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Gene therapy for mucopolysaccharidosis, type ii

A polynucleotide, HIV-1 technology, applied in the field of gene therapy composition, Hunter's syndrome, and treatment of type II mucopolysaccharidosis, can solve the problem of uncured Hunter's syndrome

Inactive Publication Date: 2019-09-03
BLUEBIRD BIO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Although treatment can increase the length and quality of life of children with Hunter syndrome, there is no cure for Hunter syndrome, and people with the severe form often die before middle age due to heart disease, airway obstruction or death from severe neurological injury

Method used

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  • Gene therapy for mucopolysaccharidosis, type ii
  • Gene therapy for mucopolysaccharidosis, type ii
  • Gene therapy for mucopolysaccharidosis, type ii

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0269] Construction of the I2S vector

[0270] The following were constructed: a third-generation lentiviral vector containing a chimeric 5′LTR; myeloproliferative sarcoma virus enhancer negative control region deleted (MND) promoter or short elongation factor 1α (EF1α) promoter with dl587rev primer binding site replacement a polynucleotide encoding an iduronate-2-sulfatase (I2S) polypeptide; and a self-inactivating (SIN) 3'LTR. See for example, figure 1 and SEQ ID NO: 1 and 2. Tables 1 and 2 show the identity, Genbank Reference, source name, and citation of each nucleotide fragment of an exemplary lentiviral vector encoding I2S.

[0271] Table 1: pMND-I2S LVV

[0272]

[0273]

[0274]

[0275] Table 2: pEF1α-I2S LVV

[0276]

[0277]

example 2

[0279] Fibroblasts transduced with a lentiviral vector encoding I2S

[0280] Human fibroblasts lacking I2S activity due to a homozygous mutation of the I2S gene (I2S - / - Cells) were cultured in Dulbecco's Modified Eagle Medium (DMEM) plus 10% fetal bovine serum (FBS) for twenty-four hours prior to transduction. The cultured I2S- / - Cells were resuspended in DMEM at 5.0E4 cells / ml plus 10% FBS, and 2 mL of this cell suspension was plated into 6-well tissue culture plates and placed at 37°C. Twenty-four hours after cell seeding, cells were transduced with 1 mL of any unpurified lentiviral vector. 1 mL of DMEM plus 10% FBS was added to control wells, and the cells were placed in a 37°C incubator. Twenty-four hours after transduction, a complete media exchange was performed. Forty-eight hours after transduction, 250 uL of supernatant from each well was removed into sterile Eppendorf tubes and frozen at -80°C. Cells were washed with 1 mL of phosphate-buffered saline and lifted w...

example 3

[0282] Protein expression in cells transduced with lentiviral vector encoding I2S

[0283] From wild-type control cells, I2S - / - Cells and I2S transduced with a lentiviral vector encoding I2S - / - Frozen cell pellets of cells (pMND-I2S and pEF1α-I2S) were thawed on ice for Western blotting. To each cell pellet was added 300 μL Mammalian Protein Extraction Reagent and 3 μL 100X HALT Protease Inhibitor Cocktail (Thermo Fisher Scientific). The pellet was resuspended by pipetting up and down slowly, and the cells were incubated on a plate shaker at room temperature for 10 minutes. Cells were centrifuged at 14,000 rpm for fifteen minutes at 4°C, and the supernatant was removed into sterile Eppendorf tubes. Loading dye was prepared by adding 25 μL of β-mercaptoethanol to 475 μL of 4X Laemmli sample buffer (Bio-Rad). Mix the samples at a 3:1 sample:loading dye ratio, 30 µL of prepared loading dye:90 µL of sample. 20 μL of each sample and 8 μL of a Precision Plus Protein Kaleidosc...

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Abstract

The invention provides compositions and methods for treating Hunter Syndrome.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 430,819, filed December 6, 2016, which is hereby incorporated by reference in its entirety. [0003] Statement Regarding the Sequence Listing [0004] The Sequence Listing related to this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into this specification. The name of the text file containing the sequence listing is BLBD_082_01WO_ST25.txt. The text file is 24KB, was created on December 6, 2017 and was submitted electronically via EFS-Web at the same time as this specification was submitted. technical field [0005] The present invention relates to gene therapy. More specifically, the present invention relates to gene therapy compositions and methods of using gene therapy compositions to treat mucopolysaccharidosis type II (MPS II), also known as Hunter syndrom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10A61K35/76A61K38/43A61P11/00A61P19/02A61P25/00
CPCA61K38/00A61K35/33A61K35/545C12N15/86C12N2740/15043C12N2740/16043C12N9/16C12Y301/06013A61P1/00A61P1/04A61P11/00A61P19/02A61P25/00A61P25/28A61P3/00A61P43/00C12N5/0656C12N2510/00A61K48/005A61K48/0083C12N5/0647C12N2740/15034
Inventor 肯德里克·A·戈斯杰弗里·B·帕森斯
Owner BLUEBIRD BIO INC
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