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Application of non-conformable lentiviral vector system in gene editor delivery

A lentiviral vector, non-integrated technology, applied in applications, genetic engineering, plant genetic improvement, etc., can solve problems such as limited packaging capacity, insertion mutation, and inability to accommodate base editors

Pending Publication Date: 2022-04-26
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the AAV virus is a widely used vector at present, but one of its disadvantages is that its packaging capacity is limited, and it can only package fragments within 4.7kb, while base editors are generally more than 5kb, so the entire vector cannot accommodate the entire vector. Base editor, the base editor needs to be split into two segments to be delivered by AAV
The packaging capacity of the lentiviral vector is large, and the base editor can be completely inserted into the same vector, but the classic lentiviral vector will integrate its genome into the host chromosome, so there is a certain risk of insertion mutation

Method used

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  • Application of non-conformable lentiviral vector system in gene editor delivery
  • Application of non-conformable lentiviral vector system in gene editor delivery
  • Application of non-conformable lentiviral vector system in gene editor delivery

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1. Non-integrating lentivirus delivery CBE realizes editing of specific sites

[0068] It is a relatively safe and efficient method to use non-integrating lentiviral packaging CBE base editors to infect target cells after harvesting virus particles to achieve gene editing at specific sites.

[0069] Experimental process: The coding genes of the currently commonly used BE4max and hyeA3ABE3max CBE base editors were respectively constructed into lentiviral vectors, and the puromycin gene was expressed in the vectors, which can be used for subsequent screening. In addition, the corresponding gRNA sequence was inserted behind the N-terminal U6 promoter through the Golden gate.

[0070] The specific construction method is as follows:

[0071] Step 1: Use the full-length plasmid of the BE4max base editor expression vector as a template, and use the following primer pair BE4max-F / BE4max-R as amplification primers to perform PCR amplification to obtain the target fragme...

Embodiment 2

[0090] Example 2, non-integrating lentivirus delivery of ABE to achieve specific site editing

[0091] Using non-integrating lentiviral packaging ABE base editors to harvest virus particles and infect target cells can achieve gene editing at specific sites, which is a relatively safe and efficient method.

[0092] Experimental process: The coding gene of the currently commonly used SpRY ABEmax base editor is constructed into a lentiviral vector. The specific construction method is as follows:

[0093] Step 1: Use the full-length plasmid of the SpRY ABEmax base editor as a template, and use the following primer pair SpRYABEmax-F / SpRY ABEmax-R as amplification primers to perform PCR amplification to obtain the target fragment.

[0094] The specific primer sequences are as follows (lowercase bases are homology arms):

[0095] SpRY ABEmax-F: 5'-aacacaggaccggttctagaATGAAACGGACAGCCGA-3';

[0096] SpRY ABEmax-R: 5'-aagtttgttgcgccggatccGACTTTCCCTCTTCTTCT-3'.

[0097] Step 2: Digest...

Embodiment 3

[0106] Example 3, non-integrated lentiviral delivery PE system realizes editing of specific sites

[0107] It is a relatively safe and efficient method to use non-integrated lentiviral packaging PE system to infect target cells after harvesting virus particles to achieve gene editing at specific sites.

[0108] Experimental process: The coding gene of the currently commonly used PE precision editing system is constructed into a lentiviral vector, and the puromycin gene is expressed in the vector, which can be used for subsequent screening. The specific construction method is as follows:

[0109] Step 1: Use the full-length plasmid of the PE base editor as a template, and use the following primer pairs as amplification primers to perform PCR amplification to obtain the target fragment.

[0110] The specific primer sequences are as follows (lowercase is the homology arm):

[0111] PE-F: 5'-aacacaggaccggttctagaATGAAACGGACAGCCGA-3';

[0112] PE-R: 5'-aagtttgttgcgccggatccGACTTTC...

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Abstract

The invention discloses an application of a non-integrated lentiviral vector system in preparation of a base editor delivery product, and the non-integrated lentiviral vector system can obtain a non-integrated lentivirus after transfecting a host cell. The non-integrated lentiviral vector system comprises a recombinant lentiviral vector for expressing a base editor, a plasmid for expressing integrase and a plasmid for expressing VSV envelope G protein. According to the method for delivering the base editor by utilizing the lentiviral vector, the base editor can be completely inserted into the same vector, and the integrase is mutated, so that the integrase cannot be integrated into a host chromosome, the base editor can be efficiently and relatively safely delivered into a target cell, and gene editing of a specific site is realized.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the application of a non-integrated lentiviral vector system in gene editor delivery. Background technique [0002] Genetic diseases are a difficult problem in clinical treatment. At present, most genetic diseases have no effective treatment. Gene editing is one of the effective ways to eliminate such diseases. Using gene editing technology, only one treatment can change the patient's DNA for a long time and achieve a permanent cure. So far, about half of the known human pathogenic mutations are point mutations (also known as single nucleotide polymorphism, single nucleotide polymorphism, SNP). Efficient and accurate correction of pathogenic SNPs is of great significance for the research and treatment of genetic diseases. [0003] Base editing (BE) does not require DNA double-strand breaks and does not require the participation of donor DNA. It can achieve precise point...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/54C12N15/38C12N15/113C12N15/62C12N5/10
CPCC12N15/86C12N9/1241C07K14/005C12N15/113C12N9/22C12N9/78C12N9/1276C12N5/0686C12Y207/07049C12Y305/04001C12Y305/04002C12N2740/15043C12N2800/107C12N2710/16722C07K2319/00C12N2510/00
Inventor 张学礼毕昌昊王玉杰
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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