weight, at least about 8 mg/kg body weight, at least about 9 mg/kg body weight, at least about 10 mg/kg body weight, at least about 15 mg/kg body weight, at least about 20 mg/kg body weight, at least about 25 mg/kg body weight, at least about 30 mg/kg body weight, at least about 40 mg/kg body weight, at least about 50 mg/kg body weight, at least about 75 mg/kg body weight, at least about 100 mg/kg body weight, at least about 200 mg/kg body weight, at least about 250 mg/kg body weight, at least about 300 mg/kg body weight, at least about 350 mg/kg body weight, at least about 400 mg/kg body weight, at least about 450 mg/kg body weight, or at least about 500 mg/kg body weight.

In one embodiment, the oligonucleotide compound can be administered to the subject one time (e.g., as a single injection or deposition). Alternatively, administration can be once or twice daily to a subject in need thereof for a period of from about 2 to about 28 days, or from about 7 to about 10 days, or from about 7 to about 15 days. It can also be administered once or twice daily to a subject for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combination thereof. For example, the dosage may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. In one embodiment, two or more combined oligonucleotide compounds, therapeutics, and the like may be used together in combination or sequentially. The dosage can vary depending upon known factors such as the pharmacodynamic characteristics of the active ingredient and its mode and route of administration; time of administration of active ingredient; age, sex, health and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment and the effect desired; and rate of excretion. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide compound is administered in maintenance doses, ranging from at least about 0.1 mg/kg body weight to about 10 mg/kg of body weight, once or more daily, to once every 2-20 years. Certain injected dosages of antisense oligonucleotides, for example, are described, in U.S. Pat. No. 7,563,884, which is hereby incorporated by reference in its entirety.

While the embodiments of the present invention are described with reference to various implementations and exploitations, it will be understood that these embodiments are illustrative and that the scope of the inventions is not limited to them. Many variations, modifications, additions, and improvements are possible. Further still, any steps described herein may be carried out in any desired order, and any desired steps may be added or deleted. Support for the present invention and additional embodiments of the present invention may be found in the attached documents all of which are expressly incorporated herein in their entirety by reference hereto. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing,” “involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

As will be apparent to one of ordinary skill in the art from a reading of this disclosure, the embodiments of the present disclosure can be embodied in forms other than those specifically disclosed above. The particular embodiments described herein are, therefore, to be considered as illustrative and not restrictive. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described herein. The scope of the invention is as set forth in the appended claims and equivalents thereof, rather than being limited to the examples contained in the foregoing description.

All publications and other references mentioned herein are incorporated by reference in their entirety, as if each individual publication or reference were specifically and individually indicated to be incorporated by reference. Publications and references cited herein are not admitted to be prior art.

EXAMPLES

Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.

Example 1
Friedreich Ataxia is Characterized by Progressive Repeat Expansion

Friedreich ataxia (FRDA) is a progressive neurodegenerative disorder caused by GAA·TTC repeat expansion in the first intron of the frataxin (FXN) gene (3). Disease severity correlates to the length of the expanded repeats and the consequent reduction of FXN gene expression. While the mechanism of repeat expansion is not yet understood, we have developed versatile human cell models with an integrated “tandem reporter” (15) that recapitulate the expansion seen in FRDA patients that have allowed us to make rapid progress in understanding the expansion process.

This model has a key advantage over authentic FXN repeat expansion: the repeats are not linked to an essential gene. Thus, the well-known problems associated with selection against frataxin knockdown cells in culture are able to be avoided (16). Further advantages of this system include a single copy genomic location, the ability to control the transcription into a repeat, and a cell environment permissive for expansion. The “tandem reporter” expansion system uses modified HEK293 cells. These cells express a large number of proteins typically expressed exclusively, or preferentially, in neurons probably due to preferential transformation of neuronal lineage by adenovirus 5 in embryonic kidney (17,18). The ability to study expansion with an accelerated time course is aided by the neuronal nature of HEK293 cells. Long uninterrupted GAA·TTC repeats cannot be propagated in bacteria. Therefore repeat arrays are built for the constructs using an in vitro ligation strategy devised (19) circumventing bacteria. This gives the system another advantage since defined, uninterrupted repeats are used.

In models, the repeats expand incrementally, continuously and nearly synchronously. Importantly, the rate of expansion is linked to the level of transcription into the repeats (20). Thus, some therapeutic strategies aimed at increasing transcription of the FXN gene will inadvertently increase repeat expansion.

These models have been used to determine that MSH3 (MutSbeta) is required for GAA·TTC repeat expansion (21). MutSbeta is comprised of MSH2 and MSH3, and binds to a site targeted for mismatch repair. Multiple lines of evidence highlight the importance of MutSbeta in repeat expansion: (i) shRNA knockdown of either MSH2 or MSH3 slowed GAA·TTC expansion in human cells, and (ii) ectopic expression of MutSbeta induced GAA·TTC repeat expansion in the native FXN gene. Once it binds to DNA to initiate repeat expansion, MutSbeta recruits a MutL complex.

The models have also been used to determine that MLH1 complexed with MLH3 (MutLgamma) is the next step required for GAA·TTC repeat expansion. Furthermore, MLH3 is involved in repeat expansion and only one of the two MLH3 alternative splicing isoforms is required.

Human MLH3 has 2 Isoforms

MLH3 is expressed in humans as two isoforms, MLH3iso1 and MLH3iso2, resulting from alternative splicing. MLH3iso1 includes exon 7, which contains a highly conserved portion of an endonuclease domain, while MLH3 isoform 2 lacks this 72 base exon (FIGS. 9B-9D). Primer pairs, MLH3 L3324 and MLH3 R3757, were used to detect MLH3iso1 and MLH3iso2 (FIG. 9A). This pair resulted in a 434 bp band for MLH3iso1 and a 362 bp band for MLH3iso2; a 16.6% difference allowing easy visualization of the presence or absence of the 72 nucleotide long exon 7. Dilutions of defined isoform templates containing or excluding exon 7 were done in 10:1, 1:1, and 1:10 respectively to demonstrate the quantitative nature of this PCR.

Forcing MLH3 Exon Choice

MLH3 expression is key to GAA·TTC expansion in human cells. Manipulation of this minor component of MMR in a repeat expansion model may be a possible therapeutic target to limit DNA repeat expansion in FRDA patients. As MLH3iso2 lacks exon 7, which contains part of its endonuclease domain, forced expression of this isoform may serve such a purpose. Splice switching oligos (SSOs) were designed to mask the acceptor and donor regions of MLH3 exon 7, inducing skipping of exon 7 and the consequent production of MLH3iso2 (FIG. 5). Forcing exclusion of exon 7 may approximate a functional knockout of the endonuclease activity of MLH3, which is critical for repeat expansion. Skipping exon 7 leaves MLH3 isoform 2 intact, so will not impact the total cellular ratios of MLH1 and its binding partners PMS2, PMS1 and MLH3. Exemplary SSOs are depicted in SEQ ID NOS: 3 and 4 below:

ML3X7acceptor6

(SEQ ID NO: 3)

5′-TCCCACctagatgagcaaggattgt-3′

ML3X7donor8

(SEQ ID NO: 4)

5′-tctggctgcaaacagatccttacCA-3′

Small Molecule Directed Skipping of MLH3 Exon 7 Slows GAA·TTC Repeat Expansion

These cells were treated with splice switching oligonucleotides designed to exclude exon 7 of MLH3, so that the cells would preferentially make MLH3iso2. Specifically, acceptor, donor or both SSOs were given twice a week to FRDA model cells in culture. After 3 weeks in culture with various treatments, RT-PCR was used to measure the relative expression of MLH3iso1 and MLH3iso2, and PCR on genomic DNA was used to measure the length of the GAA·TTC repeat. RT-PCR demonstrates that the combination of acceptor and donor SSOs at 500 nM most effectively excluded exon 7 (FIG. 6). Correlated with the preferential expression of MLH3iso2, PCR analysis of GAA·TTC expansion shows a reduced expansion rate (FIG. 7).

From these experiments, it is concluded that (i) MLH3 contributes to GAA·TTC repeat expansion in human cells, (ii) the endonuclease domain of MLH3 is needed for this effect, and (iii) a small molecule therapeutic directed at skipping of MLH3 exon 7 may be therapeutic avenue to slow the progression of repeat expansion disorders such as Friedreich ataxia.

