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DNA template for modifying primary cells by gene editing and fixed-point insertion method

A gene editing and DNA sequence technology, applied in the field of gene-directed insertion, can solve the problems of complex, uneconomical, and dangerous preparation of double-stranded linear DNA, and achieve great clinical application potential and commercial value, good safety and effectiveness. performance and cost savings

Pending Publication Date: 2021-01-12
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0002] At present, the target gene is mainly integrated into the genome of primary human T / NK cells by means of traditional lentivirus or retrovirus, but the disadvantage of this method is that the target gene will be inserted randomly. Site-specific insertion into the cell genome can improve the safety and effectiveness of genetically engineered human primary T / NK cells to a certain extent, and play a greater role in clinical practice
However, adeno-associated virus (AAV) may still cause potential danger to the body during clinical treatment, and the preparation is relatively complicated and uneconomical.
At present, there are still studies using double-stranded linear DNA as the donor DNA template for homologous recombination repair to transform primary human T / NK cells, but the preparation of double-stranded linear DNA is more complicated, and GMP preparation is immature

Method used

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  • DNA template for modifying primary cells by gene editing and fixed-point insertion method
  • DNA template for modifying primary cells by gene editing and fixed-point insertion method
  • DNA template for modifying primary cells by gene editing and fixed-point insertion method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] 1. Construction of EGFP gene and RAB11a homology arm sequence on pUC57 or transformed pMini plasmid vector 1. Transformation of pUC57 plasmid vector sequence

[0048] The pUC57 plasmid vector sequence is 2.7kb in size and mainly includes three parts: the lactose operon sequence (LacZ), the ampicillin resistance gene and its promoter region (AMP), and the prokaryotic origin of replication (ORI). On the basis of existing research, this example shrinks the pUC57 plasmid vector, removes the lactose operon sequence (LacZ) sequence by molecular cloning, and PCR clones the ORI region forward primer sequence as shown in SEQ ID NO: 1, reverse See SEQ ID NO: 2 for the primer sequence, see SEQ ID NO: 3 for the forward primer sequence of the cloned AMP region, and see SEQ ID NO: 4 for the reverse primer sequence. The pUC57 truncated mutant plasmid was constructed by homologous recombination and named pMini. See SEQ ID NO:5 for the sequence of pMini.

[0049] 2. Construction of don...

Embodiment 2

[0056] 1. Synthesize the guide RNA sequence of the first exon of RAB11a gene.

[0057] Two, the SpCas9 (2NLS and 4NLS) used in the present embodiment and the LbCpf1 or AsCpf1 protein purification used in Example 4 are mainly divided into the following two steps:

[0058] 1. Construct the Cas9 / Cpf1 plasmid expression vector, express the Cas9 / Cpf1 protein with the Escherichia coli plasmid expression vector system, and amplify the Cas9 / Cpf1 gene from the pX330 plasmid vector by PCR. The forward primer sequence is shown in SEQ ID NO: 14, and the reverse For the sequence of the forward primer, see SEQ ID NO: 15, for the sequence of the forward primer of LbCpf1, see SEQ ID NO: 16, for the sequence of the reverse primer, see SEQ ID NO: 17, for the sequence of the forward primer of AsCpf1, see SEQ ID NO: 18, for the sequence of the reverse primer See SEQ ID NO:19. Recover after PCR amplification, connect the recovered SpCas9, LbCpf1, and AsCpf1 fragments to the KS expression plasmid ...

Embodiment 3

[0067] 1. Synthesis of CAR gene and homology arm gene

[0068] Taking CD19-CAR targeting CD19 currently used in clinical treatment of leukemia as an example, the amino acid sequence of the CAR gene is shown in SEQ ID NO: 23, mainly including the ScFv that recognizes the specific tumor antigen CD19, and the extracellular segment of CD28 , the transmembrane region and the intracellular segment, and the intracellular segment of CD3ζ. The gene sequence of the left homology arm is shown in SEQ ID NO:24, and the gene sequence of the right homology arm is shown in SEQ ID NO:25. The CAR gene and homologous arm gene fragments were synthesized by PCR, the forward primer was synthesized by PCR as shown in SEQ ID NO:26, and the reverse primer was as shown in SEQ ID NO:27.

[0069] 2. Construction of CAR gene and homology arm sequence on pUC57 / pMini / pMiniZ plasmid vector

[0070] 1. Clone the homology arm sequences on both sides of the target gene CAR and TCRα into the pUC57 / pMini / pMiniZ...

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Abstract

The invention mainly discloses a DNA template for modifying primary cells by gene editing and a fixed-point insertion method. The DNA template comprises a plasmid, a target gene which is constructed on the plasmid and needs to be introduced, homologous sequences (homologous arms) at the upstream and downstream of a target sequence, and a DNA sequence recognized by Guide RNA; then, an RNP complex of a gene editing protein and the Guide RNA, and the DNA template are delivered to a cell line or a primary cell by utilizing an electroporation technology, so that a target gene is inserted into a genome of the cell line or the primary cell at a fixed point, and the cell line or the primary cell is modified. One practical application of the DNA template is that a chimeric antigen receptor T (CAR-T) cell inserted at a fixed point can be produced without depending on viruses, and therefore, the DNA template has better safety and effectiveness in the application of immune cells for treating diseases.

Description

technical field [0001] The present invention relates to the site-specific insertion of the target gene, specifically, the site-specific integration of the target gene into the genome of cell lines or primary cells by using electrotransfer technology combined with gene editing technology through a non-viral method. The chimeric antigen receptor T( CAR-T) cells can be used in the treatment of leukemia. Background technique [0002] At present, the target gene is mainly integrated into the genome of primary human T / NK cells by means of traditional lentivirus or retrovirus, but the disadvantage of this method is that the target gene will be inserted randomly. Site-specific insertion into the cell genome can improve the safety and effectiveness of genetically engineered human primary T / NK cells to a certain extent, and play a greater clinical role. At present, the CRISPR-Cas9 / Cpf1 system has been used very maturely in the realization of gene knockout. However, the technology of...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N15/85
CPCC12N15/907C12N15/85C12N2800/107
Inventor 孙洁周春荆瑞瑞
Owner ZHEJIANG UNIV
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