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An optimized acceptor splice site module for biological and biotechnological applications

A splice site, receptor technology, applied in the field of new receptor splicing regions, can solve the problem of low in vivo efficiency of rAAV dual vector system

Pending Publication Date: 2021-10-01
维格内罗有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reported in vivo efficiency of reconstituting this rAAV dual-vector system is relatively low, i.e. less than 10% (Carvalho et al., "Evaluating efficiencies of dual AAV approaches for retina targeting", (2017) Frontiers in Neuroscience, 11(503) :1-8)

Method used

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  • An optimized acceptor splice site module for biological and biotechnological applications
  • An optimized acceptor splice site module for biological and biotechnological applications
  • An optimized acceptor splice site module for biological and biotechnological applications

Examples

Experimental program
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Effect test

Embodiment 1

[0703] Example 1: Identification of optimized ASS modules

[0704] The effect of disease-associated rhodopsin mutations on mRNA splicing was analyzed using the human rhodopsin (RHO) minigene in HEK293 cells and transduced murine photoreceptor cells. Among them, we found a mutation (c.620T>G) in exon 3 of the RHO gene, which produces a new ASS ( figure 1 ). The sequence of ASS and predicted ASS elements as figure 2 A and B are shown.

[0705]In silico predictions revealed that the receptor splice site formed by the c.620T>G mutation (hereinafter referred to as ASS_620) has a similar splicing fraction to the native rhodopsin receptor splice site in exon 3 (see image 3 A). This suggests that the splicing machinery can alternately use the two splice sites. However, experimental data suggest that ASS_620 is exclusively for HEK293 cells ( figure 1 D) and mouse photoreceptors ( figure 1 E). This suggests that ASS_620 is a strong acceptor splice site.

[0706] For potential...

Embodiment 2

[0708] Example 2: Cerulein reporter assay for in vitro applications

[0709] To test this hypothesis, a splicing reporter assay was generated by splitting the coding sequence of azurein into two artificial exons interrupted by an artificial intron ( Figure 4 D). The intron contains vgASS_620 and a strong donor splice site, separated from each other by an artificial 145bp. Confocal imaging of transfected HEK293 cells reveals strong and robust azurein fluorescence when both splice sites are presented in cis ( Figure 4 F(c1)). RT-PCR experiments and sequencing of the corresponding bands confirmed that, in this configuration, the two azure exons are efficiently spliced ​​together (data not shown). Such as Figure 4 As shown in G, a specific immune signal of the expected size (27 kDa) was detected by Western blot using an antibody specific for the N-terminal portion of azurin. Taken together, these findings suggest that reporter assays are functional and highly efficient in ...

Embodiment 3

[0725] Example 3: Effect of acceptor splice site versus binding domain (BD) on remodeling efficiency.

[0726] BD length and sequence represent key determinants of remodeling efficiency. BDs most likely affect the tight binding and potential folding of mRNAs, but they are not expected to directly contribute to the efficiency or precision of the subsequent splicing process. As mentioned above, DSS is well characterized and predictions of its strength match well with its experimental performance. Therefore, there is no apparent need to optimize this splice site within the framework of split fluorophore remodeling assays. In contrast, the strength of ASS cannot be reliably predicted due to its complexity. The results showed that vgASS_620 is a particularly strong receptor splice site. Considering that splice site strength can affect remodeling efficiency for split fluorophore assays, vgASS_620 should result in higher values ​​compared to other acceptor splice sites.

[0727] To...

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Abstract

The present invention relates to an acceptor splice region, as well as uses and applications thereof.

Description

technical field [0001] The present invention relates to a new acceptor splicing region and its uses and applications. Background technique [0002] Most eukaryotic genes contain noncoding introns that must be removed from precursor messenger RNA (pre-mRNA) in a process called "splicing" to produce a translatable mature messenger RNA ( mRNA). Splicing is mediated through a large ribonucleoprotein complex (i.e., the spliceosome), which consists of five conserved small nuclear ribonucleoproteins (snRNPs) (i.e., U1, U2, U4, U5, and U6 snRNPs), and More than 300 kinds of protein composition. The spliceosome assembles and disassembles for each intron in a highly dynamic process. For this, specific splicing sequences need to be identified, and thus the boundaries of exons and introns are defined by distinct sequence motifs that are the binding sites for ribonucleoproteins involved in regulating mRNA splicing. [0003] The most relevant splicing motifs are canonical donor and ac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/86
CPCC12N15/86C12N2750/14143C12N2840/44C12N2840/445C12N15/113A61P27/02A61K35/76A61K48/0066C12N15/102C12N2320/33
Inventor 斯蒂利亚诺斯·米查拉基斯马丁·比尔埃尔维尔·贝西洛维奇
Owner 维格内罗有限责任公司
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