Lentiviral vector for delivering exogenous RNP and preparation method of lentiviral vector

A lentiviral vector and lentiviral technology, applied in the field of lentiviral vector delivery of exogenous RNP and its preparation, can solve the problems of long time and high off-target risk, and achieve the effect of improving application value

Active Publication Date: 2020-01-14
上海本导基因技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Cas9 (mRNA in nature) delivered via Cas9 mRNA usually exists longer than Cas9 (protein in nature) delivered via RNP and has a greater off-target risk

Method used

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  • Lentiviral vector for delivering exogenous RNP and preparation method of lentiviral vector
  • Lentiviral vector for delivering exogenous RNP and preparation method of lentiviral vector
  • Lentiviral vector for delivering exogenous RNP and preparation method of lentiviral vector

Examples

Experimental program
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Embodiment 1

[0032] This embodiment relates to a lentiviral vector for delivering exogenous RNP and its preparation method; including:

[0033] Step 1: Plasmids expressing membrane proteins (such as plasmids expressing VSV-G), plasmids expressing lentiviral GagPol long-chain proteins containing RNA-binding proteins, plasmids expressing lentiviral GagPol long-chain proteins, and proteins required to form RNP The plasmid and the target RNA expressing the stem-loop structure recognized by the RNA binding protein are co-transfected into virus production cells (such as 293T). in,

[0034] (1) Plasmids expressing membrane proteins include VSV-G, CD4 recognition protein, CD8 recognition protein, RD114, membrane proteins transformed from baboon endogenous retrovirus membrane proteins. In this embodiment, VSV-G is selected. The plasmid expressing VSV-G is the known plasmid pMD2G, and the map of the plasmid expressing VSV-G is shown in SEQ ID NO.1.

[0035] (2) The plasmid expressing the lentivir...

Embodiment 2

[0078] Example 2. Delivery of RNP (Cas9-gRNA) using lentiviral vectors

[0079] The specific steps are the same as in Example 1, and the exogenous target RNP is a complex of SpCas9 and gRNA. SpCas9 is a nucleic acid cleaving enzyme protein, and the exogenous RNA is selected from the gRNA targeting the AAVS1 site of the human genome, and the sequence is 5'-ggggccactagggcaggat-3' (SEQ ID NO.31); this embodiment is specifically a lentiviral vector Delivery of RNP (Cas9-gRNA) to 293T cells.

[0080] Depend on figure 2 It can be seen that after 293T cells were infected with lentiviral particles carrying RNP (Cas9-gRNA) for 72 hours, the AAVS1 site was amplified by PCR (primer F: 5'-ttcgggtcacctctcactcc-3' (SEQ ID NO.32), primer R: 5' -ggctccatcgtaagcaaacc-3' (SEQ ID NO. 33)) and sequenced with primer F. The gene knockout efficiency of TIDE analysis was as high as 83.1%.

Embodiment 3

[0081] Example 3. Gene editing of different animal cells using lentiviral vector delivery of RNP (Cas9-gRNA)

[0082] The specific steps are the same as in Example 1, and the exogenous target RNP is a complex of SpCas9 and gRNA. SpCas9 is a nucleic acid cleaving enzyme protein, and the exogenous RNAs were selected from gRNA targeting the vegfa site of the mouse genome, gRNA targeting the vegfa site of the green monkey genome, and gRNA targeting the AAVS1 site of the human genome. The gRNA sequences targeting the vegfa site of the mouse and green monkey genes are both 5'-ctcctggaagatgtccacca-3' (SEQ ID NO.34).

[0083] Depend on image 3 It can be seen that, 72 hours after mouse-derived NIH3T3 cells, green monkey-derived Cos7 cells and human-derived 293T cells were infected with lentiviral particles carrying RNP (Cas9-gRNA), the vegfa or AAVS1 sites were amplified by PCR and sequenced. Primer F: 5'-gatgagtggctgttggcctg-3' (SEQ ID NO.35) for PCR amplification of the vegfa si...

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Abstract

The invention discloses a lentiviral vector for delivering exogenous RNP and a preparation method thereof. The lentiviral vector is prepared by transfecting plasmids containing lentiviral vector genome sequences into virus production cells. The genome sequences are respectively positioned on plasmids for expressing membrane proteins; expressing plasmids of lentivirus GagPol long-chain protein containing RNA binding protein, expressing plasmids of lentivirus GagPol long-chain protein, expressing plasmids of protein required for forming RNP and expressing plasmids of exogenous target RNA containing an RNA stem-loop structure recognized by the RNA binding protein. In the process of packaging the RNP-containing lentiviral particles, the lentivirus GagPol long-chain protein is supplemented, sothat the negative influence of the exogenous RNA binding protein contained in the modified lentivirus GagPol long-chain protein on the normal form of the lentiviral particle is reduced, and the integrity of the lentiviral particle shell and the stability of the cell infection activity are ensured.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a lentiviral vector delivering exogenous RNP and a preparation method thereof. Background technique [0002] RNP has important application potential in gene editing, gene therapy, drug development and other fields. The application of RNP is limited by the following factors: (1) RNP itself cannot enter cells and requires a high-efficiency carrier system; (2) RNA is poorly stable and easily degraded by nucleases in the environment, and after the formation of RNP , RNA becomes more stable; (3) Existing RNP delivery technologies such as gold nanoparticles, liposomes, and electrotransduction are difficult to be directly used in vivo. [0003] Currently, RNP delivery options include electroporation, gold nanoparticles, liposomes, and engineered lentiviral particles. In the prior art, there is an RNP (Lu, B., et al., Delivering SaCas9 mRNA by lentivirus-like bionanoparticles f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867
CPCC12N15/86C07K14/00C12N2740/15043Y02A50/30
Inventor 蔡宇伽凌思凯
Owner 上海本导基因技术有限公司
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