Methods for treating progeroid laminopathies using oligonucleotide analogues targeting human LMNA
Inventors
Kole, Ryszard • Collins, Francis S. • Erdos, Michael R. • Cao, Kan • Gordon, Leslie B.
Assignees
University of Maryland Baltimore • Progeria Research Foundation Inc • Sarepta Therapeutics Inc • US Department of Health and Human Services
Publication Number
US-10398721-B2
Publication Date
2019-09-03
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 by administering antisense oligonucleotides targeted to human LMNA pre-mRNA to reduce expression of aberrantly spliced LMNA mRNA isoforms that encode progerin. These antisense oligonucleotides are composed of morpholino subunits with phosphorus-containing intersubunit linkages, have a substantially uncharged, nuclease resistant backbone, are capable of uptake by mammalian cells, and contain targeting sequences complementary to regions of the human LMNA pre-mRNA including exon 10, intron 10, exon 11, or combinations thereof.
The technical problem addressed is that Hutchinson-Gilford progeria syndrome (HGPS) and other progeroid laminopathies are caused by aberrant splicing of LMNA pre-mRNA due to a mutation that activates a cryptic splice site producing progerin, a dominant negative mutant lamin A protein. Existing treatments are insufficient, and there is a need for improved oligonucleotide-based therapies capable of specifically modulating the aberrant splicing to reduce progerin expression.
The disclosed methods include antisense oligonucleotides that hybridize near but not overlapping the LMNA exon 11 cryptic splice site, targeting sequences complementary to specific bases of LMNA exons and introns, and the use of specific backbone linkages such as uncharged phosphorodiamidate morpholino oligonucleotides (PMOs) and related chemistries. These oligonucleotides can be conjugated to cell-penetrating peptides, enhancing cellular uptake and therapeutic efficacy. The invention also contemplates pharmaceutical compositions comprising such oligonucleotides for treating progeroid laminopathies like HGPS and related diseases.
Claims Coverage
The patent presents two independent claims directed to methods of treating atherosclerosis using antisense oligonucleotides targeting human LMNA pre-mRNA, with detailed structural and compositional features.
Use of morpholino-based antisense oligonucleotides targeting human LMNA pre-mRNA to treat atherosclerosis
Administering to a subject an antisense oligonucleotide composed of morpholino subunits with phosphorus-containing intersubunit linkages, containing about 12-40 bases, and having a targeting sequence comprising specific SEQ ID NOs, to modulate aberrant splicing of human LMNA pre-mRNA.
Attachment of cell-penetrating peptide with linker to antisense oligonucleotide
The antisense oligonucleotide is covalently attached to a cell-penetrating peptide and linker moiety (such as glycine, cysteine, proline, 6-aminohexanoic acid, β-alanine, and combinations thereof), where the cell-penetrating peptide is selected from specific SEQ ID NOs sequences.
Specific phosphorus-containing intersubunit linkages in morpholino oligonucleotides
Morpholino subunits are joined by phosphoroamidate or phosphorodiamidate linkages according to specific structural formulas including uncharged type (a) linkages and cationic type (b1) linkages with defined R groups and linkers.
Structural and positional details of linkages
Details such as R1 and R2 being methyl in linkages of type (a); presence of at least one type (b1) linkage with R groups as hydrogen, methyl, or specific guanidino derivatives; linkers L being hydrocarbons of defined chain length; and other structural characteristics.
Positioning of cell-penetrating peptides on oligonucleotides
The cell-penetrating peptide can be attached either at the 5' end or the 3' end of the antisense oligonucleotide via its C-terminus.
Oligonucleotide backbone comprising morpholino ring structures with defined linkages
Use of oligonucleotides comprising morpholino rings joined by intersubunit linkages with specified general structures, where a defined proportion of these linkages are type (B) and share the same structure.
Targeting sequence specificity to SEQ ID NOs
The targeting sequences comprise any one of SEQ ID NOs: 3-8, 10-18, and 20-34, including specific embodiments where the sequence consists essentially of SEQ ID NO:4 or SEQ ID NO:11.
The independent claims collectively cover methods of treating atherosclerosis utilizing specifically structured antisense morpholino oligonucleotides that modulate LMNA pre-mRNA splicing. The claims emphasize the chemical composition of the oligonucleotides, their specific targeting sequences, and the conjugation to cell-penetrating peptides for enhanced delivery.
Stated Advantages
Reduced expression of pathogenic progerin protein by modulating aberrant LMNA pre-mRNA splicing in progeroid diseases.
Use of nuclease-resistant, substantially uncharged morpholino antisense oligonucleotides facilitating cellular uptake and stability.
Enhanced cellular delivery and uptake through conjugation with cell-penetrating peptides.
Ability to target LMNA pre-mRNA regions near but not overlapping the cryptic splice site, improving therapeutic specificity.
Applicability to treating progeroid laminopathies as well as age-related and cardiovascular conditions such as atherosclerosis.
Documented Applications
Treatment of progeroid diseases and related conditions including Hutchinson-Gilford progeria syndrome (HGPS).
Treatment of progeroid laminopathies generally.
Treatment of age-related conditions and cardiovascular diseases including atherosclerosis.
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