REFERENCES

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14. Cannavo, E., Marra, G., Sabates-Bellver, J., Menigatti, M., Lipkin, S. M., Fischer, F., Cejka, P. and Jiricny, J. (2005) Expression of the MutL homologue hMLH3 in human cells and its role in DNA mismatch repair. Cancer Res, 65, 10759-10766.

15. Banerjee, A., Sammarco, M. C., Ditch, S., Wang, J. and Grabczyk, E. (2009) A novel tandem reporter quantifies RNA polymerase II termination in mammalian cells. PloS one, 4, e6193.

16. Calmels, N., Seznec, H., Villa, P., Reutenauer, L., Hibert, M., Haiech, J., Rustin, P., Koenig, M. and Puccio, H. (2009) Limitations in a frataxin knockdown cell model for Friedreich ataxia in a high-throughput drug screen. BMC Neurol, 9, 46.

17. Graham, F. L., Smiley, J., Russell, W. C. and Nairn, R. (1977) Characteristics of a human cell line transformed by DNA from human adenovirus type 5. The Journal of general virology, 36, 59-74.

18. Shaw, G., Morse, S., Ararat, M. and Graham, F. L. (2002) Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells. The FASEB journal: official publication of the Federation of American Societies for Experimental Biology, 16, 869-871.

19. Grabczyk, E. and Usdin, K. (1999) Generation of microgram quantities of trinucleotide repeat tracts of defined length, interspersion pattern, and orientation. Analytical biochemistry, 267, 241-243.

20. Ditch, S., Sammarco, M. C., Banerjee, A. and Grabczyk, E. (2009) Progressive GAA·TTC repeat expansion in human cell lines. PLoS genetics, 5, e1000704.

21. Halabi, A., Ditch, S., Wang, J. and Grabczyk, E. (2012) DNA mismatch repair complex MutSbeta promotes GAA·TTC repeat expansion in human cells. The Journal of biological chemistry, 287, 29958-29967.

Example 2

MLH3 expression is key to GAA·TTC expansion in human cells and forms the basis for the first therapeutic to slow the expansion rate in Friedreich ataxia and perhaps other repeat expansion diseases. This minor component of mismatch repair (MMR) will be developed as a therapeutic target to limit repeat expansion in FRDA patients. in vivo efficacy and safety studies will be conducted in a mouse model. Without being bound by theory, selective expansion of GAA·TTC repeats in disease relevant tissues to a critical size drives disease onset and progression in FRDA. Somatic expansion of GAA·TTC repeats requires transcription through the repeat then the sequential actions of MutSp (MSH2/MSH3 heterodimer) and MutLγ (MLH1/MLH3 heterodimer). MSH3 expression was linked to active GAA·TTC expansion in FRDA patient primary cells (G1). Similarly, MMR or MSH3 expression has been linked to region specific expansion of CAG·CTG repeats in the Huntington's disease (HD) “R6” mouse model (G2-G4). MLH3 operates downstream of MSH3. Without being bound by theory, MLH3, while a minor player in canonical MMR, is a major force in DNA repeat expansion. MLH3 has also recently been identified as a component of CAG·CTG expansion in the HD “R6” mouse (G5).

Like the HD “R6” mouse, the FRDA “YG22” mouse model exhibits region specific GAA·TTC repeat expansion (G6). Splice-switching oligonucleotides (SSOs) will be used to block this expansion as a first step leading to human trials. The SSOs will be targeted to the mismatch repair protein MLH3. MLH3 is expressed in humans as two isoforms, MLH3 isoform 1 and MLH3 isoform 2, due to alternative splicing. MLH3 isoform 1 includes exon 7, which contains a conserved endonuclease domain, while MLH3 isoform 2 lacks exon 7. It has been recently found that MLH3 isoform 1 is required for GAA·TTC expansion, while isoform 2 is not. Skipping exon 7 by use of SSOs effectively shifts MLH3 to isoform 2 and stops repeat expansion in human cells. Finally, skipping exon 7 leaves MLH3 isoform 2 intact, the total cellular ratios of MLH1 and its binding partners PMS2, PMS1 and MLH3 will not be impacted. The mouse MLH3 exon structure parallels that of humans.

This approach targets a central mechanism that is likely shared by all repeat expansion diseases. Therefore, it has the potential to treat many, if not all of the diseases in this class. The SSOs to be used are the same type already in human trials for Duchene muscular dystrophy (G7-G9). Consequently, this project has great translational potential.

MMR has been implicated in repeat expansions of numerous disorders including Huntington's disease (HD) and myotonic dystrophy (DM) (G10-G13). Although somatic mosaicism of GAA·TTC allele size in FRDA patients has long been known (G14-G16) consideration of a role for MMR in the underlying GAA·TTC repeat expansion is more recent (G1,G17-G19).

In the MMR pathway, MutS heterodimers are responsible for identifying and binding mismatched bases and/or insertion/deletion loops of varying size (G20). MSH2 (MutS Homologue 2) is a component of both MutS complexes and has consequently been implicated in DNA repeat expansion (G10, G17, G19). Upon mismatch recognition by a MutS complex, a MutL heterodimer is recruited to make an incision near the lesion recognition site (G21-G23). Under physiologic conditions, binding of MutL initiates recruitment of the necessary machinery that will excise the lesion and synthesize the DNA patch.

While the mechanism of repeat expansion is not yet fully understood, that GAA·TTC expansion rate is associated with transcription within the repeat (FIGS. 1A-1F) and requires the action of mismatch repair (MMR) complex MutSβ rather than MutSα (G1, G24). Much of the accumulated evidence agrees that the MutSβ complex, and/or the MSH3 subunit in particular, is rate limiting for expansion seen in Huntington's disease (HD), myotonic dystrophy (DM) (G12, G13, G25, G26) as well as in FRDA (G1).

Analogous to the role of MSH2 in MutS complexes, MLH1 (MutL Homologue 1) is the core subunit of known MutL complexes. MLH1 combines with one of three partners called PMS1 (post-meiotic segregation increased 1), PMS2, and MLH3 to form MutLβ, MutLα, and MutLγ, respectively. It is estimated that about 90% of MLH1 in most human cells is bound to PMS2 (MutL(a) (G28, G29); further, PMS1 and PMS2 are estimated to be present in 10 fold and 60 fold molar excess of MLH3 (G29). MutLα and MutLγ appear to have a role in MMR while MutLβ does not. As with MSH2, MLH1 depletion is strongly associated with hereditary nonpolyposis colorectal cancer (HNPCC) and sporadic gastric and endometrial carcinomas (G30-G32); PMS2 depletion is also associated with HNPPC albeit to a lesser extent than MLH1. Currently, evidence for MLH3 indicates that it rarely, if ever, contributes to cancer development (G33-G35).

PMS1, PMS2, and MLH3 have all been reported to compete for the same binding site on the C-terminal of MLH1 (G36); as with the MutS homologues, it is possible that the abundance of these proteins in relation to one another regulates their ability to compete for MLH1 and hence, their stability. Expression of MLH1 and PMS2 correlate strongly; however, evidence for MLH3 implicates that in addition to its already low abundance, MLH3 expression may not be tethered to the expression of other MutL proteins (G29). Generally, MLH3 is better understood for its role in meiotic recombination and the repair of frame-shift mutations than in canonical MMR (G37, G38). Interactions between MLH3, MLH1, and MSH3 have all been reported (G36-G38).

In the current working model shown in FIGS. 1A-1F, resolution of a structure formed by transcription causes an out-of-register re-annealing of the two strands that leads to loop-outs. Without being bound by theory, the MMR pathway is aberrantly activated by these small loop-outs in the repeat. The contribution of MutL subunits to GAA·TTC repeat expansion will be assessed in the human cellular model of FRDA. Expression of MLH1, PMS2, and MLH3 was depleted in the cellular model and changes in expansion rate over time were quantified.

MLH3 Expression is Key to GAA·TTC Expansion in Human Cells.

