Compositions and methods for treating non-age-associated hearing impairment in a human subject
Inventors
Simons, Emmanuel John • Reisinger, Ellen • KÜGLER, Sebastian • Al-Moyed, Hanan
Assignees
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Publication Number
US-11781145-B2
Publication Date
2023-10-10
Expiration Date
Abstract
Provided herein are compositions that include at least two different nucleic acid vectors, where each of the at least two different vectors includes a coding sequence that encodes a different portion of an otoferlin protein, and the use of these compositions to treat hearing loss in a subject.
Core Innovation
The invention provides therapeutic compositions for non-age-associated hearing impairment by delivering human otoferlin isoform 5 gene portions using a plurality of AAV vectors. Each vector contains only a portion of a gene encoding human otoferlin isoform 5 polypeptide rather than full-length otoferlin sequence in a single vector, and the vectors are configured to be capable of constituting full-length otoferlin messenger RNA in a target cell.
The split gene across the first and second AAV vectors is arranged so that full-length otoferlin messenger RNA is produced in mammalian target cells, including cochlear inner hair cells, by recombination/concatemerization and/or RNA splicing to form full-length otoferlin in the target cell. The disclosed configurations include use of inverted terminal repeats and, in at least one vector, a polyadenylation sequence, to support full-length otoferlin messenger RNA formation.
AAV dual-vector strategies are described with packaging capacity constraints in which each vector independently contains packaging capacity of less than about 6 kb. The invention addresses subjects with defective otoferlin, including non-syndromic sensorineural hearing loss associated with defective otoferlin, and includes AAV vector/capsid embodiments used for delivery to the cochlea and inner ear.
Claims Coverage
The independent claims cover three related inventive arrangements: a plurality of AAV vectors configured to reconstitute full-length otoferlin messenger RNA in a target cell, a similar plurality in which both vectors use AAV2 ITRs, and a composition comprising two recombinant AAV vectors. Across the independent claims, the main inventive features relate to split-gene delivery of human otoferlin isoform 5 using two separately packaged AAV vectors with ITRs and a polyadenylation sequence enabling constituting full-length otoferlin messenger RNA.
Split-gene dual-vector AAV reconstituting full-length otoferlin messenger RNA
A plurality of AAV vectors comprising a first AAV vector having an ITR and a promoter operably linked to a portion of a gene encoding a human otoferlin isoform 5 polypeptide and a second AAV vector having an ITR, a portion of the gene encoding the human otoferlin isoform 5 polypeptide and a polyadenylation sequence, where the first and second AAV vectors independently contain packaging capacity of less than about 6 kb and are capable of constituting a full-length otoferlin messenger RNA in a target cell.
Dual-vector split AAV using AAV2 ITRs to constitute full-length otoferlin messenger RNA
A plurality of AAV vectors comprising a first AAV vector having a promoter operably linked to a portion of a gene encoding a human otoferlin isoform 5 polypeptide and a second AAV vector having a portion of the gene encoding the human otoferlin isoform 5 polypeptide and a polyadenylation sequence, where the first and second AAV vectors each comprise an AAV2 ITR and are capable of constituting a full-length otoferlin messenger RNA in a target cell.
Composition of first and second recombinant AAV vectors forming full-length otoferlin messenger RNA
A composition comprising a first recombinant AAV vector packaged into a capsid protein and comprising a promoter operably linked to a portion of a gene encoding a human otoferlin isoform 5 polypeptide, a 5′ ITR and a 3′ ITR, and a second recombinant AAV vector packaged into a capsid protein and comprising a portion of the gene encoding the human otoferlin isoform 5 polypeptide, a polyadenylation sequence, a 5′ ITR and a 3′ ITR, where the first rAAV and the second rAAV are capable of constituting a full-length otoferlin messenger RNA in a target cell.
Collectively, the independent claims require a dual-vector split-gene design for human otoferlin isoform 5 using AAV vector elements so that the two separately packaged vectors together are capable of constituting full-length otoferlin messenger RNA in a target cell, including an embodiment constrained by packaging capacity less than about 6 kb and an embodiment using AAV2 ITRs.
Stated Advantages
Enables constituting full-length otoferlin messenger RNA in a target cell from two separate AAV vectors using split portions of a gene encoding human otoferlin isoform 5.
Supports therapeutic compositions for non-age-associated hearing impairment in subjects with defective otoferlin.
Restored ABR waveforms in the described otoferlin gene-reconstitution context.
Restored otoferlin expression localized to inner hair cells (IHCs) in the described otoferlin gene-reconstitution context.
Restored exocytosis/vesicle replenishment as shown by electrophysiology in the described otoferlin gene-reconstitution context.
Documented Applications
Treatment of non-syndromic sensorineural hearing loss associated with defective otoferlin by delivering split gene portions to mammalian target cells including cochlear inner hair cells.
In vivo cochlear delivery in juvenile sheep, with ABR/DPOAE monitoring and histology/qPCR for otoferlin expression plus apoptosis assessment in the context of otoferlin gene reconstitution.
Generation of CRISPR-engineered large-animal OTOF models in sheep, with genotyping and expression analyses in the context of otoferlin gene-reconstitution strategy evaluation.
Human clinical delivery example using round-window infusion with AAV-OTOF in the context of otoferlin gene-reconstitution.
Broader vector strategy evaluation including trans-splicing dual-AAV design elements, hybrid recombination concepts, and alternative modalities including single viral vectors and non-viral delivery in the described otoferlin reconstitution context.
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