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Recombinant adeno-associated virus for efficient tissue-specific expression of RS1 protein and application

A technology for protein expression and purpose, applied in the biological field, can solve problems such as inability to achieve tissue-specific expression of target protein, limitation of therapeutic effect, and increase in the amount of AAV vector

Pending Publication Date: 2021-08-31
成都金唯科生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Most of the existing technologies for XLRS gene therapy use broad-spectrum promoters such as CMV promoters, which cannot achieve tissue-specific expression of the target protein, and have certain limitations on the therapeutic effect
In addition, non-tissue-specific expression will increase the amount of AAV vector, which will increase the risk of immunity

Method used

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  • Recombinant adeno-associated virus for efficient tissue-specific expression of RS1 protein and application
  • Recombinant adeno-associated virus for efficient tissue-specific expression of RS1 protein and application
  • Recombinant adeno-associated virus for efficient tissue-specific expression of RS1 protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1 expression vector construction

[0043] The present invention first optimizes the codon of the RS1 gene, and the nucleotide sequence is shown as 645-1319bp in SEQ ID NO.4. On the basis of optimizing RS1, the present invention designs four expression cassettes, the schematic diagram of the AAV virus vector genome and the cis plasmid map of AAV virus packaging are as follows Figure 1-4 shown. Full sequence synthesis of four viral packaging cis plasmid vectors.

[0044] ssAAV8.CB7.EGFP.bGH viral packaging plasmid (referred to as pAAVss.CB7.EGFP.bGH), the full sequence of the plasmid is shown in SEQ ID NO.1, the main functional element site: 1-168 is the 5' inverted terminal repeat Sequence (ITR), 241-899 is the CB7 promoter sequence, 900-1916 is the intron sequence, 1982-2701 is the EGFP gene sequence, 2717-2941 is the bGHpolyA sequence, and 2992-3159 is the 3' end ITR sequence.

[0045] scAAV8.RK.EGFP.bGH viral packaging plasmid (pAAVsc.RK.EGFP.bGH for sh...

Embodiment 2

[0048] The specificity comparison of embodiment 2 viral packaging plasmid expressed in vitro

[0049] The pAAVss.CB7.EGFP.bGH and pAAVsc.RK.EGFP.bGH in Example 1 were transfected with the same amount of plasmids (2 μg) into HEK293 cells and optic neuroblastoma cells Y79 cultured in a six-well plate, after 48 hours The expression of EGFP was observed by immunofluorescence photography, and the results showed that pAAVss.CB7.EGFP.bGH could express EGFP in both HEK293 cells and Y79 cells, while pAAVsc.RK.EGFP.bGH could only express EGFP in Y79 cells. It shows that the RK promoter can realize tissue-specific expression of the target protein ( Figure 5 ).

Embodiment 3

[0050] Example 3 AAV virus preparation and purification

[0051] Referring to the method of packaging and purifying recombinant AAV virus reported by Martin Lock et al., the Rep of AAV2 and the Cap protein expression plasmid of AAV8 (pAAV2 / 8), helper plasmid (pAdΔF6) and AAV target gene vector cis plasmid (pAAVss. .cohRS1.rBG / pAAVsc.RK.cohRS1.bGH) were co-transfected into HEK293 cells to package and prepare virus ssAAV8.CB7.cohRS1.rBG and virus scAAV8.RK.cohRS1.bGH. After 144 hours of transfection, the cell culture supernatant was harvested and cut. After concentrating the virus liquid to the filter (TFF), use iodixanol ultrafast gradient centrifugation to purify the AAV virus, collect the purified AAV virus, use Amicon Ultra 100K centrifuge to desalt, and finally place the AAV virus in 20mM Tris (pH8.0) ,1mMMgCl 2 , 200mM NaCl and 0.001% PF68 preparations for use. The prepared AAV virus was tested for virus purity by SDS-PAGE staining, and the endotoxin content was detected...

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Abstract

The invention discloses a recombinant adeno-associated virus for efficient tissue-specific expression of RS1 protein and application. An RS1 protein expression cassette consists of a promoter-a target gene sequence-polyA, wherein the promoter is selected from an RK promoter, a target gene is selected from a wild-type RS1 gene or the codon-optimized RS1 gene, and the polyA is selected from any vector of rBG or bGH, and contains the RS1 protein expression cassette. According to the invention, a gene expression cassette is optimized, and the recombinant adeno-associated virus is used as a gene delivery vector, so that tissue-specific efficient and continuous expression of the RS1 protein is realized in retina or ganglion cells, thereby achieving the purpose of treating X-linked juvenile retinoschisis. Through tissue-specific expression of the target gene, the use amount of a virus vector can be reduced, and then the immune risk is reduced, and the purpose of long-acting treatment is achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a recombinant adeno-associated virus for highly efficient tissue-specific expression of RS1 protein and its application. Background technique [0002] X-Linked juvenile retinoschisis (XLRS) is an X-linked rare hereditary blinding eye disease, usually with low vision and serious complications starting from childhood in males, and has been Developed into old age, and the condition gradually deteriorated. Globally, the incidence of XLRS is about 1:5000-1:25000. XLRS primarily involves the bilateral retina, with a split cavity between the retinal nerve fiber layer and the ganglion cell layer. In the early electroretinogram (ERG) response of XLRS patients, the dark-adapted b-wave amplitude decreased, while the a-wave amplitude usually remained normal. Since the a wave originates from the photoreceptor, this indicates that the activity of the early photoreceptor is relatively normal, whil...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/864C12N15/12C12N5/10C12N7/01A61K38/17A61K48/00A61P27/02
CPCC12N15/86C12N7/00C07K14/47A61K38/17A61K48/0008A61K48/005A61P27/02C12N2800/22C12N2750/14121C12N2750/14143
Inventor 杨阳魏于全
Owner 成都金唯科生物科技有限公司
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