Lentiviral mediated shRNA knockdown of MLH1, PMS2 and MLH3 was carried out in four independent clones of HEK293 cells carrying a single copy of the tandem reporter vector bearing 176 GAA·TTC repeats (G24). Each knockdown used a pool of four shRNA-expressing lentivirus. After 4 weeks in culture, DNA and protein extracts were prepared as described previously (G1). A representative PCR sizing gel is shown in FIG. 2A. DNA from MLH1 knockdown cells (MLH1sh) can be seen to have reduced expansion compared to the empty vector control cells (pLKO) at week 4. DNA from MLH3 knockdown cells exhibit the least expansion (MLH3sh). The number of triplet repeats gained in 4 weeks was calculated for all four cell lines and presented in graphical form in FIG. 2B. MLH1 knockdown and MLH3 knockdown each showed a substantial and statistically significant reduction in expansion rate. In contrast, PMS2 knockdown samples showed a trend towards greater expansion. Thus shRNA knockdown of MMR proteins MLH1 and MLH3, but not PMS2 slows GAA·TTC expansion in human cells, indicating a role for MutLγ.

Like MutS, MutL partners are more stable as heterodimers (G28). Western blot analysis of protein extracts from the cells showed the expected reduction of both PMS2 and MLH1 in the MLH1 knockdown cells as compared to controls (FIG. 3 compare lanes MLH1sh and lanes C). When PMS2 was knocked down, the protein level of MLH1 decreased, but was still evident (FIG. 3 lanes PMS2sh). In contrast with MLH1 and PMS2, MLH3 knockdown led to a slight decrease in MLH1 protein but PMS2 protein did not change. Because PMS2 stability depends on binding to MLH1, the lack of change in PMS2 with MLH3 knockdown indicates that MutLα levels are unaffected by the loss of MLH3 in these cells.

Taken together these data indicate that the necessary complex for GAA·TTC expansion is MutLγ, a heterodimer of MLH1 and MLH3. The lack of connection between MLH3 and cancer (G33-G35), and that reducing MLH3 levels did not affect levels of MutSα, which is linked to cancer (G30-G32), suggested MLH3 as a possible therapeutic target to limit DNA repeat expansion in FRDA patients.

MLH3 Exon Skipping as an Alternative to MLH3 Knockdown.

MLH3 is expressed in humans as two isoforms (FIG. 4). MLH3 isoform 1 includes exon 7, which contains a highly conserved portion of an endonuclease domain, while MLH3 isoform 2 lacks this 72 base exon (FIG. 4). Without being bound by theory, if the endonuclease activity of MLH3 is critical to repeat expansion, then exclusion of exon 7 would stop repeat expansion. Skipping exon 7 leaves MLH3 isoform 2 intact, and therefore does not impact the cellular ratios of MLH1 and its binding partners PMS2, PMS1 and MLH3.

MLH3 exon 7 was skipped using oligonucleotide analogues that would bind and mask the splice donor and acceptor signals flanking exon 7 in the unspliced pre-mRNA (FIG. 5). DNA oligonucleotides were also designed to assay the ratio of MLH3 isoform 1 and MLH3 isoform 2 mRNA via reverse transcription PCR (RT-PCR) in order to quantify the efficacy of the SSOs.

Morpholinos are oligonucleotide analogues that bind their complementary target DNA or RNA very tightly due to their uncharged backbone of morpholino subunits, which also makes them resistant to nucleases and proteases (G39). The SSOs used in tissue culture are “vivo-morpholinos” that have an octaguanidine moiety conjugated to the morpholino to enhance cellular uptake (G40).

SSOs Effectively Changed MLH3 from Isoform 1 to Isoform 2 and Reduced Repeat Expansion.

Different concentrations of acceptor SSO, donor SSO and combinations of the two were examined. An example of the experiments conducted with the SSOs is shown in FIG. 6. The acceptor SSO gave a graded concentration dependent effect on exon skipping and the donor SSO was somewhat more effective, showing a steeper gradient. However, efficacy of splice switching was greatly enhanced when donor and acceptor SSOs were used in combination (FIG. 6, arrow).

The effect that the SSOs had on MLH3 isoforms was mirrored by the effect the SSOs had on repeat expansion (FIG. 7). The SSO combinations and concentrations that effectively switch MLH3 to isoform 2, also slowed GAA·TTC repeat expansion (compare FIGS. 6 and 7).

That splice-switching oligonucleotide directed skipping of MLH3 exon 7 slows GAA·TTC repeat expansion in our model system provides proof of principle. The SSOs were then studied in FRDA patient-derived cells. In light of reports of sustained morpholino action in neuronal rescue models in the mouse (G41, G42), whether the effect from a single exposure to the SSOs could be sustained in post mitotic cells was examined.

A Single Dose of MLH3 SSOs Slows Repeat Expansion Over 4 Weeks in FRDA Fibroblasts.

FRDA patient-derived fibroblastic cells do not exhibit repeat expansion at the GAA·TTC repeats in the FXN gene under normal circumstances. However, ectopic expression of the DNA mismatch repair protein MSH3 will cause the GAA·TTC repeats to expand whether the fibroblasts are passaged and actively dividing, or confluent and not dividing (G1). MSH3 was expressed in FRDA fibroblasts via lentivirus transduction as has been done in the past, and then it was demonstrated that expansion by a one-time treatment with MLH3 SSOs can be reduced.

FRDA fibroblasts GM04078 (Coriell) were transduced with lentivirus expressing MSH3 at time zero (T₀). Transduced cells were plated at high density and allowed to reach confluence. At week 1, some cells were exposed to 750 nM SSOs (acceptor and donor) for 24 hours. After that, all cells were fed with normal growth media (DMEM+10% FBS). At 5 weeks post transduction (4 weeks post SSO treatment), DNA was prepared and repeats were sized with PCR. The results of one such experiment are shown in FIG. 8. Despite the small differences, it is apparent that the treated cells expanded less than did the untreated cells. The results of experiments such as this indicate that the SSOs used have a sustained effect on non-dividing cells, and this effect can be seen in as little as one month in culture. Such experiments also inform our power analysis for the planned mouse study.

Research

The “YG22” FRDA mouse model exhibits region specific GAA·TTC repeat expansion (G6). MLH3-specific splice-switching oligonucleotides (SSOs) will be used to block this expansion. MLH3 has recently been identified as a component of CAG·CTG expansion in the Huntington's disease “R6” mouse model (G5) and region specific CAG·CTG expansion in the same model has been linked to MSH3 (G2-G4). The parallels are considerable; without being bound by theory, MLH3 is key to GAA·TTC repeat expansion in the FRDA YG22 mouse, as well as in human FRDA cells.

However, whether the SSOs will penetrate where needed and whether the vivo-morpholino versions will cause clotting will be examined in mice. Therefore, SSOs will be tested in C57BL/6J mice before moving to the much longer experiments in the Tg(FXN)YG22 Pook mice.

The following will be tested: two types of SSO, two ages of application, and two delivery protocols in mice. Simple or “naked” morpholinos have long been used in model organisms, including mice, and have been in human trials for the past several years (G7-G9). Their safety and efficacy are well known. One drawback is the consistent penetration in adult tissue.

The conjugated “vivo-morpholino” is designed to have better tissue penetration in the mouse (G40, G43). Without being bound by theory, retrograde transport of vivo-morpholinos will get them into dorsal root ganglia. The vivo-morpholinos are superior for tissue culture compared to naked morpholinos (G41, G44).

Design and test of MLH3 exon skipping SSOs in mouse cells. The mouse MLH3 exon structure parallels that of humans. The SSOs and splice assay nucleotides needed will parallel those used for the human MLH3 locus, although they will have somewhat different nucleotide sequences. These reagents will be tested in mouse cell lines in culture, including cells from the C57BL/6 mouse, just as has been done with human MLH3. A small synthesis of vivo-morpholinos will be used for the tissue culture assays. Once mouse MLH3 isoform switching has been optimized, a larger synthesis of the vivo-morpholinos and the corresponding simple morpholinos will be ordered. When SSOs for the mouse are discussed in this example, either naked or vivo-morpholinos, it will most likely refer to a cocktail of donor and acceptor blocking sequences as shown in FIGS. 6 and 7.

SSOs that are effective for mouse MLH3 will be identified quickly. Confidence in the bioinformatic analysis of the MLH3 gene in mice, the design of the SSOs and the splice detection oligonucleotide pairs has been established.

