BR-112019000061-B1 - Switching and splicing modulation of pre-mRNA comprising bicyclic framework portions, with enhanced characteristics for the treatment of genetic disorders.

Abstract

The present invention provides antisense splicing switching oligonucleotides with improved characteristics that enhance clinical applicability for the treatment, improvement, prevention, and/or delay of neuromuscular disorders, such as DMD.

Inventors

• JUDITH CHRISTINA THEODORA VAN DEUTEKOM
• PETER CHRISTIAN DE VISSER

Assignees

• BIOMARIN TECHNOLOGIES B.V

Dates

Publication Date

20260310

Application Date

20170705

Priority Date

20160705

Claims (10)

1. 1. Oligonucleotide characterized in that said oligonucleotide comprises: i) at least one 5-methylcytosine base, and ii) at least one blocked nucleotide acid (LNA) of the formula wherein L is a phosphorothioate backbone linkage and B is a nucleobase, and iii) a 2'-O-methyl substitution on all monomers not comprising an LNA, wherein all monomers are linked by phosphorothioate backbone linkages, and wherein said oligonucleotide consists of SEQ ID Nos: 455, 459, 4528, 4531, 4532, 4533, 4535, 4542, 4548 or 4568.
2. 2. Oligonucleotide, according to claim 1, characterized in that it consists of the nucleotide sequence of SEQ ID NO: 4568.
3. 3. Oligonucleotide, according to any one of claims 1 to 2, characterized in that said oligonucleotide induces the modulation of pre-mRNA splicing.
4. 4. Oligonucleotide, according to claim 3, characterized in that said modulation of pre-mRNA splicing alters the production or composition of a protein.
5. 5. Oligonucleotide, according to claim 4, characterized in that said pre-mRNA splicing modulation comprises exon skipping or exon inclusion.
6. 6. Oligonucleotide, according to claim 5, characterized in that said pre-mRNA splicing modulation comprises exon skipping.
7. 7. Oligonucleotide, according to any one of claims 1 to 6, characterized in that said oligonucleotide has an improved parameter by comparison with a corresponding oligonucleotide that does not comprise an LNA.
8. 8. Composition characterized in that it comprises an oligonucleotide as defined in any one of claims 1 to 7.
9. 9. Composition according to claim 8, characterized in that said composition comprises at least one excipient that may further assist in enhancing the targeting and/or dispensing of said composition and/or said oligonucleotide to a tissue and/or a cell and/or into a tissue and/or a cell.
10. 10. Use of an oligonucleotide as defined in any one of claims 1 to 7, or a composition as defined in claim 8 or 9, characterized in that it is for the production of a medicament for the treatment, prevention and/or delay of Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD), or Spinal Muscular Atrophy (SMA).

