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A gene therapy carrier and its use

A gene therapy and vector technology, applied in gene therapy, genetic engineering, plant genetic improvement, etc., can solve problems such as inability to cure diseases

Active Publication Date: 2022-04-08
BEIJING IMMUNOCHINA MEDICAL SCI & TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no effective clinical treatment for HDLS. Generally, symptomatic treatment mainly includes general care, nutritional support, antiepileptic drugs and antibiotics.
Symptomatic treatment does not cure the disease, but only slightly relieves the patient's symptoms

Method used

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  • A gene therapy carrier and its use
  • A gene therapy carrier and its use
  • A gene therapy carrier and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Example 1: Construction of CSF1R lentiviral vector

[0107] In this example, the vectors pMND-CSF1R-LV and pEF1α-CSF1R-LV were respectively constructed as follows: a third-generation lentiviral vector containing a chimeric 5′LTR; the myeloproliferative sarcoma virus enhancer negative control region was deleted and dl587rev primer binding site substituted (MND) promoter or short elongation factor 1α (EF1α) promoter; polynucleotide encoding CSF1R polypeptide; and self-inactivating (SIN) 3′LTR (see figure 1 ). Specifically, the pMND-CSF1R-LV vector mainly includes: chimeric 5'LTR, Psi (ψ) packaging signal, human immunodeficiency virus (HIV) rev response element (RRE), cPPT / FLAP, myeloproliferative sarcoma virus enhancer Negative control region deleted and dl587rev primer binding site replacement (MND) promoter, polynucleotide encoding CSF1R polypeptide, truncated epidermal growth factor receptor (EGFRt) tag, woodchuck hepatitis virus post-transcriptional regulatory elemen...

Embodiment 2

[0138] Example 2: Transduction of human CD34 with a lentiviral vector encoding CSF1R + hematopoietic stem cells

[0139] (1) Treat the cell plate with recombinant human fibronectin (Retronectin)

[0140] The recombinant human fibronectin solution was diluted to 60 μg / ml with buffer. Add the recombinant human fibronectin dilution to the cell culture plate (0.2 mL in a 48-well plate). Place at room temperature for 4 hours, or overnight at 4°C in the dark. Aspirate the recombinant human fibronectin solution, and use an appropriate amount of 2% BSA (bovine serum albumin) to block with DPBS (Duchenne's phosphate buffered saline, without calcium and magnesium). Place at room temperature for 30min. Aspirate the BSA blocking solution and wash the cell plate twice with DPBS. Add appropriate amount of DPBS for later use.

[0141] (2) HSCs pre-stimulation

[0142] The isolated hematopoietic stem cells were cultured in cell plates pretreated with recombinant human fibronectin, cell...

Embodiment 3

[0145] Example 3: Detection of protein expression in cells transduced by a lentiviral vector encoding CSF1R (Coomassie Brilliant Blue staining detection)

[0146] 1. Three days after infecting hematopoietic stem cells with lentivirus encoding CSF1R, the cells were collected and the expression of related proteins was detected.

[0147] 2. Prepare a separating gel with a concentration of 8% and a volume of 15ml

[0148] h 2 O: 6.9ml

[0149] 30% gel stock solution: 4.0ml

[0150] 1.5mol / L Tris-HCl (pH8.8): 3.8ml

[0151] 10% SDS (sodium dodecyl sulfate): 0.15ml

[0152] 10% ammonium persulfate: 0.15ml

[0153] TEMED (tetramethylethylenediamine): 0.01ml

[0154] 3. Once TEMED is added, immediately after mixing, carefully inject the separating gel into the prepared gap between the glass plates, leaving enough space for the layering glue. Use a capillary to gently add a few milliliters of n-butanol to its top layer to prevent the inhibitory effect of oxygen in the air on gel...

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Abstract

The present invention relates to a gene therapy carrier and its application. The gene therapy carrier includes: a) left (5') LTR; b) Psi (ψ) packaging signal; c) retrovirus export element; d) central polypurine region / central termination sequence; e) a promoter operably linked to a polynucleotide encoding a colony-stimulating factor 1 receptor polypeptide; f) a polynucleotide encoding a colony-stimulating factor 1 receptor polypeptide; g) a post-transcriptional regulatory element; and h) Right (3') LTR. The gene therapy vector can transform cells in vitro to make the cells express CSF1R protein, thereby treating HDLS and improving the survival status of HDLS patients.

Description

technical field [0001] The invention relates to the technical field of gene therapy, in particular to a gene therapy carrier and its application. Background technique [0002] Hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS, hereditary diffuse leukoencephalopathy with spheroids) was first described by Swedish doctors in 1984. Clinical manifestations include cognitive impairment (memory decline, speech impairment, etc.), personality and behavior changes (depression, apathy, anxiety, irritability, etc.), motor dysfunction (difficulty walking, movement disorders, ataxia, etc.), and are easily misdiagnosed as AIDS. Alzheimer's disease, frontotemporal dementia, Parkinson's syndrome, multiple sclerosis and leukoencephalopathy. The onset age was 47.2±14.5 years old, the average disease duration was 6.0±3.1 years, and the death age was 57.2±13.1 years old. Pathological manifestations include atrophy of the frontal and frontoparietal lobes, dilation of the ventri...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N15/12C12N5/10A61K48/00A61K38/17A61P25/00A61P25/28A61P25/14
CPCC12N15/86C07K14/7153A61K48/0008A61K38/1793A61P25/00A61P25/28A61P25/14C12N2740/15043
Inventor 鲁薪安何霆齐菲菲刘光华胡雪莲
Owner BEIJING IMMUNOCHINA MEDICAL SCI & TECH CO LTD
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