Compositions and methods for treating non-age-associated hearing impairment in a human subject

A composition, mammalian technology, applied in the directions of biochemical equipment and methods, chemical instruments and methods, drug combinations, etc., capable of solving problems such as hearing loss

Pending Publication Date: 2020-12-01
アコーオスインコーポレイテッド
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Pathogenic variants in MT-RNR1 are associated with predisposing to hearing loss and/or with delayed sensorineural hearing loss in the setting of their exposure to certain...

Method used

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  • Compositions and methods for treating non-age-associated hearing impairment in a human subject
  • Compositions and methods for treating non-age-associated hearing impairment in a human subject
  • Compositions and methods for treating non-age-associated hearing impairment in a human subject

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0500] Example 1: Characterization of Human Otoferin Gene, Homologs, Orthologs.

[0501] The otoferlin gene and corresponding mRNA are provided below.

Embodiment 2

[0502] Embodiment 2: Construction of viral vector

[0503] Recombinant AAV was produced by transfection with an adenovirus-free method as used by Xiao et al., J Virol 1999, 73(5):3994-4003. A cis plasmid with AAV ITR, a trans plasmid with AAV Rep and Cap genes, and a helper plasmid with essential regions from the adenovirus genome were co-transfected in 293 cells at a ratio of 1:1:2. The AAV vectors used herein express human or mouse otoferrin under various dual vector strategies using the constructs described below. AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, rh8, rh10, rh39, rh43, and Anc80 were each prepared to encapsulate three sets of otoferlin constructs to test (i) concatenation - Trans-splicing strategy, (ii) heterozygous intron-homologous recombination-trans-splicing strategy, and (iii) exonic homologous recombination strategy, as summarized in Pryadkina et al., Meth Clin Devel 2015, 2:15009 of.

Embodiment 3

[0504] Example 3: Cloning OTOF components

[0505] Oligo-dT and random primed cDNA libraries were constructed from poly(A)+ mRNA from human total fetus, adult brain, heart, kidney and mouse fetal heart as described by Yasunaga et al., Am J Genet 2000, 67:591-600 . These libraries were subjected to RACE-PCR experiments using adapter primers and a series of primers selected from the otoferlin cDNA sequence. The PCR product was directly cloned into pGEM-TEasy vector and sequenced. To isolate the long form of human cDNA (7 kb), a reverse primer (5'-TTCACCTGGGCCCGCA-GCATCCT- 3' (SEQ ID NO:29)).

[0506]Total RNA was extracted from mouse cochlea using the method described in Strenzke et al., EMBO J.35(23):2519-2535, 2016. RT-PCR experiments were performed in various murine and human RNA sources according to the GeneAmp RNA PCR kit. Two primer pairs were used to reconstruct murine cDNA from brain and cochlea, one from exon 1 5'-UTR (5'-AGGCGTGTGAGCCACACTCCACCA-3'(SEQ ID NO:30)) ...

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PUM

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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 portionof an otoferlin protein, and the use of these compositions to treat hearing loss in a subject.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 634,088, filed February 22, 2018; the entire contents of said U.S. Provisional Patent Application are incorporated herein by reference. [0003] sequence listing [0004] This application contains a Sequence Listing, which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy was created on February 21, 2019 and is named SequenceListing.txt. technical field [0005] The present invention generally relates to the use of nucleic acids to treat hearing loss in a human subject. [0006] Background of the invention [0007] The ear is a complex organ classically described as comprising the outer ear, middle ear, inner ear, auditory (hearing) nerve, and auditory system (which processes sound as it travels from the ear to the brain). In addition to detecting sound, the ear al...

Claims

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Application Information

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IPC IPC(8): C12N15/86C12N15/65C07K14/435A61K9/00A61K38/17A61K48/00A61P25/00A61P27/16
CPCA61P25/00A61P27/16C12N15/65C12N15/86C12N2750/14143C12N2800/40C12N2830/50C12N2840/44C12N2830/48A61K48/005A61K48/0075A01K2217/075A01K2227/105A01K2267/0306A01K67/0276A01K2227/103C07K14/705A61K9/19A61K9/5153A61K38/17A61K48/00A61K9/00C07K14/435A61K9/0046C12N2750/14141
Inventor E.J.西蒙斯R.吴E.雷辛格H.阿尔-莫耶德S.库格勒
Owner アコーオスインコーポレイテッド
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