Tricyclo-DNA antisense oligonucleotides, compositions, and methods for the treatment of disease
Provided are tricyclo-DNA (tc-DNA) AON and methods employing tc-DNA AON for modifying splicing events that occur during pre-mRNA processing. Tricyclo-DNA (tc-DNA) AON are described that may be used to facilitate exon skipping or to mask intronic silencer sequences and/or terminal stein-loop sequences during pre-mRNA processing and to target RNase-mediated destruction of processed mRNA. Tc-DNA AON described herein may be used in methods for the treatment of Duchenne Muscular Dystrophy by skipping a mutated exon 23 or exon 51 within a dystrophin gene to restore functionality of a dystrophin protein; in methods for the treatment of Spinal Muscular Atrophy by masking an intronic silencing sequence and/or a terminal stem-loop sequence within an SMN2 gene to yield modified functional SMN2 protein, including an amino acid sequence encoded by exon 7, which is capable of at least partially complementing a non-functional SMN1 protein; and in methods for the treatment of Steinert's Myotonic Dystrophy by targeting the destruction of a mutated DM1 mRNA comprising 3′-terminal CUG repeats.
1. A method for the delivery of an antisense oligonucleotide (AON) into a human patient comprising administering to the patient a tricyclo-DNA (tc-DNA) antisense oligonucleotide (AON),
wherein the tc-DNA AON has a length of 10 to 18 nucleotides, wherein all the nucleotides of the tc-DNA AON are tc-DNA nucleotides;
wherein the tc-DNA AON comprises a sequence of nucleotides that is complementary to an intron-exon junction; and
wherein the administration of the tc-DNA AON to the patient results in increased exon skipping at the intron-exon junction in the patient.
2. The method of claim 1 , wherein the tc-DNA AON comprises a sequence of nucleotides that is complementary to an intron-exon junction within a mutated dystrophin pre-mRNA.
3. The method of claim 2 , wherein the administration of the tc-DNA AON to the patient results in increased skipping of exon 23 in the patient.
4. The method of claim 2 , wherein the wherein the administration of the tc-DNA AON to the patient results in increased skipping of exon 51 in the patient.
5. The method of claim 1 , wherein the tc-DNA AON has a length of 12 to 16 nucleotides.
6. The method of claim 1 , wherein the tc-DNA AON has a length of 13 to 15 nucleotides.
7. The method of claim 1 , wherein the administration comprises intramuscular (i.m.), intravenous (i.v.), intraperitoneal (i.p.), intra-arterial, sub-cutaneous or transdermic injection of the tc-DNA AON.
8. The method of claim 1 , comprising administering 1 mg/kg to 100 mg/kg body weight/day to the patient.
9. The method of claim 1 , comprising injecting the tc-DNA AON directly into the muscle of the patient.
10. The method of claim 1 , wherein 8-16 nucleotides of said tc-DNA AON are complementary to a dystrophin pre-mRNA intronic splice donor site, wherein 2-8 nucleotides of said tc-DNA AON are complementary to a dystrophin pre-mRNA exonic region, and wherein said exonic region is contiguous with and 3′ to said intronic splice donor site.
11. The method of claim 1 , wherein the tc-DNA AON comprises the nucleotide sequence 5′-AACCTCGGCTTACCT-3′ (SEQ ID NO: 1).
12. The method of claim 1 , wherein the tc-DNA AON is complementary to the nucleotide sequence 5′-AGAAATGCCATCTTC-3′ (SEQ ID NO: 2).
13. The method of claim 1 , wherein the tc-DNA AON is complementary to the nucleotide sequence 5′-AAATGCCATCTTCCT-3′ (SEQ ID NO: 3).
14. The method of claim 1 , wherein said tc-DNA AON is complementary to the nucleotide sequence 5′-TGCCATCTTCCTTGA-3′ (SEQ ID NO: 4).
15. The method of claim 1 , wherein the patient has Duchene Muscular Dystrophy (DMD).