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Modified ube3a gene for a gene therapy approach for angelman syndrome

A composition and sequence technology, applied in gene therapy, chemical instruments and methods, biochemical equipment and methods, etc., can solve problems such as preventing the overall distribution of proteins

Pending Publication Date: 2020-03-06
UNIV OF SOUTH FLORIDA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, only a small number of neurons are successfully transduced and thus express the protein, preventing the overall distribution of the protein in the brain as required for effective therapy

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
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1 - Efficiency of secretion signaling

[0096] To test the efficacy of the secretion signal, GFP (SEQ ID No: 1 ) (XM 013480425.1 ) was cloned in frame with human insulin, GDNF (SEQ ID No: 2) (AB675653.1 ) or the IgK signal peptide.

[0097] ATGGCTCGTC TTTCTTTTGT TTCTCTTCTT TCTCTGTCAC TGCTCTTCGG GCAGCAAGCAGTCAGAGCTC AGAATTACAC CATGGTGAGC AAGGGCGAGG AGCTGTTCAC CGGGGTGGTG CCCATCCTGGTCGAGCTGGA CGGCGACGTA AACGGCCACA AGTTCAGCGT GTCCGGCGAG GGCGAGGGCG ATGCCACCTACGGCAAGGAC TGCCTGAAGT TCATCTGCAC CACCGGCAAG CTGCCCGTGC CCTGGCCCAC CCTCGTGACCACCTTCGGCT ACGGCCTGAT GTGCTTCGCC CGCTACCCCG ACCACATGAA GCAGCACGAC TTCTTCAAGTCCGCCATGCC CGAAGGCTAC GTCCAGGAGC GCACCATCTT CTTCAAGGAC GACGGCAACT ACAAGACCCGCGCCGAGGTG AAGTTCGAGG GCGACACCCT GGTGAACCGC ATCGAGCTGA AGGGCATCGA CTTCAAGGAGGACGGCAACA TCCTGGGGCA CAAGCTGGAG TACAACTACA ACAGCCACAA CGTCTATATC ATGGCCGACAAGCAGAAGAA CGGCATCAAG GTGAACTTCA AGATCCGCCA CAACATCGAG GACGGCAGCG TGCAGCTCGCCGACCACTAC CAGCAGAACA CCCCCATCGG CGACGGCCCC GTGCTGCTGC CCGACAAC...

Embodiment 2

[0105] Example 2 - Mouse UBE3A Vector Constructs

[0106] The pTR plasmid was used to generate mouse UBE3A vector constructs. The mouse (Mus musculus) UBE3A gene is formed by cDNA (U82122.1);

[0107] ATGAAGCGAG CAGCTGCAAA GCATCTAATA GAACGCTACT ACCATCAGTT AACTGAGGGCTGTGGAAATG AGGCCTGCAC GAATGAGTTT TGTGCTTCCT GTCCAACTTT TCTTCGTATG GATAACAATGCAGCAGCTAT TAAAGCCCTT GAGCTTTATA AAATTAATGC AAAACTCTGT GATCCTCATC CCTCCAAGAAAGGAGCAAGC TCAGCTTACC TTGAGAACTC AAAAGGTGCA TCTAACAACT CAGAGATAAA AATGAACAAGAAGGAAGGAA AAGATTTTAA AGATGTGATT TACCTAACTG AAGAGAAAGT ATATGAAATT TATGAATTTTGTAGAGAGAG TGAGGATTAT TCCCCTTTAA TTCGTGTAAT TGGAAGAATA TTTTCTAGTG CTGAGGCACTGGTTCTGAGC TTTCGGAAAG TCAAACAGCA CACAAAGGAG GAATTGAAAT CTCTTCAAGA AAAGGATGAAGACAAGGATG AAGATGAAAA GGAAAAAGCT GCATGTTCTG CTGCTGCTAT GGAAGAAGAC TCAGAAGCATCTTCTTCAAG GATGGGTGAT AGTTCACAGG GAGACAACAA TGTACAAAAA TTAGGTCCTG ATGATGTGACTGTGGATATT GATGCTATTA GAAGGGTCTA CAGCAGTTTG CTCGCTAATG AAAAATTAGA AACTGCCTTCCTGAATGCAC TTGTATATCT GTCACCTAAC GTGGAATG...

Embodiment 3

[0116] Example 3 - In Vitro Testing of Mouse UBE3A Vector Constructs

[0117] 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% penicillin / streptomycin at 37°C in 5% CO 2 Grow and subculture at 80% confluency.

[0118] The vector (2 μg / well in a 6-well plate) was transfected into cells using the PEI transfection method. Two days before transfection, cells were incubated with 0.5 × 10 6 Cells / well were subcultured in DMEM medium in 6-well plates. The medium was changed the night before transfection. Endotoxin-free dH 2 O was heated to 80°C and polyethyleneimine (Sigma-Aldrich Co. LLC, St. Louis, MO) was dissolved. The solution was cooled to about 25°C, and the solution was neutralized with sodium hydroxide. For each transfection well, add AAV4-STUb vector or negative control (m...

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Abstract

The present invention provides a novel vector, composition and method of treating a neurological disorder characterized by deficient UBE3A. The UBE3A gene, which encodes for E6-AP, a ubiquitin ligase,was found to be responsible for Angelman syndrome (AS). 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. A UBE3A protein construct was generated withadditional sequences that allow the secretion from cells and uptake by neighboring neuronal cells. The UBE3A vector may be used in gene therapy to confer a functional E6-AP protein into the neurons and rescue disease pathology.

Description

[0001] Cross References to Related Applications [0002] This application is the U.S. Provisional Patent Application Serial No. 62 entitled "Modified UBE3A Gene for a Gene Therapy Approach for Angelman Syndrome" filed on June 28, 2017 / 525,787, the contents of which are hereby incorporated by reference into the present disclosure. 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 approximately 1 in 15,000 births (Clayton-Smith, Clinical Study of Angelman Syndrome, UK: A Study of 82 Affected Individuals observations (Clinical research on Angelman syndrome in the United Kingdom: observations on82 affected individuals), Am J Med Genet. 1993 Apr 1; ​​46(1):12-5), but the actual number of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/711C12N15/85C12P19/34
CPCA61K31/711C12Y603/02019C12N9/93A61K48/005C12N15/86C12N2750/14143A61K2300/00A61P25/28A61K35/761A61K48/0058A61K48/0066C07K14/62C12N2810/40C12N2810/854C12N2840/007
Inventor 凯文·罗恩·纳什埃德温·约翰·韦贝尔
Owner UNIV OF SOUTH FLORIDA
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