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Modified UBE3A gene for a gene therapy approach for angelman syndrome

A genome, seqidno.13 technology, applied in gene therapy, chemical instruments and methods, biochemical equipment and methods, etc., can solve problems such as unsolved diseases

Pending Publication Date: 2018-01-02
UNIV OF SOUTH FLORIDA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, work in this field and proposed therapies, as with steroids, do not address the underlying condition, or in the case of demethylated compounds may lead to other conditions such as autism

Method used

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  • Modified UBE3A gene for a gene therapy approach for angelman syndrome
  • Modified UBE3A gene for a gene therapy approach for angelman syndrome
  • Modified UBE3A gene for a gene therapy approach for angelman syndrome

Examples

Experimental program
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Effect test

Embodiment 1

[0057] To test the efficacy of the secretion signal, GFP was cloned in frame with human insulin, GDNF or IgK signal peptides. The construct was inserted into pTR plasmid and transfected into HEK293 cells (American Type Culture Collection, Manassas, VA). HEK293 cells were cultured in Dulbecco's Modified Essential Medium (DMEM) containing 10% FBS and 1% Pen / Strep at 37°C in 5% CO 2 Grow and subculture at 80% confluency.

[0058] The vector (2 μg / well in a 6-well plate) was transfected into the cells using the PEI transfection method. Two days before transfection, the cells were plated in 6-well plates with DMEM medium at 0.5 x 10 6 cells / well subcultured. Change the medium the night before transfection. Endotoxin-free dH 2 O was heated to about 80°C and polyethyleneimine (Sigma-Aldrich Co. LLC, St. Louis, MO) was dissolved. The solution was allowed to cool to about 25°C, and the solution was neutralized with sodium hydroxide. For each transfected well, add AAV4-STUb vecto...

Embodiment 2

[0062] The mouse-UBE3A vector construct was generated using the pTR plasmid. The mouse (Mus musculus) UBE3A gene was formed from the following cDNA (U82122.1):

[0063]

[0064]

[0065] The cDNA was subcloned and sequenced. The mouse UBE3A gene (SEQ ID No. 1) was fused to the DNA sequence (SEQ ID No. 2) encoding the secretion signal peptide and the HIV TAT sequence (SEQ ID No. 4). The secretion signal peptide has the DNA sequence:

[0066] atg gcc ctg ttg gtg cac ttc cta ccc ctg ctg gcc ctg ctt gcc ctc tgggag ccc aaa ccc acc cag gct ttt gtc (SEQ ID No.2), its encoded protein sequence:

[0067] MALLVHFLPLLALLALWEPKPTQAFV (SEQ ID No. 3);

[0068] At the same time the HIV TAT sequence is:

[0069] tac ggc aga aag aag agg agg cag aga agg aga (SEQ ID No.4), its encoded protein sequence:

[0070] YGRKKRRQRRR (SEQ ID No. 5).

[0071] The construct sequence of SEQ ID No. 1 fused to SEQ ID No. 2 and SEQ ID No. 4 was inserted into the pTR plasmid. The plasmid was cleaved w...

Embodiment 3

[0074] The mouse vector properties of the constructs generated in Example 2 were tested in HEK293 cells (American Type Culture Collection, Manassas, VA). HEK293 cells were cultured in Dulbecco's Modified Essential Medium (DMEM) containing 10% FBS and 1% Pen / Strep at 37°C in 5% CO 2 Grow and subculture at 80% confluency.

[0075] The vector (2 μg / well in a 6-well plate) was transfected into the cells using the PEI transfection method. Two days before transfection, the cells were plated in a 6-well plate at 0.5 x 10 6 cells / well were subcultured in DMEM medium. Change the medium the night before transfection. Endotoxin-free dH 2 O was heated to about 80°C and polyethyleneimine (Sigma-Aldrich Co. LLC, St. Louis, MO) was dissolved. The solution was allowed to cool to about 25°C, and the solution was neutralized with sodium hydroxide. For each transfected well, add AAV4-STUb vector or negative control (medium only) at 2 μg per 200 μL in serum-free DMEM, and add 9 μL of 1 μg / μ...

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Abstract

Angelman Syndrome (AS) is a genetic disorder occurring in one in every 15,000 births. It is characterized by severe mental retardation, seizures, difficulty speaking and ataxia. The gene responsible for AS was discovered to be UBE3A and encodes for E6-AP, an ubiquitin ligase. A unique feature of this gene is that it undergoes maternal imprinting in a neuron-specific manner. In the majority of AS cases, there is a mutation or deletion in the maternally inherited UBE3A gene, although other cases are the result of uniparental disomy or mismethylation of the maternal gene. While most human disorders characterized by severe mental retardation involve abnormalities in brain structure, no gross anatomical changes are associated with AS. We have generated a Ube3a protein with additional sequencesthat should allow the secretion from cells and uptake by neighboring neuronal cells. This would confer a functional E6-AP protein into the neurons and rescue disease pathology.

Description

[0001] Cross References to Related Applications [0002] This application claims U.S. Provisional Application No. 62 entitled "Modified UBE3A Gene for a Gene Therapy Approach for Angelman Syndrome" filed on May 7, 2015 / 158,269, which provisional application is incorporated herein by reference. technical field [0003] The present invention relates to the treatment of Angelman syndrome. More specifically, the present invention provides therapeutic methods and compositions for the treatment of Angelman syndrome. Background technique [0004] Angelman Syndrome (AS) is a genetic disorder affecting neurons that is estimated to occur in about 1 in 15,000 births (Clayton-Smith, Angelman Syndrome Clinical Study, UK: Observations of 82 Affected Individuals (Clinical research on Angelman syndrome in the United Kingdom: observations on 82 affected individuals). Am J Med Genet. 1993 Apr 1;46(1):12-5), but the actual number of AS cases diagnosed may be higher due to misdiagnosis. [...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00C12N9/00C12N15/52C12N15/85
CPCC12N15/52C12N9/104C12N9/93C07K2319/01C07K2319/036C12Y203/02C12Y603/02019C07K2319/02C07K2319/10C12N2750/14143A61P15/08A61P21/00A61P25/00A61P3/04C12N15/85A61K48/005
Inventor 凯文·罗恩·纳什埃德温·约翰·韦贝尔
Owner UNIV OF SOUTH FLORIDA
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