Breeding of mice during the design and testing period. B6.Cg-Fxn^tm1Mkn-Tg(FXN)YG22Pook/J and C57BL/6J mice will be ordered from the Jackson Laboratory. The strain B6.Cg-Fxn^tm1MknTg(FXN)YG22Pook/J is a double mutant: hemizygous for a human FXN locus with expanded GAA·TTC repeats and heterozygous for a knockout of the mouse FXN locus. Through selective breeding, mice homozygous for the human FXN locus, but lacking the knockout of mouse FXN, will be produced. Subsequent crosses with C57BL/6J mice will produce offspring bearing a human FXN transgene and normal mouse FXN alleles to avoid possible selection against repeat expansion due to insufficient frataxin.

Initial testing of SSO safety and efficacy in C57BL16 mice. It can be quickly ascertained in wild type mice whether the SSOs are safe and effective for MLH3 isotype switching. After a single injection of candidate SSOs in newborn (PND0, in facial vein) or adult (8 weeks, in tail vein) C57BL/6J mice, the mice will be humanely killed and dissected after intervals (see Table 3 for numbers, dosing and intervals). RNA will be isolated from brain, cerebellum, dorsal root ganglia, heart, and liver, and will be assayed for MLH3 isotype via reverse transcription PCR (RT-PCR). Safety, tissue penetration, and persistence of action by high or low doses of naked morpholino and vivo-morpholino SSOs will be determined in both newborn and adult mice before any experiments in the “YG22” mice are conducted.

TABLE 3

Mouse distribution and end points in research protocol. DNA

and RNA will be isolated from brain, cerebellum, dorsal root ganglia, heart and liver.

Mice
Regimen
Reagent
Dose
Collection time points

66 wt mice
Post natal day 0
12 vivo-
50 mg/kg
RNA will be isolated at weeks

C57BL/6J
(PND0) Cohort
morpholino

1, 2, 4 and 8 for splice

One-time injection
12 morpholino
50 mg/kg
switching efficacy RT-PCR

assay (N = 3 for each time point

12 control
Saline
& condition).

Young adult Cohort
12 vivo-
50 mg/kg

Single injection at 8
morpholino
5 mg/kg

weeks
12 morpholino
50 mg/kg

5 mg/kg

6 control
Saline
RNA will be isolated 1 and 2

weeks after injection for splice

switching efficacy RT-PCR

assay (N = 3 for each time &

condition).

96 ″YG22″
Post natal day 0
16 vivo-
50 mg/kg
DNA & RNA will be isolated

Transgenic
(PND0) Cohort
morpholino

at 3 months (n = 8) and

mice
One-time injection
16 morpholino
50 mg/kg
at 6 months (n = 8)

carrying

16 control
Saline

Tg(FXN)YG
Young adult Cohort
16 vivo-
50 mg/kg
8 DNA & RNA @6 months

22Pook
Bimonthly injection
morpholino
5 mg/kg
8 DNA & RNA @6 months

starting at 8 weeks
16 morpholino
50 mg/kg
8 DNA & RNA @6 months

5 mg/kg
8 DNA & RNA @6 months

16 control
Saline
16 DNA & RNA @6 months

Two types of SSO and two delivery protocols to be tested in YG22 mice. A single application at birth (post natal day 0, PND0) will be administered via facial vein injection. Application of naked morpholino or vivo-morpholino SSOs at birth has been shown to penetrate into the central nervous system (CNS), possibly due to a leaky blood brain barrier at birth (G41, G42). For the PND0 cohort, a one-time injection will be followed by an interval of normal mouse rising with no further experimental manipulations until the mouse is humanely killed at the appropriate time point (see Table 3).

Bi-monthly application in adult mice, starting at 8 weeks will be administered via tail vein injection. The “YG22” cohort of GAA·TTC repeat bearing adult mice will get a tail vein injection every two weeks starting at 8 weeks before the mice are humanely killed at 26 weeks of age. Further, in adult mice we will test two concentrations of SSO, 5 mg/kg and 50 mg/kg (see Table 3 for distribution). A chronic low dose may be safer than a high dose, and these experiments will help determine if the low dose is sufficient to get into tissues such as the heart (G45, G46).

SSO efficacy will be assayed in two ways. The MLH3 isoform 1 to isoform 2 ratios will be measured via RT-PCR. GAA·TTC repeat length in “YG22” mice will be measured via PCR. Tissue from the CNS and PNS, as well as somatic tissues, will be further assayed.

Tissues to be dissected from mice for RNA and DNA isolation will include 1) brain, 2) cerebellum, 3) dorsal root ganglia (DRG), 4) heart and 5) liver. Tail DNA will also have been prepared separately for genotyping, and may serve as an additional control.

Without being bound by theory, reduced expansion in the cerebellum and DRG of PND0 injected YG22 mice will be observed. Without being bound by theory, persistent SSO activity will be observed in tissues containing long lived post-mitotic cells such as brain, cerebellum and heart, but less so in liver due to dilution by cell division.

Without being bound by theory, robust SSO activity will be observed in the livers of the young adult YG22 repeated high dose morpholino and vivo-morpholino cohorts, and a little less activity will be observed in the heart.

Without being bound by theory, little or no SSO activity will be observed in the heart and little activity will be observed in the livers of low dose morpholino YG22 adult cohort, but moderate to robust activity will be observed in the low dose vivo-morpholino YG22 adult cohort due to chronic accumulation of the vivo-morpholino.

Without being bound by theory, SSO activity will be observed in the DRG of vivo-morpholino, but not naked morpholino treated adults. If sufficient GAA·TTC expansion occurs in the DRG, an effect of SSO treatment, will be observed, particularly in the high dose vivo-morpholino cohort.

Without being bound by theory, reduced expansion will not be observed in the cerebellum or brain of the young adult YG22 cohort due to poor penetration of the blood brain barrier no matter the dose.

Statistics

Statistical analysis will predominantly use one-way or two-way ANOVA. We used power analysis to arrive at the number of animals for the study, based upon our ability to separate repeats differing by 1-2% of length. The transgenic repeat lengths detected by PCR will range between 1000 to 1300 base pairs (bp). FRDA patient derived cells we have worked with can gain 4 to 6 repeats (12-18 bp) a month. We assume that if the mouse cerebellar samples gain comparably, we should detect a 50% effect of the treatment in 2 months with 8 (morpholino) 8 (vivo-morpholino) to 16 controls for each injection/dosing condition with 80% power. If mouse GAA·TTC repeat expansion exceeds the human rate, we will have more power. Our long-term experiments should have a larger expansion differential producing more power and allowing for some dropout. The RT-PCR based mRNA splicing assay is more easily measured, has a large delta and requires a smaller “n” when not accompanied by the DNA repeat sizing assay. Hence the wild-type C57BL/6J mice used in early tests have reduced numbers compared to “YG22.”

Example 3

Monitoring mice for long-term consequences of the treatments described in these examples can be done. The longest experiments will look at 6-month-old mice, well before even a complete knockout from birth would show an effect. Changing the ratio of MLH3 isoforms, not knocking MLH3 out, will be changed. Furthermore, morpholino oligomers are lost or diluted by cell division. Consequently, cells at risk for a cancer phenotype such as intestinal epithelia or lymphoblasts will only transiently be deficient in MLH3 isoform 1.

In one embodiment, the frataxin deficient phenotype of the Pook mice can be studied at a later stage. The mFXN knockout allele will be bred out to avoid potential interference with repeat expansion by a frataxin deficient phenotype. Here, frataxin replete Tg(FXN)YG22Pook mice will be used solely to examine repeat expansion.

As discussed in the preceding examples, SSO efficacy will be assayed in two ways: 1) the MLH3 isoform 1 to isoform 2 ratios will be measured via RT-PCR. This will be done in cultured cells, C57BL/6J and YG22Pook mice; and 2) GAA·TTC repeat length in YG22Pook mice will be measured via PCR (conventional and small-pool).

It will be determined whether morpholino SSO mediated MLH3 isoform switching can reduce the rate of GAA·TTC repeat expansion in the YG22Pook mice. Because morpholino SSOs only need to bind their target to abrogate splicing, an SSO or cocktail of SSOs effective in mice will be identified. Without being bound by theory, the most effective SSO or SSO cocktail found in tissue culture can be toxic to mice in the vivo-morpholino form, but the unmodified, or simple morpholino oligomers, will also be used as a backup. The simple morpholino SSOs will have reduced penetration in adult tissue. But, simple morpholino SSOs have been shown to be effective from a single PND0 injection in mice for at least 65 days (2) and are safe enough that several are in human trials (11-13).