Description

Field [001] The invention relates to the field of antisense oligonucleotides, more specifically, splicing-switching oligonucleotides for the treatment of genetic disorders, more specifically neuromuscular disorders. The invention relates, in particular, to the use of oligonucleotides with improved characteristics that enhance clinical applicability, as defined herein. Fundamentals of the invention [002] Antisense oligonucleotides (AONs) are under (pre)clinical development for many diseases and conditions, including cancer, inflammatory conditions, cardiovascular diseases, and neurodegenerative and neuromuscular disorders. Their mechanism of action is directed in several directions, such as RNaseH-mediated degradation of target RNA in the nucleus or cytoplasm, modulation of splicing (exon inclusion or skipping) in the nucleus, or inhibition of translation by steric hindrance of ribosomal subunit binding in the cytoplasm. Splicing modulation oligonucleotides or splicing switching (SSOs) were first described for correcting aberrant splicing in human β-globin pre-mRNAs (Dominski and Kole, 1993), and are currently being studied for a variety of genetic disorders including, but not limited to, cystic fibrosis (CFTR gene, Friedman et al., 1999), breast cancer (BRCA1 gene, Uchikawa et al., 2007), prostate cancer (FOLH1 gene, Williams et al., 2006), inflammatory diseases (IL-5Ralfa and MyD88 genes, Karras et al., 2001, Vickers et al., 2006), ocular albinism type 1 (OA1 gene, Vetrini et al., 2006), ataxia telangiectasia (ATM gene, Du et al., 2007), and syndromes of Nevoid basal cell carcinoma (PTCH1 gene, Uchikawa et al., 2007), methylmalonic acidemia (MUT gene, Rincon et al., 2007), preterm labor (COX-2 gene, Tyson-Capper et al., 2006), atherosclerosis (APOB gene, Khoo et al., 2007), propionic acidemia (PCCA, PCCB genes, Rincon et al., 2007), leukemia (c-myc and WT1 genes, Renshaw et al., 2004, Giles et al., 1999), dystrophic epidermolysis bullosa (COL7A1 gene, Goto et al., 2006), familial hypercholesterolemia (APOB gene, Disterer et al., 2013), laser-induced choroidal neovascularization and corneal graft rejection (KDR gene, Uehara et al., 2013). al., 2013), hypertrophic cardiomyopathy (MYBPC3 gene, Gedicke-Hornung et al., 2013), Usher syndrome (USH1C gene, Lentz et al., 2013), Fukuyama-type congenital muscular dystrophy (FKTN gene, Taniguchi-lkeda et al., 2011), laser-induced choroidal neovascularization (FLT1 gene, Owen et al., 2012), cancer (STAT3 and bcl-X genes, Zammarchi et al., 2011, Mercatante et al., 2002) and Hutchinson-Gilford progeria (LMNA gene, Osorio et al., 2011), Miyoshi myopathy (DYSF gene, Wein et al., 2010), spinocerebellar ataxia type 1 (gene ATXN1, Gao et al., 2008), Alzheimer's disease/FTDP-17 tauopathies (MAPT gene, Peacey et al., 2012), myotonic dystrophy (CLC1 gene, Wheeler et al., 2007), and Huntington's disease (Evers et al., 2014). However, AONs with desplicing switching have progressed the furthest in the treatment of neuromuscular disorders such as Duchenne muscular dystrophy (DMD), a spinal muscular dystrophy (type of spinal muscular atrophy (SMA)). [003] Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are the most common forms of muscular dystrophy in childhood. DMD is a severe and lethal neuromuscular disorder that results in wheelchair frame dependence before the age of 12, and patients usually die before the age of 30 due to respiratory or cardiac failure. It is caused by frameshift deletions (~67%) or duplications (~7%) of one or more exons, or by point mutations (~25%) in the 2.24 Mb DMD gene, resulting in the absence of functional dystrophin. BDM is also caused by mutations in the DMD gene, but these maintain the open reading frame, produce semi-functional dystrophin proteins, and result in a typically much milder phenotype and a longer lifespan. Over the past decade, specific splicing modification to restore the disrupted reading frame of the transcript has emerged as a promising therapy for DMD (van Ommen et al., 2008; Yokota et al., 2007; van Deutekom et al., 2007; Goemans et al., 2011; Voit et al., 2014; Cirak et al., 2011). Using highly sequence-specific splicing-switching antisense oligonucleotides (AONs) that bind to the exon flanking or containing the mutation and interfere with its splicing signals, skipping that exon can be induced during the splicing of DMD pre-mRNAs. Despite the resulting truncated transcript, the open reading frame is restored and a protein is produced that is similar to those found in BMD patients. AON-induced exon skipping provides a mutation-specific and therefore personalized therapeutic approach for DMD patients. Since most mutations cluster around exons 45 to 55, skipping a specific exon can be therapeutic for many patients with different mutations. Exon 51 skipping applies to the largest subset of patients (-13%), including those with deletions from exons 45 to 50, 48 to 50, 50, or 52. The applied AONs are chemically m