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-9326992-B2
Publication Date
2016-05-03
Expiration Date
2032-12-07
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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 in subjects by administering antisense oligonucleotides that target human LMNA pre-mRNA. These oligonucleotides modulate aberrant splicing of LMNA pre-mRNA to reduce the expression of aberrantly spliced isoforms encoding progerin, the toxic mutant protein responsible for Hutchinson-Gilford progeria syndrome (HGPS). The antisense oligonucleotides are composed of morpholino subunits linked by phosphorus-containing intersubunit linkages and have specific targeting sequences complementary to exons and introns of the LMNA gene.
The primary problem addressed is the aberrant splicing caused by a single-point mutation (1824C>T) in the LMNA gene activating a cryptic splice donor site that produces progerin. Progerin accumulates on the nuclear membrane causing characteristic nuclear defects and premature aging symptoms in patients with HGPS. Prior art describes antisense oligonucleotides such as splice-switching oligonucleotides (SSOs) that correct aberrant splicing for other diseases, but improved LMNA-targeted oligonucleotide-based treatments for HGPS are needed.
The invention specifically outlines antisense oligonucleotides that are morpholino-based, nuclease resistant, capable of cell uptake, and comprise 12-40 nucleotide bases with targeting sequences complementary to regions within exon 10, intron 10, exon 11, or combinations thereof of human LMNA pre-mRNA. These oligonucleotides may target sequences upstream or downstream of the exon 11 cryptic splice site, and are designed to avoid overlapping the mutation site itself while still effectively modulating splicing. Variants of the oligonucleotides include phosphorodiamidate morpholino oligonucleotides (PMO), PMOs with piperazine-containing linkages (PMOplus), and PMO-X oligonucleotides.
Claims Coverage
The patent contains several independent claims directed to methods of treating Hutchinson-Gilford progeria syndrome using antisense oligonucleotides targeting human LMNA pre-mRNA. These claims specify the chemical structure, targeting sequences, and modifications of the oligonucleotides used.
Use of morpholino-based antisense oligonucleotides modulating aberrant LMNA splicing
Methods for treating HGPS by administering antisense oligonucleotides composed of morpholino subunits and phosphorus-containing intersubunit linkages. These oligonucleotides modulate aberrant splicing of human LMNA pre-mRNA and contain up to 40 nucleotide bases with specific targeting sequences comprising SEQ ID NOs: 3-8, 10-18, and 20-34.
Specific targeting sequences consistent with SEQ ID NOs
The oligonucleotides have targeting sequences comprising or consisting essentially of certain SEQ ID NOs, including 3-7, 11, 14-16, especially SEQ ID NO: 4 or SEQ ID NO: 11.
Oligonucleotide backbone and linkage structure
The oligonucleotides include phosphorodiamidate morpholino oligonucleotides (PMO) and variants having piperazine-containing or 4-aminopiperidinyl-containing intersubunit linkages. The backbone linkages have specific chemical structures with a range of uncharged and cationic linkages, including type (a) and (b1) linkages with defined substituents.
Inclusion of cationic intersubunit linkages in the oligonucleotide backbone
Oligonucleotide backbones may contain about 10%-50% cationic intersubunit linkages to enhance properties, with specific chemical characterizations and distribution of such cationic linkages.
Conjugation to cell-penetrating peptides
The antisense oligonucleotides may be covalently attached to cell-penetrating peptides, preferably arginine-rich peptides. The peptides can be linked at the C-terminus to either the 5′ or 3′ end of the oligonucleotide through one- or two-amino acid linkers.
Pharmaceutical compositions comprising the antisense oligonucleotides
Methods include administering pharmaceutical compositions containing the described antisense oligonucleotides for treatment of HGPS.
Preparation of oligonucleotides with defined chemical and structural features
Claims include oligonucleotides with a backbone comprising morpholino rings joined by intersubunit linkages of defined types (A and B) with detailed chemical structures, presence of at least 5% to 50% linkage (B), and specific substituents ensuring stability, uptake, and specificity.
Use of compounds of formula formulations targeting specific SEQ ID NOs
Claims cover administering compounds of specified formulae with targeting sequences from SEQ ID NOs: 3-8, 10-18, 20-34 with specific chemical variations of linkages and terminal groups.
The claims cover the inventive use of morpholino-based antisense oligonucleotides with defined chemical backbones and targeting sequences complementary to human LMNA pre-mRNA for treating Hutchinson-Gilford progeria syndrome. They include oligonucleotides with mixtures of charged/uncharge linkages, conjugation to peptides for enhanced delivery, and pharmaceutical compositions containing such oligonucleotides.
Stated Advantages
Methods using the oligonucleotides provide improved treatment of diseases associated with LMNA aberrant splicing including progeroid laminopathies like HGPS.
The targeted antisense molecules are nuclease resistant and have enhanced cellular uptake.
Use of oligonucleotides with cationic backbone linkages and/or conjugation to cell-penetrating peptides enhances delivery into mammalian cells.
Oligonucleotides of the invention can modulate splicing of LMNA pre-mRNA without overlapping the mutation site, providing specificity and efficacy.
Documented Applications
Treatment of progeroid diseases and related conditions, including progeroid laminopathies such as Hutchinson-Gilford progeria syndrome (HGPS).
Treatment of age-related conditions and cardiovascular diseases, including atherosclerosis.
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