Testing of the SSOs can be done quickly and relatively inexpensive in cultured mouse cells and wild-type C57BL/6J mice, while the YG22Pook mice are bred. Without being bound by theory, both immediate toxicity and the efficacy in MLH3 splice switching in C57BL/6J tissues will be determined in a matter of days. The longer time points are there to determine the staying power of the morpholinos in various tissues to determine the dosing regimen needs to be adjusted before starting long-term experiments in YG22Pook mice.

MLH3 isoform 1->2 switching is a direct measure of SSO efficacy. Consequent effects of a decrease in MLH3 isoform 1 on GAA·TTC repeat expansion rate will be measured via PCR across the repeat (both conventional and small pool PCR).

The mice available from Jackson lab are heterozygous for mFXN knockout and hemizygous for Tg(FXN)YG22Pook. Breeders will be developed with normal mFXN genes that are homozygous for Tg(FXN)YG22Pook for two reasons: (1) Breeding with C57BL/6J will produce offspring that are all hemizygous for YG22 so that mice can be efficiently treated at birth without wasting reagent or waiting for genotyping; and (2) Normal mouse frataxin expression will ensure that expansion of the repeat will not be counter-selected by frataxin insufficiency.

RT-PCR determination of MLH3 isoform 1->2 switching is a direct measure of SSO efficacy, and comparison of MLH3 isoforms to those in control tissues will be informative regarding tissue penetration of the SSOs.

Mice available through the Jackson Laboratory, which are heterozygous for mFXN knockout and hemizygous for Tg(FXN)YG22Pook, will be used. Breeders will be developed with normal mFXN genes that are homozygous for Tg(FXN)YG22Pook for two reasons: 1) Breeding with C57BL/6J will produce offspring that are all hemizygous for YG22 so that mice can be efficiently treated at birth without wasting reagent or waiting for genotyping; and 2) Normal mouse frataxin expression will ensure that expansion of the repeat is not counter-selected by frataxin insufficiency.

Without being bound by theory, the activity of the SSOs will diminish over a shorter time course in rapidly dividing tissues as the mouse grows and the SSOs will be diluted. Furthermore, cells that are post-mitotic at birth should retain SSOs and their activity far longer. For instance, Porensky et al. showed that a single injection of simple morpholino SSOs at birth lead to splice switching that remained robust in brain and spinal cord at 65 days (2). Consequently, there will also be sustained activity of PND0 administered SSOs in brain and spinal cord for at least 8 weeks. If the splice switching activity declines thereafter, the aggregate effects of diminished and partially diminished MLH3 isoform 1 expression on GAA·TTC repeat expansion at 3 months or greater will be able to be assessed.

The heart will be examined because it is an affected tissue in FRDA. Without being bound by theory, the working model will show that the repeat focally expands in heart leading to stochastic loss of fibers (along with their expanded repeats). Thus, it is nevertheless of great interest to determine whether therapeutic SSOs can be administered into the heart.

Changes in the rate of expansion will be assessed by comparing the rate of expansion in tissues that typically exhibit expansion with those that typically do not in YG22 mice. In addition, the rate of expansion will be compared in tissues from untreated littermates to tissues in the treatment groups. Conventional PCR has been shown to be sufficient to see gross changes in size in the YG22 repeat, particularly in the cerebellum (A14, A15). Small pool PCR will have to be used to assess more rare events in other tissues, as well as to simplify the smear obtained from expanded repeats seen with conventional PCR in the cerebellum (A16).

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A8. Lipkin, S. M., Moens, P. B., Wang, V., Lenzi, M., Shanmugarajah, D., Gilgeous, A., Thomas, J., Cheng, J., Touchman, J. W., Green, E. D. et al. (2002) Meiotic arrest and aneuploidy in MLH3-deficient mice. Nature genetics, 31, 385-390.

A9. Chen, P. C., Dudley, S., Hagen, W., Dizon, D., Paxton, L., Reichow, D., Yoon, S. R., Yang, K., Arnheim, N., Liskay, R. M. et al. (2005) Contributions by MutL homologues Mlh3 and Pms2 to DNA mismatch repair and tumor suppression in the mouse. Cancer Res, 65, 8662-8670.

A10. Chen, P. C., Kuraguchi, M., Velasquez, J., Wang, Y., Yang, K., Edwards, R., Gillen, D., Edelmann, W., Kucherlapati, R. and Lipkin, S. M. (2008) Novel roles for MLH3 deficiency and TLE6-like amplification in DNA mismatch repair-deficient gastrointestinal tumorigenesis and progression. PLoS genetics, 4, e1000092.

A11. Kinali, M., Arechavala-Gomeza, V., Feng, L., Cirak, S., Hunt, D., Adkin, C., Guglieri, M., Ashton, E., Abbs, S., Nihoyannopoulos, P. et al. (2009) Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in Duchenne muscular dystrophy: a single-blind, placebo-controlled, dose-escalation, proof-of-concept study. Lancet Neurol, 8, 918-928.

A12. Goemans, N. M., Tulinius, M., van den Akker, J. T., Burm, B. E., Ekhart, P. F., Heuvelmans, N., Holling, T., Janson, A. A., Platenburg, G. J., Sipkens, J. A. et al. (2011) Systemic administration of PRO051 in Duchenne's muscular dystrophy. The New England journal of medicine, 364, 1513-1522.

A13. Cirak, S., Arechavala-Gomeza, V., Guglieri, M., Feng, L., Torelli, S., Anthony, K., Abbs, S., Garralda, M. E., Bourke, J., Wells, D. J. et al. (2011) Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study. Lancet, 378, 595-605.

A14. Anjomani Virmouni, S., Sandi, C., Al-Mahdawi, S. and Pook, M. A. (2014) Cellular, Molecular and Functional Characterisation of YAC Transgenic Mouse Models of Friedreich Ataxia. PloS one, 9, e107416.

A15. Ezzatizadeh, V., Sandi, C., Sandi, M., Anjomani-Virmouni, S., Al-Mahdawi, S. and Pook, M. A. (2014) MutLalpha heterodimers modify the molecular phenotype of Friedreich ataxia. PloS one, 9, e100523.
A16. Clark, R. M., De Biase, I., Malykhina, A. P., Al-Mahdawi, S., Pook, M. and Bidichandani, S. I. (2007) The GAA triplet-repeat is unstable in the context of the human FXN locus and displays age-dependent expansions in cerebellum and DRG in a transgenic mouse model. Human genetics, 120, 633-640.

Example 4
Exploring DNA Mismatch Repair Complexes Involved in Repeat Expansion.

Friedreich ataxia (FRDA) is a progressive neurodegenerative disorder caused by GAA·TTC repeat expansion in the first intron of the frataxin (FXN) gene. Disease severity correlates to the length of the expanded repeats and the reduction of FXN mRNA. The mechanism of repeat expansion is not yet completely understood; however, it has previously been shown that the expansion rate is associated to transcription within the repeats. Without being bound by theory, DNA repair enzymes are attracted to structures formed within the GAA·TTC repeat during transcription and the subsequent actions of these repair enzymes then promote the expansion process. It has been shown that shRNA knockdown of either MSH2 or MSH3, components of mismatch repair complex MutSβ, slowed GAA·TTC expansion in the model and FRDA patient fibroblasts. Furthermore, ectopic expression of MutSβ induced GAA·TTC repeat expansion in the native FXN gene. Other components of DNA mismatch repair complexes are being examined to elucidate their role in FRDA (for example, see FIGS. 2A-2B and FIG. 3 for MLH1, MLH3, and PMS2 data). For example, components of the heterodimeric complexes in mismatch repair's human homolog MutL are of interest, which participate in the repair of a subset of mismatches, recognized by the MSH2-MSH3 complex. Due to alternative splicing and alternative AUG start codon usage there are many possible variations in MutL complexes (See FIGS. 9B-9D). Without being bound by theory, only a few particular isoforms of these complexes are responsible for repeat expansion. Presently, these components of DNA mismatch repair are being examined in order to elucidate their role in FRDA and discover possible therapeutic targets, exploring the role of MLH3 isoforms in FRDA repeat expansion. The heterodimer of MutL Homologue one (MLH1) with MLH3 forms the necessary MutL complex, which is known as MutLγ. MLH3, a component of the MutLγcomplex, has 2 isoforms due to alternative splicing. MLH3 isoform 1 (MLH3 iso1) has exon 7 and MLH3 iso2 lacks exon 7 (FIGS. 9B-9D). Exon 7 contains the endonuclease domain. Without being bound by theory, excluding exon 7 can approximate a functional knock out if MLH3 is critical to repeat expansion. The expression levels of the spliced variants were correlated to the GAA·TTC repeat expansion rate in a human cell model.

