Gene medicine for preventing and treating neovascular eye diseases

A technology for eye diseases and genes, which is applied in the fields of cardiovascular system diseases, gene therapy, sensory diseases, etc., can solve the problems that subjects are prone to infection and inflammation, difficult to apply, etc., to achieve inhibition of proliferation and migration activity, inhibition of proliferation, The effect of inhibiting angiogenesis ability

Pending Publication Date: 2021-10-08
厦门朔望医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the use of adenoviral vectors, the subjects are prone to symptoms such as infection and inflammation, so it is difficult to obtain further applications (Mori K, Gehlbach P, Yamamoto S, Duh E, Zack DJ, Li Q, Berns KI, Raisler BJ, Hauswirth WW, Campochiaro PA. AAV-mediated gene transfer of pigment epithelium-derived factor inhibits choroidal neovascularization. Invest Ophthalmol Vis Sci. 2002 Jun; 43(6):1994-2000; Haurigot V, Villacampa P, Ribera A, Bosch A, Ramos D , Ruberte J, Bosch F. Long-Term Retinal PEDF Overexpression Prevents Neovascularization in a Murine Adult Model of Retinopathy. PLoS One. 2012; 7(7):e41511. doi:10.1371 / journal.pone.0041511. Epub 2012Jul 20. Askou AL , Alsing S, Benckendorff JNE, Holmgaard A, Mikkelsen JG, Aagaard L, Bek T, Corydon TJ. Suppression of Choroidal Neovascularization by AAV-Based Dual-Acting Antiangiogenic Gene Therapy MolTher Nucleic Acids. 2019 Jun 7; 16:38-50.doi: 10.1016 / j.omtn.2019.01.012.Epub2019Feb 2.)
[0007] However, there is no report on a gene drug for the treatment of neovascular eye diseases based on the synergy between VEGF and PEDF.

Method used

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  • Gene medicine for preventing and treating neovascular eye diseases
  • Gene medicine for preventing and treating neovascular eye diseases
  • Gene medicine for preventing and treating neovascular eye diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1: VEGFA shRNA sequence design and PEDF overexpression adeno-associated virus plasmid construction

[0073] (1) Construction of VEGFA interference vector

[0074] First search the sequence information of human VEGFA (NM_001025366.3) on NCBI, and then use the online design software http: / / rnaidesigner.thermofisher.com / rnaiexpress / design.do to predict the shRNA interference sequence of VEGFA. Add the sequence corresponding to the restriction site XhoI in the middle of the sequence as a hairpin structure, and assemble according to "EcoRI restriction site sticky end-sense strand-XhoI-antisense strand-MluI restriction site sticky end. Synthesize and anneal method, constructing it into plasmid pAAVE2099 (digested by EcoRI and MluI), verified by enzyme digestion, and successfully sequenced, and finally obtained pAAVE2099-shVEGFA-1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 totaled 12 VEGFA interference vectors.

[0075] The transcribed DNA sequences of shVEGFA-1 to 12 a...

Embodiment 2

[0135] Example 2: Packaging of Adeno-Associated Virus Particles

[0136] 5% CO at 37°C 2 293T cells were cultured in a cell incubator, and the medium used was DMEM medium supplemented with 10% fetal bovine serum. Subculture 293T cells in a 10cm-diameter petri dish. After 24 hours (the cells are in the algebraic growth phase), select a petri dish with a cell confluence of about 50%, and replace it with DMEM medium containing 2% fetal bovine serum to culture the cells.

[0137]Prepare the transfection mixture of target plasmids (pAAVE2099, pAAVE2099-shVEGFA-9, pAAVE2099-PEDF, AAV-PEDF-shVEGFA-9), packaging plasmids (pHelper, pAAV-RC2) and liposome transfection reagent. The mixture was added to the cells that had been exchanged for transfection, and the DMEM medium containing 5% fetal calf serum was replaced with fresh DMEM medium after 12 hours. Cells were harvested 72 h after transfection. Add PBS to resuspend the cell pellet, freeze and thaw three times at -80°C / 37°C, centr...

Embodiment 3

[0139] Example 3: Detection of VEGFA mRNA level

[0140] HUVEC cells were divided into four groups (AAV-NC group, AAV-shVEGFA-9 group, AAV-PEDF group, AAV-PEDF-shVEGFA-9 group). The cells in each group were infected with the corresponding adeno-associated virus (the multiplicity of infection was 10), and cultured for 4 days. Total cellular RNA was extracted by the Trizol method, and RNA was reverse-transcribed into cDNA using random primers. The qPCR reaction system included 5 μL of 2×SYBRGreen Mix, 0.2 μL of upstream and downstream primers (5 pmol / mL), 0.2 μL of cDNA template, ddH 2 O supplemented to 10 μL. The reaction program was [95°C 2min; (95°C 15s, 60°C 30s, 72°C 30s)×40 cycles; melting curve]. With GAPDH as internal reference, use 2 -△△CT Calculate the relative expression level. The primer sequences involved in the experiment are as follows:

[0141] VEGF-F: 5'-AGGGCAGAATCATCACGAAGT-3' (SEQ ID NO. 33);

[0142] VEGF-R: 5'-AGGGTCTCGATTGGATGGCA-3' (SEQ ID NO. 34);...

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Abstract

The invention discloses a gene medicine for preventing and treating neovascular eye diseases. The invention relates to the technical field of gene medicines, in particular to a recombinant vector which comprises a PEDF or PEDF functional fragment gene and a shRNA fragment targeting human VEGFA (vascular endothelial growth factor A). The invention also provides application of the recombinant vector in preparation of medicines for preventing or treating angiogenesis-related eye diseases. The PEDF overexpression and VEGFA interference adeno-associated virus involved in the invention can interfere with the expression level of the VEGFA in vascular endothelial cells, effectively down-regulate the protein level of the VEGFA, further inhibit the proliferation and migration activity of the vascular endothelial cells, and inhibit the angiogenesis ability of the vascular endothelial cells, so that a new target and approach for the treatment of wet maculopathy is provided.

Description

technical field [0001] The invention relates to the technical field of gene medicine, in particular to a gene medicine for preventing or treating eye diseases related to angiogenesis. Background technique [0002] Angiogenic diseases are mainly caused by an imbalance between pro-angiogenic and anti-angiogenic factors. Stimulating factors such as biological force, hyperoxia, thrombus, immune and inflammatory responses, and genetic mutations can all cause abnormalities in angiogenesis. Angiogenesis in the eye can occur in the retina, choroid, and cornea, and uninhibited angiogenesis can lead to severe visual impairment. The new blood vessels are secretory, and the accumulation of secretions in the long-term will eventually lead to the damage of retinal function. In addition, abnormal growth of blood vessels can lead to abnormal tissue structure and impaired corneal transparency. Ophthalmic diseases associated with neovascularization include wet maculopathy, diabetic retinop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/12C12N15/113A61K38/17A61K48/00A61P9/10A61P27/02
CPCC12N15/85C07K14/4702C12N15/1136A61K48/005A61K38/1709A61P27/02A61P9/10C12N2310/141C12N2310/531
Inventor 李程何彰华黄红军张春乐祖勇
Owner 厦门朔望医药科技有限公司
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