Oligonucleotides for treating expanded repeat diseases
Inventors
Linsley, Peter • Leppert, Brian James • Hanson, Gunnar J.
Assignees
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Publication Number
US-11674139-B2
Publication Date
2023-06-13
Expiration Date
Abstract
The invention provides for a method for selectively reducing the expression of a mutant mRNA and/or protein having an expanded nucleotide repeat relative to a wild-type mRNA, comprising contacting a cell with an antisense oligonucleotide of sufficient length and complementarity to the expanded nucleotide repeat. More particularly it relates to selectively reducing the expression of mutant Huntington protein associated with Huntington's disease. The antisense oligonucleotide comprising either a nucleotide or a repeated three nucleotide sequence as defined in the claims.
Core Innovation
The invention relates to antisense oligonucleotides of 10-40 nucleotides having sequences complementary to expanded DNA repeat(s) associated with human disease. It focuses on expanded nucleotide repeat disorders such as Huntington’s disease, including expanded HTT CAG repeats, and also addresses ALS, including C9ORF72 GGGGCC repeats. The oligonucleotides are directed to mutant transcripts/proteins produced from the expanded repeat-containing allele.
The antisense oligonucleotides are presented in multiple chemistries and structural forms, including morpholino oligomer variants and additional defined backbone/intersubunit linkage options, including APN and related linkage selections, and cationic linkages such as “plus,” “etpip,” and “mapT.” Nucleobase/base-pairing moieties and backbone/intersubunit linkages are extensively defined using structural options for substituents, including selections for R1, Rx, Ry, and Rz.
The document further specifies that the antisense sequences are selected from repeat-derived motifs, including (CAG)7, (CCG)n, (CTG)n, (TTC)n, (NGC)n, (GNC)n, (AGAAT)n, and (CGCG4CG4)n, with n from 3 to 10, and includes exemplary repeat-complementary sequences such as (GCT)7G. It describes improved selectivity and potency for specific chemistries in terms of reducing mutant-versus-wild-type allele expression, and it also provides diagnostic and assessment approaches using Northern blotting with an oligonucleotide probe and determining cellular responsiveness by measuring mutant versus wild-type mRNA/protein levels after introduction of the oligonucleotide.
Claims Coverage
The provided claim set includes three independent claims. Across these independent claims, the coverage centers on antisense oligonucleotides of defined length having repeat-complementary sequences for human disease-associated expanded DNA repeats, specific construction from repeated three-nucleotide sequence motifs with constrained repeat counts and nucleobase/backbone substituent options, and a narrowly defined exemplar sequence and substituent configuration for a specific repeated motif.
Antisense oligonucleotide with disease-associated expanded DNA repeat complementary sequence and defined nucleotide structure
An antisense oligonucleotide of 10-40 nucleotides having a sequence complementary to an expanded DNA repeat associated with a human disease, where each nucleotide has a structure defined by Nu and selectable substituent options R1, Rx, Ry, and Rz, and where the sequence is selected from (CAG)7, (CCG)n, (CTG)n, (TTC)n, (NGC)n, (GNC)n, (AGAAT)n, and (CGCG4CG4)n with n from 3 to 10.
Antisense oligonucleotide from repeated three-nucleotide sequence with constrained repeat count and selectable substituents
An antisense oligonucleotide comprising a repeated three nucleotide sequence consisting of a formula where Nu1, Nu2, and Nu3 are nucleobases selected from adenine, guanine, thymine, uracil, cytosine, and hypoxanthine; n is from about 3 to about 10; and R1, Rx, Ry, and Rz are defined by selectable groups, wherein the nucleotide sequence is selected from (CAG)7, (CCG)n, (CTG)n, (TTC)n, (NGC)n, and (GNC)n with n from 3 to 10.
Narrowly defined antisense oligonucleotide with repeated (CAG)7 and fixed substituent identities
An antisense oligonucleotide consisting of a repeated three nucleotide sequence having the formula, wherein the repeated three nucleotide sequence is (CAG)7, n is 7, R1 is —N(Me)2, Rx is HO—, Ry is hydrogen, and Rz is null.
The independent claims broadly cover antisense oligonucleotides designed to bind disease-associated expanded DNA repeat sequences using defined nucleotide structural options and repeat-derived motifs, with additional narrower coverage for antisense oligonucleotides constructed from repeated three-nucleotide motifs and for a specific fixed configuration corresponding to (CAG)7 with defined substituent identities.
Stated Advantages
Improved selectivity and potency for APN/PMOX versus other chemistries based on reduction of mutant-versus-wild-type allele expression.
Enhanced activity in an in vivo eGFP-654 reporter assay for APN-modified oligomers targeting expanded HTT repeats.
Selective reduction of mutant HTT mRNA and mutant HTT protein relative to wild-type in assays using RT-PCR and Western blot.
Demonstrated selectivity differences among modified antisense oligonucleotides and alternative cationic intersubunit linkages in expanded repeat targeting.
Documented Applications
Assessment of cellular responsiveness by measuring mutant versus wild-type mRNA/protein levels using Northern blotting with an oligonucleotide probe after introduction of the oligonucleotide.
Reducing mutant HTT mRNA and mutant HTT protein versus wild-type in expanded HTT repeat targeting assays using RT-PCR and Western blot.
In vivo testing in eGFP-654 mice using an ICV reporter assay for expanded HTT repeat targeting activity.
Therapeutic use for nucleotide repeat disease animal models as concluded in the document.
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