Detecting MLH3 Isoforms 1 and 2. To study the functional diversity of MLH3 protein isoforms, the altered expression levels of the spliced variants were measured to evaluate their effects on GAA·TTC repeat expansion. Primer pairs, MLH3 L3324 and MLH3 R3757, resulted in a 434 bp band for MLH3 iso1 and a 362 bp band for MLH3 iso2. A 16.6% difference allowing for visualizing the presence or absence of the 72 nucleotide long exon 7. Dilutions of defined isoform templates containing or excluding exon 7 were done in 10:1, 1:1, and 1:10 respectively to demonstrate the quantitative nature of this PCR (FIG. 9A).

Forcing Exon Choice. Splice switching oligos (SSOs) were designed to mask the acceptor and donor regions of MLH3 exon 7 to induce skipping of exon 7 and the consequent production of MLH iso2 (FIG. 5).

RT-PCR demonstrates exon skipping. 100 nM and 500 nM dilutions of acceptor, donor, and a combination of both SSOs were given twice a week to FRDA model cells to examine the effect of MLH3 SSOs on MLH3 isoforms. The control received media only. Cells were assessed for MLH3 isoform variant expression with RT-PCR using the designed primer pair (FIG. 6). The control resembles the 10:1 (MLH1 iso1:MLH3 iso2) ratio of defined templates of known concentration shown in FIG. 9A. The combination of acceptor and donor SSOs caused the effective exclusion of exon 7.

MLH3 iso1 Required for Expansion. PCR analysis of GAA·TTC expansion was conducted at week 3 with 100 nM and 500 nM dilutions of acceptor, donor, and a combination of both SSOs to examine the effect of MLH3 SSOs on MLH3 isoforms. Repeat growth assay PCR product equals 500 bp flanking sequence plus 3×'s number of repeats. Sample 6 with 500 nM of both acceptor and donor SSOs slowed expansion (FIG. 7). Sample 4 with 500 nM of the donor SSO also had a slowing effect but not as substantially as the combination of the SSOs (FIG. 7).

MLH3 contributes to GAA·TTC repeat expansion in human cells. Specifically, MLH3 exon 7 is necessary for GAA·TTC repeat expansion in human cells. Lack of exon 7 slows GAA·TTC repeat expansion in FRDA model cells. The endonuclease domain of MLH3 is needed for this effect.

Targeting both the splice donor and acceptor region of exon 7 excluded MLH3 iso1. Such a method as splice skipping can have potential as a future therapeutic avenue for FRDA. In one embodiment, MLH3 is useful as a therapeutic target to slow the progression of repeat expansion disorders such as Friedreich ataxia. On another embodiment, small molecule directed skipping of MLH3 exon 7 is a useful therapeutic approach.

Future studies will include: (1) Observation of intrinsic expression of MLH3 variants iso1 and iso2 in different cell lines, including FRDA patient cells; (2) Observation of variants in the CNS and heart tissue; and (3) Explore MLH1, which has 22 isoforms, and is the MutLγ partner of MLH3.

Example 5

The core innovation of this technology is a therapeutic oligonucleotide for the treatment of DNA repeat expansion diseases, which include Friedreich ataxia, ALS, and Huntington's disease, among others. This innovation will likely be given orphan drug designation, highlighting a commercialization strategy associated with a number of commercial benefits.

Currently there is no effective treatment and no cure for any of the many DNA repeat expansion diseases. The core innovation of this technology aims to markedly shift the therapeutic focus from purely symptomatic to one that directly tackles the underlying disease mechanism. By slowing the expansion rate of the disease-causing DNA repeat, this therapeutic aims to slow the progression of the disease and extend a high quality of life for the individual.

Without being bound by theory, the approach targets a central mechanism that is shared by a number of repeat expansion diseases. Therefore, it has the potential to treat many, if not all of the diseases in this class. The gene-specific exon skipping in Duchenne muscular dystrophy (DMD) is more limited; SSO mediated exon skipping in DMD can only treat a fraction of the DMD patient population (G3-G5). Nonetheless, at least two startup companies (Sarepta Therapeutics and Prosensa) have been formed around exon skipping in DMD. Diseases that can benefit from this technology include Friedreich ataxia, ALS (c9orf72), Huntington's disease, Fragile X syndrome, Myotonic dystrophy Types I and II, Spino Cerebellar Ataxias (SCAs) currently including SCA1, SCA2, SCA3, SCA6, SCA7, SCA8, SCA0, SCA12, SCA17, SCA31 and SCA36 among others.

Explanation of Specific Experiments and Concepts to be Proven

The mismatch repair protein MLH3 is key to GAA·TTC repeat expansion. This minor component of MMR will be developed as a therapeutic target to limit repeat expansion in FRDA patients, and all repeat expansion patients. The SSOs used are the same type already in human trials for Duchenne muscular dystrophy (G3-G5). The work shown in FIG. 6 and FIG. 7 were carried out using the “tandem reporter” model system (G10).

The work will be expanded upon, verifying it in primary patient cells, adding additional disease causing repeats to the “tandem reporter” expansion model such as CAG·CTG (DM and the polyglutamine disorders), CGG·CCG (fragile X) and CCGGGG·CCCCGG (ALS due to C9ORF72), and designing and testing additional SSOs to achieve greater efficacy. The approach will also be studied using a mouse model of Friedreich ataxia repeat expansion, as discussed in the Examples herein.

MLH3 Splice Skipping Stops Repeat Expansion in Patient-Derived Cells

FRDA patient-derived cells do not exhibit repeat expansion at the GAA·TTC repeats in the FXN gene under normal circumstances. However, ectopic expression of the DNA mismatch repair protein MSH3 can cause the GAA·TTC repeats to expand (G2). MSH3 works upstream of MLH3 in a minor arm of mismatch repair (see FIGS. 1A-1F). MSH3 will be expressed in target cells as has been done in the past, and then it will be demonstrated that the repeat can be stopped from expanding by treating the cells with MLH3 specific SSOs. Friedreich ataxia patient cells gain an average of 1 repeat a week, so after 6 to 8 weeks of continuous culture. Without being bound by theory, a positive result in the repeat size profiles of the collected DNA, as in the “tandem reporter” model (FIG. 7), will be observed.

Myotonic dystrophy (DM1) patient-derived cells will be obtained from the Coriell cell repositories, and ectopic expression of MSH3 will be used to encourage the repeats to expand as outlined in Halibi et al (G2). The long CTG·CAG repeats in the DMPK gene of DM1 are fairly unstable in patients. Expansion will be detected, although the exact time course is not yet known. A recent publication on a mouse model of DM1 suggests that the rate of CTG·CAG expansion in mouse cells (G11) exceeds what we found for GAA·TTC in FRDA patient-derived cells. However, the bona fide DM1 patient-derived cells may expand more slowly, so the cells will be cultured for 60 to 120 days before collecting DNA.

Fragile X syndrome patient and carrier-derived cells will be obtained from the Coriell cell repositories, and ectopic expression of MSH3 will be used to encourage the CGG·GGC repeats to expand as outlined in Halibi et al (G2). Several mouse models of Fragile X syndrome have been developed (G12,G13) and PCR techniques incorporating 2M to 3M betaine are known (G12) that will get through these templates to size the repeats accurately after 60 to 120 days of continuous culture.

