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A recombinant adeno-associated virus gene therapy vector and medicine for treating patients with type 2 achromatopsia

A viral vector and gene technology, applied in gene therapy, the introduction of foreign genetic material using vectors, viruses/phages, etc., can solve the problems of incurable diseases and ineffectiveness of inherited retinal diseases.

Active Publication Date: 2020-06-12
沈阳伟景生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are no exact statistics, it has been observed in recent years that the proportion of type 2 caused by CNGA3 gene mutation in Asian countries, including China, is significantly higher than that of European and American populations, accounting for 60-90% of total color blindness.
[0004] Traditional medical methods have no effect on inherited retinal diseases, so until now these diseases are still in a state of no cure

Method used

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  • A recombinant adeno-associated virus gene therapy vector and medicine for treating patients with type 2 achromatopsia
  • A recombinant adeno-associated virus gene therapy vector and medicine for treating patients with type 2 achromatopsia
  • A recombinant adeno-associated virus gene therapy vector and medicine for treating patients with type 2 achromatopsia

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Experimental program
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Effect test

Embodiment 1

[0045] Construction of embodiment 1.pTR-CBA-AT2R intron 1-hCNGA3 plasmid

[0046] The cDNA (SEQ ID NO:1) of human CNGA3 (hCNGA) was cloned downstream of the CBA (chicken beta actin) promoter (SEQ ID NO:3) with the CMV IE enhancer (SEQ ID NO:5) in between And the unique rat AT2R (angiotensin II type 2 receptor) Intron 1 (SEQ ID NO: 2), can enhance the long-term expression of the transgene hCNGA. The hCNGA cDNA was followed by the SV40 polyadenylation signal (SEQ ID NO:8). The expression cassette is flanked by inverted terminal repeats: ITR5' (SEQ ID NO: 7), ITR3' (SEQ ID NO: 9), and the viral vector also includes ColE1ori (SEQ ID NO: 10), Amp(r) (SEQ ID NO:11) and f1(+) origin (SEQ ID NO:12).

[0047] The characteristic map of pTR-CBA-AT2R intron 1-hCNGA3 plasmid is as follows figure 1 As shown, the starting and ending points of each component are as follows:

[0048] ITR 5' Start: 19 End: 161

[0049] CMV ie enhancer start: 162 end: 542

[0050] Chiken beta-actin promote...

Embodiment 2

[0059] Construction of embodiment 2.pTR-PR2.1-AT2R intron 1-hCNGA3 plasmid

[0060] The method was the same as that in Example 1, the only difference being that the cone cell-specific promoter PR2.1 (SEQ ID NO: 4) was used instead of the CBA promoter.

Embodiment 3

[0061] Embodiment 3. Construction of AAV2-CBA-AT2R intron 1-hCNGA3 viral vector

[0062]Vectors were obtained by plasmid co-transfection method. AAV2-CBA-AT2R intron 1-hCNGA3 recombinant adeno-associated virus vector was initially formed by co-transfecting HEK 293T cells with the helper plasmid containing the AAV2 coat protein gene and the gene that can help AAV replication and the pTR-CBA-AT2R Intron 1-hCNGA3 plasmid . After initial purification with iodine butanol, further purification by ion-exchange chromatography using 5 ml Hitrap Q Sepharose as a filler for fast protein liquid chromatography (the instrument used is Pharmacia AKTA FPLC system (Amersham Biosciences, Piscataway, NJ) .Use the 215mM NaCl of pH8.0 afterwards, rinse the agarose gel column, the recombinant viral vector of peak is collected.After the liquid of collection passes through concentrator (100K concentrater, Millipore), rinse with Tween 20 containing 0.014% The washing concentrator concentrates the re...

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Abstract

The invention relates to a gene therapy vector. The gene therapy vector is used for gene therapy of type II achromatopsia. The gene therapy vector provided by the invention includes a human cyclic nucleotide gated channel alpha3 gene and a rat angiotensin type II receptor intron 1. The invention further relates to a medicine and a composition, which include the gene therapy vector.

Description

technical field [0001] The invention relates to the field of gene therapy, in particular to a recombinant adeno-associated virus gene therapy vector and medicine for treating type 2 achromatopsia patients. Background technique [0002] The human retina contains approximately 6 million cones and 100 million rods. Cone cells are mainly responsible for color vision and central and fine vision in the bright room, mainly distributed in the macula, especially the central fovea (accounting for 100%); on the contrary, rod cells are mainly responsible for peripheral vision in the dark environment, mainly distributed in the Peripheral parts of the retina, the closer to the macula, the less. Panchromatopsia is a rare recessive genetic disorder characterized by loss of cone function, leaving only rod vision. The clinical manifestations of achromatopsia are: pendulum nystagmus, photophobia, severe vision impairment, no color vision and no central vision, manifested as day blindness. E...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/864A61K48/00A61P27/02
CPCA61K38/1709A61K48/005C12N15/86C12N2750/14143
Inventor 庞继景
Owner 沈阳伟景生物科技有限公司
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