Methods for treating progeroid laminopathies using oligonucleotide analogues targeting human LMNA
Inventors
Kole, Ryszard • Collins, Francis S. • Erdos, Michael R. • Cao, Kan
Assignees
University of Maryland Baltimore • Progeria Research Foundation Inc • Sarepta Therapeutics Inc • US Department of Health and Human Services
Publication Number
US-9833468-B2
Publication Date
2017-12-05
Expiration Date
2032-12-07
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
Provided are methods of treatment in subjects having progeroid diseases and related conditions which rely upon LMNA-targeted antisense oligonucleotides for reducing expression of one or more aberrantly spliced LMNA mRNA isoforms that encode progerin.
Core Innovation
The invention provides methods for treating progeroid diseases and related conditions through the use of antisense oligonucleotides targeted to the human LMNA pre-mRNA. Specifically, these oligonucleotides modulate aberrant splicing events that generate progerin, a truncated, mutant form of lamin A protein implicated in disorders such as Hutchinson-Gilford progeria syndrome (HGPS). The oligonucleotides comprise morpholino subunits joined by phosphorus-containing intersubunit linkages, possess a substantially uncharged, nuclease-resistant backbone, and contain targeting sequences complementary to exons and introns around LMNA exon 11, including the cryptic splice site activated by the 1824C>T point mutation.
The problem addressed by this invention arises from HGPS being caused primarily by a single-point mutation in the LMNA gene. This mutation activates a cryptic splice donor site leading to the production of progerin, which accumulates abnormally on the nuclear membrane causing cellular and tissue dysfunction manifesting as premature aging, cardiovascular problems, and early death. Prior attempts to correct aberrant splicing using antisense oligonucleotides such as phosphorodiamidate morpholino oligonucleotides (PMOs) have demonstrated potential but there remains a need for improved oligonucleotide-based therapeutic strategies targeting LMNA pre-mRNA to effectively reduce progerin expression in affected subjects.
This invention advances such therapeutic approaches by providing a structurally optimized class of antisense oligonucleotides that are actively taken up by mammalian cells, resistant to nuclease degradation, and specifically designed to hybridize to regions surrounding the exon 11 cryptic splice site, or adjacent exonic or intronic sequences, thereby modulating splicing to reduce mutant LMNA mRNA and progerin protein levels. Additionally, embodiments include oligonucleotides conjugated to cell-penetrating peptides to enhance delivery. The methods also extend to treatment of other progeroid laminopathies, related age-associated conditions, and cardiovascular diseases associated with LMNA aberrations, broadening their clinical applicability.
Claims Coverage
The patent includes two independent claims covering methods of treating HGPS by administering antisense oligonucleotides and oligonucleotide compositions with defined structural features.
Therapeutic method using antisense oligonucleotide targeting LMNA pre-mRNA
A method for treating HGPS by administering an antisense oligonucleotide composed of morpholino subunits linked by phosphorus-containing intersubunit linkages, containing about 12-40 bases, with targeting sequences chosen from specific SEQ ID NOs: 3-8, 10-18, and 20-34. The oligonucleotide is covalently attached to a cell-penetrating peptide and linker of the formula G-CPP, where G is a linker selected from glycine, cysteine, proline, 6-aminohexanoic acid (Ahx), β-alanine (B), and Ahx-B, and CPP is selected from SEQ ID NOS: 39-54.
Oligonucleotide backbone linkage chemistry
The morpholino subunits are joined by phosphorus-containing intersubunit linkages of specified structures, with linkages of type (a) (uncharged) and at least one linkage of type (b1) (cationic piperazinyl modifications with defined substituents including guanidino and hydrocarbon linkers) for enhanced activity and delivery.
Oligonucleotide backbone with defined targeting sequences
An antisense oligonucleotide comprising a backbone of morpholino ring structures joined by intersubunit linkages, binding sequence-specifically via base-pairing moieties to LMNA pre-mRNA target sequences encompassing SEQ ID NOS: 3-8, 10-18, and 20-34, with a specified general structure of intersubunit linkages and defined percentage of linkage (B).
Specific sequence embodiments and peptide conjugates
Methods specify antisense oligonucleotides having targeting sequences consisting essentially of particular SEQ ID NOs such as 4 and 11 and oligonucleotides covalently linked to cell-penetrating peptides attached via linkers at either the 5′ or 3′ termini of the oligonucleotide.
The claims cover therapeutic methods of treating HGPS using antisense morpholino oligonucleotides with optimized backbone linkages and specific targeting sequences, optionally conjugated to cell-penetrating peptides, focused on modulating aberrant LMNA pre-mRNA splicing to reduce progerin expression.
Stated Advantages
The oligonucleotides have a substantially uncharged, nuclease-resistant backbone enabling active uptake by mammalian host cells.
The antisense compounds specifically modulate aberrant splicing of LMNA pre-mRNA, thereby reducing mutant progerin mRNA and protein expression.
Conjugation to cell-penetrating peptides improves cellular delivery and reduces toxicity, enhancing therapeutic windows.
Methods apply broadly to progeroid laminopathies, age-related conditions, and cardiovascular diseases associated with LMNA abnormalities.
Documented Applications
Treatment of progeroid diseases and related conditions such as Hutchinson-Gilford progeria syndrome using LMNA-targeted antisense oligonucleotides.
Therapy for age-related conditions and cardiovascular diseases including atherosclerosis linked to LMNA dysfunction.
Interested in licensing this patent?