Repeat Tracts Representative of Repeat Expansion Diseases of Interest into Tandem Reporter Cell Lines Will be Cloned to Test MLH3 Exon Skipping

Most of the repeat expansion diseases do not have suitable patient derived cell lines with which to perform expansion studies. The rapid expansion system with a repeat cloned between tandem reporters expands GAA·TTC repeats at a rate ten- to twenty-fold faster than the patient derived cells that have been enhanced with MSH3 expression (G2). The cell lines are permissive for GAA·TTC expansion. Therefore, tandem reporter constructs will be made as have been done previously for GAA·TTC tracts (G1) using the serial ligation method previously developed (G14). Huntington's CAG·CTG lines, DM1 CTG·CAG lines, Fragile X CGG·CCG lines and ALS CCGGGG·CCCCGG lines will be generated. The techniques for making the cell lines are routine in the inventor's laboratory (e.g., G1 and G14 describe the processes in detail, which are herein incorporated by reference in their entireties).

Generation of these additional disease model cell lines will demonstrate the generality of the expansion model to other repeat expansion diseases. Comparing the rates of expansion of these various repeats in an identical environment will go far in elucidating the underlying mechanisms. The rapid expansion system with the tandem reporter carrying various disease specific DNA repeats may be desired as discovery platforms by companies seeking to develop additional therapies for repeat expansion.

Design and Test of Additional SSOs to Optimize MLH3 Exon Skipping

The results shown in FIG. 6 indicate that while the initial choices for acceptor and donor SSOs are very effective in combination, there is room for improvement. This is particularly true for the acceptor blocking SSO. New SSOs will be designed, particularly at the acceptor site, to improve efficacy. Safety and efficacy studies will also be performed in a mouse model of Friedreich ataxia GAA·TTC repeat expansion. Several sets of mouse-specific SSOs will be tested to optimize mouse MLH3 exon skipping. The data obtained from the mouse studies will inform the search for a better human set of SSOs.

Goals

Demonstration of the efficacy of MLH3 exon skipping in bona fide FRDA patient cells will be conducted. Cell lines will be obtained, grown for 60 days plus or minus ectopic MSH3 and SSOs, and subsequently will performing the RT-PCR assays on the MLH3 splice isoforms and the PCR reaction to determine repeat lengths.

Goals will be: Preparing the plasmids for the in vitro constructions. Selecting for the site-specific integration of the constructs, amplifying the cells in tissue culture and then assaying the initial size of the repeats before freezing the cell lines in liquid nitrogen. If the patient derived cells for myotonic dystrophy or Fragile X syndrome do not produce results, the corresponding repeat will be used in the rapid expansion system with the MLH3 SSOs to provide the data.

REFERENCES

G1. Ditch, S., Sammarco, M. C., Banerjee, A. and Grabczyk, E. (2009) Progressive GAA·TTC repeat expansion in human cell lines. PLoS genetics, 5, e1000704.

G2. Halabi, A., Ditch, S., Wang, J. and Grabczyk, E. (2012) DNA mismatch repair complex MutSbeta promotes GAA·TTC repeat expansion in human cells. The Journal of biological chemistry, 287, 29958-29967.

G3. Kinali, M., Arechavala-Gomeza, V., Feng, L., Cirak, S., Hunt, D., Adkin, C., Guglieri, M., Ashton, E., Abbs, S., Nihoyannopoulos, P. et al. (2009) Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in Duchenne muscular dystrophy: a single-blind, placebo-controlled, dose-escalation, proof-of-concept study. Lancet Neurol, 8, 918-928.

G4. Goemans, N. M., Tulinius, M., van den Akker, J. T., Burm, B. E., Ekhart, P. F., Heuvelmans, N., Holling, T., Janson, A. A., Platenburg, G. J., Sipkens, J. A. et al. (2011) Systemic administration of PRO051 in Duchenne's muscular dystrophy. The New England journal of medicine, 364, 1513-1522.

G5. Cirak, S., Arechavala-Gomeza, V., Guglieri, M., Feng, L., Torelli, S., Anthony, K., Abbs, S., Garralda, M. E., Bourke, J., Wells, D. J. et al. (2011) Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study. Lancet, 378, 595-605.

G6. The New Economics of Orphan Diseases. Genetic Engineering & Biotechnology News, Jan. 1, 2013.

G7. The Economic Power of Orphan Drugs. Thomson Reuters, 2012.

G8. Top 20 orphan drugs by 2018. FiercePharma, Jul. 23, 2013.

G9. Global Market for Orphan Drugs is Expected to Reach $112 Billion in 2017. Drugs.com, August 2013.

G10. Banerjee, A., Sammarco, M. C., Ditch, S., Wang, J. and Grabczyk, E. (2009) A novel tandem reporter quantifies RNA polymerase II termination in mammalian cells. PloS one, 4, e6193.

G11. Gomes-Pereira, M., Hilley, J. D., Morales, F., Adam, B., James, H. E. and Monckton, D. G. (2014) Disease-associated CAG·CTG triplet repeats expand rapidly in non-dividing mouse cells, but cell cycle arrest is insufficient to drive expansion. Nucleic acids research.

G12. Lavedan, C., Grabczyk, E., Usdin, K. and Nussbaum, R. L. (1998) Long uninterrupted CGG repeats within the first exon of the human FMR1 gene are not intrinsically unstable in transgenic mice. Genomics, 50, 229-240.

G13. Entezam, A., Biacsi, R., Orrison, B., Saha, T., Hoffman, G. E., Grabczyk, E., Nussbaum, R. L. and Usdin, K. (2007) Regional FMRP deficits and large repeat expansions into the full mutation range in a new Fragile X premutation mouse model. Gene, 395, 125-134.

G14. Grabczyk, E. and Usdin, K. (1999) Generation of microgram quantities of trinucleotide repeat tracts of defined length, interspersion pattern, and orientation. Analytical biochemistry, 267, 241-243.

G15. BioMarin buys Prosensa for up to $840M, shoots for quick OK of Duchenne drug. FierceBiotech, Nov. 24, 2014.

Example 6
Summary

Without being bound by theory, the GAA·TTC repeats that cause Friedreich ataxia (FRDA) continue to grow in length over time in the tissues that are affected by the disease. Without being bound by theory, this is what causes the gradual onset of Friedreich ataxia, and also what causes its progressive nature. Data indicate that this continued expansion of GAA·TTC repeats requires transcription through the repeat then the sequential actions of several DNA mismatch repair proteins called MutSbeta (MSH2/MSH3 heterodimer) and then MutLgamma (MLH1/MLH3 heterodimer). Transcription is the process of copying the double stranded DNA into RNA so that protein can be made. Subjects, such as humans, need transcription of the frataxin gene. However, during transcription of the GAA·TTC repeat, the repetitive DNA can become misaligned. A small loop in the misaligned DNA can be mistaken for a mismatch by MutSbeta, which binds it, and then attracts MutLgamma. MutLgamma is the protein complex that cuts the DNA in the repeat to start the expansion. Without the cut, there is no expansion. One small part of MLH3, called exon 7, is the knife that does the cutting. In people there are two forms of MLH3, one carries the knife (exon 7), one does not. As described herein, compositions and methods designed to skip exon 7 have been identified, using splice-switching oligonucleotides (SSOs), so that little or no MLH3 carries a knife. The repeat stops expanding in cells that are treated with SSOs. The mouse MLH3 gene (mMlh3) is like that of humans (hMLH3). There is a mouse model of FRDA called “YG-22” that shows tissue specific GAA·TTC repeat expansion. As described herein, testing SSOs to block this expansion in mice serves as a first step heading to human trials.

First, the mouse MLH3 gene was targeted with a panel of SSOs specific to the mouse. This experiment was completed, and the SSOs were tested for efficiency of splice switching in mouse cell lines. The mouse cell lines that were initially used turned out to express little mMlh3, which made the experiments difficult and time consuming. Subsequently, a number of mouse cell lines were tested to find ones that were more like neurons. One of these neuron-like cell lines expressed sufficient mMlh3 to determine that an SSO pair flanking the knife exon of mMlh3 would work much like the human SSOs for hMLH3. Subsequent experiments tested these SSOs in the mice. The first hurdle in the mice was a safety concern. In rare cases that are sequence-specific, SSOs can clump together and cause a blood clot in mice. Initial tests in mice demonstrate that this has not happened, and that the morpholinos were all well tolerated. Subsequent, tests will show how well these SSOs are at splice switching mMlh3 in different mouse tissues and organs, and also the ability of the SSOs to slow repeat expansion in FRDA model mice.

