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siRNAs, expression vectors and viral particles targeting synaptotagmin-11 and their pharmaceutical applications

A technology for combining proteins and carriers, applied in the direction of retroRNA viruses, DNA/RNA fragments, viruses, etc.

Active Publication Date: 2021-01-29
XI AN JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, the results of genome-wide association analysis show that Syt11 itself has a close genetic correlation with PD, and may be an important pathogenic gene of PD (2011, Lancet). However, whether Syt11 is directly involved in the pathological process of PD and its pathogenic mechanism has not yet been seen. to report

Method used

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  • siRNAs, expression vectors and viral particles targeting synaptotagmin-11 and their pharmaceutical applications
  • siRNAs, expression vectors and viral particles targeting synaptotagmin-11 and their pharmaceutical applications
  • siRNAs, expression vectors and viral particles targeting synaptotagmin-11 and their pharmaceutical applications

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Embodiment 1

[0050] siRNA against synaptotagmin Syt11 and its expression vector and virus preparation:

[0051] Retrieve synaptotagmin Syt11 gene sequence from Genebank, predict its siRNA target, synthesize effective siRNA sequence against Syt11; synthesize siRNA double-stranded DNA oligo, insert expression vector multiple cloning site region to construct shRNA expression plasmid; shRNA The virus expression plasmid and the helper plasmid were co-transfected into human embryonic kidney 293T cells to produce virus particles. After recovery and concentration, the shRNA expression virus that efficiently silences Syt11 was obtained.

[0052] 1. According to the Syt11 nucleotide sequence reported in Genebank (NM_152280 Homo sapiens, NM_031667 Rattusnorvegicus, NM_018804 Musmusculus), select 19-21 nucleotides for the coding region of Syt11 gene as the siRNA target, and select a 19nt random oligo The polynucleotide sequence served as a control. The target sequence of each siRNA on the Syt11 gene ...

Embodiment 2

[0115] Syt11 shRNA silencing efficiency of Syt11

[0116] 1. Liposome transfection of human kidney embryo HEK293 cells

[0117] Human kidney embryo HEK293A cells were cultured in DMEM (Gibco) medium containing 10% fetal bovine serum (FBS). The transfection reagent VigoFect (Vigorous Biotechnology Beijing Co.) was used to transfect HEK293A cells. When verifying the silencing efficiency of Syt 11, its expression plasmids were co-transfected with 5 shRNA expression plasmids randomly selected into 293A cells, and transfected for 72 hours. Then used for real time PCR detection.

[0118] 2. Extraction of mRNA

[0119] HEK cells were taken and lysed with TRIZOL at room temperature for 5 min. Add 0.2ml chloroform for every 1ml TRIZOL used, vortex shake vigorously for 15s, mix thoroughly and centrifuge to take the upper RNA aqueous phase, add an equal volume of isopropanol to precipitate RNA, centrifuge at 16000g for 10min at 4°C, remove the supernatant and wash the precipitate with...

Embodiment 3

[0123] Transfection of Syt11 shRNA expression plasmid into neurons

[0124] 1. Liposomal transfection of hippocampal neurons

[0125] Take the hippocampus tissue of mice on day P0-1, digest it with 0.25% trypsin at 37°C for ~12min, and blow the digested tissue to obtain a cell suspension. The cell suspension was dropped onto poly-L-lysine (Sigma)-coated glass slides / petri dishes, and cultured with Neurobasal (Gibco) containing 2% B27, 0.5 mM L-glutamine and 5 μM cytosine arabinoside. After 5 days of culture, Lipofectamine 2000 (Invitrogen) was used to transfect the shRNA expression plasmid in Example 2 into hippocampal neurons, and the expression of shRNA in neurons was observed after 12-14 days. The result is as Figure 4 As shown, it can be seen that after liposome transfection, a large number of hippocampal neurons expressing green fluorescent protein can be found, and the cells positive for green fluorescent protein are neurons that are successfully transfected and expre...

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Abstract

The invention provides an siRNA targeting synaptotagmin-11, an expression vector, a virus particle and a pharmaceutical application thereof. It belongs to the technical field of biomedicine, and specifically relates to an siRNA inhibiting the expression of the synaptophytic protein Syt11 gene, a pharmaceutical composition containing the siRNA as an active ingredient, a recombinant vector and an expression virus expressing the siRNA, and their expression in Parkinson's disease and Syt11 related to other diseases. The present invention designs and synthesizes small molecule interfering RNA for the nucleotide sequence of the Synaptotagmin-11 (Syt11) gene, constructs shRNA expression vectors and expression viruses for the target sequence, the siRNA specifically targets the Syt11 gene at the mRNA level, and can It effectively inhibits the expression of Syt11 and is used for the treatment of Parkinson's syndrome and other diseases related to the expression of Syt11. After the siRNA is transferred into the neurons in the substantia nigra region of the midbrain, the expression of Syt11 can be effectively silenced, and the pathological process of Parkinson's syndrome can be reversed.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to siRNA inhibiting the expression of the synaptophytic protein Syt11 gene, a pharmaceutical composition containing the siRNA as an active ingredient, a recombinant vector expressing the siRNA and an expression virus, and their treatment in Parkinson's disease and related diseases Applications in other diseases related to Syt11 expression. Background technique [0002] Parkinson's disease (PD) and Alzheimer's disease are the most important neurodegenerative diseases in humans today. The incidence of PD in people over 65 years old is more than 2%, and the incidence rate in people over 85 years old can be as high as 5%. The clinical syndrome of PD mainly manifests as motor deficits such as bradykinesia, resting tremor, rigidity of action, and posture disturbance. Most PD patients also have depression, dementia, sleep deprivation, olfactory disturbance, pain, urinary in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/867A61K31/7088A61P25/16
CPCC12N15/113C12N15/86C12N2310/14C12N2740/15043
Inventor 王昌河康新江
Owner XI AN JIAOTONG UNIV
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