Methods and compositions for increasing protein expression and/or treating a haploinsufficiency disorder

A dose-and-deficient technology for use in biochemical devices and methods, drug combinations, gene therapy, etc., to address problems such as language and motor dysfunction

Pending Publication Date: 2021-07-30
CASE WESTERN RESERVE UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This leads to impaired language

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  • Methods and compositions for increasing protein expression and/or treating a haploinsufficiency disorder
  • Methods and compositions for increasing protein expression and/or treating a haploinsufficiency disorder
  • Methods and compositions for increasing protein expression and/or treating a haploinsufficiency disorder

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preparation example Construction

[0141] Preparation of viral vectors

[0142] Methods of preparing viral vectors are known in the art. Generally, known viruses are prepared in suitable host cell lines using conventional techniques, including culturing transfected or infected host cells under suitable conditions so as to produce infectious virus particles. Nucleic acids encoding viral genes and / or tRNAs can be integrated into plasmids and introduced into host cells by conventional transfection or transformation techniques. Examples of host cells for producing known viruses include human cell lines such as HeLa, Hela-S3, HEK293, 911, A549, HER96 or PER-C6 cells. Specific preparation and purification conditions will vary depending on the virus and production system used.

[0143] In certain embodiments, producer cells can be administered directly to a subject, however, in other embodiments, following production, infectious viral particles are recovered from culture and optionally purified. Typical purificatio...

Embodiment

[0173] The following examples are illustrative only and are not intended to limit the scope or content of the invention in any way.

[0174] Example 1 - tRNA expression that enhances expression of wild-type SCN1A transcript

[0175] In this example, the least desirable and most overexpressed codons in SCN1A were determined. Expression of cognate tRNAs to these codons could enhance SCN1A expression and treat Dravet syndrome.

[0176] First, the percent usage of each codon in the SCN1A gene was determined. Subsequently, the optimality of each codon in the SCN1A gene was determined using the tRNA fitness index (tAI). tAI is an estimate of translation efficiency for each codon, which takes into account estimates of tRNA cellular concentration (based on tRNA gene copy number) and decoding efficiency, with higher tAI values ​​indicating more optimal codons. For example, tAI is described in dos Reis et al., (2004) NUCLEIC ACIDS RES. 32(17):5036-5044 and Mahlab et al., (2014) PLoS ...

Embodiment 2

[0182] Example 2 - tRNA expression that enhances expression of wild-type SCN1A transcript

[0183] In this example, the ability of ectopic delivery of selection transfer RNAs (tRNAs) to enhance wild-type SCN1A expression in cultured cells was assessed.

[0184] ATA (Ile), GTA (Val) and AGA (Arg) were chosen as exemplary non-optimal codons in the SCN1A gene. Described in SEQ ID NO: 1 coding ATA tRNA (tRNA ATA ), described in SEQ ID NO: 2 encoding GTA tRNA (tRNA GTA ) of the nucleotide sequence described in SEQ ID NO: 3 encoding AGA tRNA (tRNA AGA ) nucleotide sequence.

[0185] The SCN1A open reading frame was amplified from human brain RNA (Clontech) by RT-PCR using an upstream primer containing a KpnI site, a downstream primer containing a NotI site. The SCN1A open reading frame was then cloned between the promoter and the SV40 poly(A) site of pTRE-Tight (Clontech) using KpnI and NotI sites.

[0186] tRNA ATA , tRNA GTA and tRNA AGA The gene sequence (including 200 ba...

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Abstract

A tRNA that hybridizes to a non-optimal codon can be used to increase expression in a mammalian cell of a gene product encoded by a gene containing the non-optimal codon or to treat a haploinsufficiency disorder in a subject having a haploinsufficient gene containing the non-optimal codon.

Description

[0001] priority statement [0002] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 736,847, filed September 26, 2018, which is hereby incorporated by reference in its entirety. technical field [0003] The present disclosure generally relates to methods and compositions for increasing expression in a subject of a gene product encoded by a gene comprising a non-optimal codon and / or treating haploinsufficiency in the subject. Background technique [0004] Haploinsufficiency occurs when one allele is inactive and the amount of gene product expressed by the remaining active allele is insufficient to maintain normal gene function. Many diseases are associated with, or are caused by, haploinsufficiency. [0005] An example of haploinsufficiency is Dravet syndrome. Dravet syndrome is a rare catastrophic intractable epilepsy that begins in infancy. Initially, patients experience prolonged seizures. During the following year, other types of s...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/63C12N15/67
CPCC12N15/67C12N2320/34C12N2330/51C12N15/113A61K48/00A61P25/08C12N2750/14143A61P43/00A61K31/7088A61K45/06A61K48/0066C12N15/11C12N15/86
Inventor 杰弗里·M·科莱尔托马斯·斯威特哈维·洛迪什
Owner CASE WESTERN RESERVE UNIV
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