MLH3 is expressed in humans as two isoforms, MLH3 isoform 1 and MLH3 isoform 2, due to alternative splicing. MLH3 isoform 1 includes exon 7, which contains a conserved endonuclease domain, while MLH3 isoform 2 lacks exon 7. It was recently determined that the MLH3 isoform 1 is required for GAA·TTC expansion, while isoform 2 is not. Skipping exon 7 by use of SSOs effectively shifts MLH3 to isoform 2 and stops repeat expansion in human cells. Finally, skipping exon 7 leaves MLH3 isoform 2 intact, so the SSOs will not impact the total cellular ratios of MLH1 and its binding partners PMS2, PMS1 and MLH3.

Forced Exclusion of the Exon Coding for the Mouse MLH3 Endonuclease Domain, as Well as Neighboring Exons as a Backup.

The mouse MLH3 exon structure parallels that of humans except that the endonuclease domain is contained in exon six rather than exon seven. In FIG. 11 this exon is circled in red to highlight it. Although mouse MLH3 is not reported to have isoforms lacking this exon, the exon is 72 bases long, and skipping it leaves the downstream exons in the same translational reading frame just like human MLH3 isoform 2. Consequently, without being bound by theory, it was anticipated that the SSOs targeting this exon to produce results like we found in hMLH3.

Due, in part, to the lack of reported mMlh3 isoform 2 homologues, other exons were also targeted in order to have several viable candidate SSOs to put into the mouse. This provides a backup: 1) in case mMlh3 exon 6 was not as easy to skip as hMLH3 exon 7 and 2) as mentioned in the original grant, the literature indicate that rare adverse effects of vivo-morpholinos may be mediated by sequence-specific interactions of the morpholinos that cause them to aggregate in the bloodstream (1). Consequently, the initial strategy was to target exons 5 and 7 as well as exon 6 in the mouse MLH3 pre-mRNA. The graphic in FIG. 12 serves to visualize that strategy. Optimally, each SSO would serve to exclude the exon it targets, however, in practice a range in efficacy has been identified in the case of human MLH3, and the same is expected in mice. Previously experiments demonstrate that using a pair of SSOs was more effective than a single SSO at the same total morpholino concentration (see FIG. 6 and FIG. 7).

Primary fibroblasts derived from the C57BL/6 mouse were originally proposed to be used to test the SSOs. Part of the reasoning for using an isogenic line was to avoid the possibility that private point mutations in a cell line would interfere with results. Unfortunately, the primary fibroblasts expressed little mMlh3, making the determination of splice switching much more difficult than it had been in the HEK293 cells used in human experiments. NIH3T3 cell line was used, as this cell line might work better. The primary and transformed mouse fibroblastic cells gave similar, if not presentable, results, allowing for decisions to be made about SSO efficacy and excluded the mMlh3 exon 7-targeted SSO mMlh3dr8 from further consideration.

Without be bound by theory, the ability to study repeat expansion and hMLH3 isoforms in HEK293 cells was aided by the neuronal nature of the HEK293 line (2,3). Therefore, after working with fibroblasts, mouse cell lines with a neuronal nature were sought for future experiments. A mouse neuroblastoma cell line called Neuro-2A (4) expressed sufficient mMlh3 for us to complete the testing of the candidate SSOs. For example, experiments such as that shown in FIG. 13 were used to refine doses in order to look at possible synergies between the SSOs. In general, the SSO treatments resulted in the discrete fragments predicted.

For instance, in lanes 4 and 5 in FIG. 13, there are 3 bands corresponding to the 4 possible fragments indicated to the right of the gel image. The bands within each lane are fairly quantitative relative to one another because they are in competition for the same primers, but we did not control for loading between lanes. However, the consistently lower yield of products with use of mMlh3ac4 was verified with real-time PCR quantification of mMlh3 message. Without being bound by theory, this reproducible several fold reduction was related to nonsense-mediated decay due to the frame-shift caused by loss of the 73 base long exon 5. In contrast, skipping exon 6 leaves the reading frame intact.

Although the morpholino mMlh3ac4 was more effective than any other single morpholino in reducing the amount of full-length mMlh3 mRNA, it was not used in mice for several reasons. First, and foremost, the combination of SSOs mMlh3ac5 and mMlh3dr7 produced a reliable switch to the mouse equivalent of hMLH3 isoform 2. This pair has the dual advantages of closely mimicking what will be accomplish in FRDA patients, and not causing degradation of the mMlh3 mRNA, so that the ratio of the isoforms can more readily be detected. In contrast, use of mMlh3ac4 complicated the assays because of the degradation of mMlh3 exon 5-skipped mRNA. In mouse fibroblasts, the products became difficult to detect.

Morpholinos can Sustain mMlh3 Splice Switching in the Mouse.

The FRDA mouse model work is underway and results demonstrate that the mouse versions of the SSOs penetrate tissue and change the mouse MLH3 splicing pattern (FIG. 14). Ongoing studies in the mouse model will track and correlate the expansion of FRDA repeats over time to provide proof of therapeutic efficacy, which will enhance pharmaceutical interest. The results of a test for splice-switching activity in C57BL/6 mice are shown in FIG. 14.

Injection of Higher Doses of In Vivo Morpholinos in Mice Demonstrate No Noticable Adverse Reactions.

A rare, but serious side effect was the possibility that one of the In Vivo-morpholinos would clump and cause a toxic clot in the animals. In the literature, such an adverse event is usually fatal within a few minutes (1). Such a reaction is more likely at a dose above 12 μg/g. So far, no mice have exhibited such adverse events at even 50 μg/g.

The higher dose was more effective, switching a relatively greater fraction of the mMlh3 to isoform 2. Coupled with the lack of adverse events, future experiments will use the higher dose. Finally, because current formulations do not cross the blood brain barrier, the SSOs will be injected directly into brain for future experiments.

Murine SSO Sequences Tested in this Example

Oligo

Identifier
name
Nucleotide Sequence

SEQ ID NO: 45
mMlh3ac4
GAACCTGCGATTCACGGAGATAAGT

SEQ ID NO: 46
mMlh3ac5
TCCACCTACAAAATAATCCAGGATT

SEQ ID NO: 47
mMlh3dr7
AACTACAGACAGATACTTACCAGTA

SEQ ID NO: 48
mMlh3dr8
CATGTCCTCAGGCTACTGACCGTAA

Murine Mlh3

The Mus musculus mutL homolog 3 (Mlh3) gene comprises approximately 36,126 bp contained within the genomic region (GRCm38/mm10) Assembly chr12:85,234,466-85,270,591. The chromosomal location of murine Mlh3 is Accession No: NC_000078.6.

The two major murine Mlh3 variants (mMlh3) comprise:

- 1) Mus musculus mutL homolog 3 (Mlh3), transcript variant 1, mRNA NCBI Reference Sequence: NM_175337.2
- 2) Mus musculus mutL homolog 3 (Mlh3), transcript variant 2, mRNA NCBI Reference Sequence: NM_001304475.1

The two major human Mlh3 variants comprise:

- 1) RefSeq: NM_001040108.1 Homo sapiens mutL homolog 3 (MLH3), transcript variant 1, mRNA.
- 2) RefSeq: NM_014381.2 Homo sapiens mutL homolog 3 (MLH3), transcript variant 2, mRNA.

References Cited in this Example

1) Ferguson, D. P., Dangott, L. J. and Lightfoot, J. T. (2014) Lessons learned from vivo-morpholinos: How to avoid vivo-morpholino toxicity. BioTechniques, 56, 251-256.

2) Graham, F. L., Smiley, J., Russell, W. C. and Nairn, R. (1977) Characteristics of a human cell line transformed by DNA from human adenovirus type 5. The Journal of general virology, 36, 59-74.

3) Shaw, G., Morse, S., Ararat, M. and Graham, F. L. (2002) Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells. The FASEB journal: official publication of the Federation of American Societies for Experimental Biology, 16, 869-871.

4) Olmsted, J. B., Carlson, K., Klebe, R., Ruddle, F. and Rosenbaum, J. (1970) Isolation of microtubule protein from cultured mouse neuroblastoma cells. Proceedings of the National Academy of Sciences of the United States of America, 65, 129-136.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims.

	Number	Date	Country
Parent	15345058	Nov 2016	US
Child	16889050		US

	Number	Date	Country
Parent	16889050	Jun 2020	US
Child	18927273		US

	Number	Date	Country
Parent	PCT/US2015/029724	May 2015	WO
Child	15345058		US

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