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Gene therapy for mucopolysaccharidosis, type I

A polynucleotide and promoter technology, applied in the field of gene therapy composition, treatment of type I mucopolysaccharidosis, can solve the problem of no cure and the like

Inactive Publication Date: 2019-09-06
BLUEBIRD BIO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] While treatment can improve the length and quality of life of individuals with MPS I, there is no cure

Method used

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  • Gene therapy for mucopolysaccharidosis, type I
  • Gene therapy for mucopolysaccharidosis, type I
  • Gene therapy for mucopolysaccharidosis, type I

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0285] Construction of IDUA vector

[0286] The following were constructed: 3rd generation lentiviral vector containing 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 sub; a polynucleotide encoding an α-L iduronidase (IDUA) polypeptide; and a self-inactivating (SIN) 3'LTR. See for example, figure 1 and SEQ ID NO: 1 and 2. Table 1 and Table 2 show the identity, gene bank reference (Genbank Reference), source name and citation of each nucleotide fragment of the exemplary lentiviral vector encoding IDUA.

[0287] Table 1: pMND-IDUA LVV

[0288]

[0289]

[0290] Table 2: pEF1α-IDUA LVV

[0291]

[0292]

example 2

[0294] Fibroblasts transduced with a lentiviral vector encoding IDUA

[0295] Human fibroblasts lacking IDUA activity due to a homozygous mutation of the IDUA gene (IDUA - / - cells) and the human fibroblasts were cultured in Dulbecco's Modified Eagle Medium (DMEM) plus 10% fetal bovine serum (FBS) for twenty-four hours before transduction. The trained IDUA - / - 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 phosphat...

example 3

[0297] Protein expression in cells transduced with a lentiviral vector encoding IDUA

[0298] From wild-type control cells, IDUA - / - Cells (GM0798 and GM06214) and IDUA transduced with a lentiviral vector encoding IDUA - / - Frozen cell pellets of cells (MND.IDUA and EF1α(EFS).IDUA) 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 ...

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PUM

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Abstract

The invention provides compositions and methods for treating MPS I.

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 / 430,795, 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_081_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 I (MPS I). Background technique [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01A61K35/76A61K38/43C12N15/867C12N5/10A61P3/00A61P27/02A61P27/16A61P25/00A61P11/00A61P9/00A61P19/00
CPCA61K48/005C12Y302/01076C12N9/2402C12N15/86C12N2740/16043A61K38/00A61P11/00A61P19/00A61P25/00A61P25/28A61P27/02A61P27/16A61P3/00A61P43/00A61P9/00
Inventor 肯德里克·A·戈斯杰弗里·B·帕森斯
Owner BLUEBIRD BIO INC
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