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
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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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