SH2D1A gene, sgRNA and application of sgRNA

A DNA molecule and gene technology applied in the field of T cell molecular biology

Active Publication Date: 2019-05-10
NANJING DRUM TOWER HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the application of SH2D1A gene in restoring invasively exhausted CD8+ T cells in liver cancer tissue has not been reported

Method used

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  • SH2D1A gene, sgRNA and application of sgRNA
  • SH2D1A gene, sgRNA and application of sgRNA
  • SH2D1A gene, sgRNA and application of sgRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Embodiment 1 screening SH2D1A gene

[0066] After the project passed the review of the ethics committee, the applicant systematically studied the transcriptome expression profile of infiltrating CD8+ T cells in human liver cancer tissues, discovered new genes that determine the exhaustion of CD8+ T cells, and analyzed liver cancer tissues by flow cytometry. Infiltrating CD8+ T cells were sorted according to the expression level of inhibitory surface receptors (PD-1, TIM3) on the cell surface: 3 cases of non-exhausted CD8+ T cells (PD1-TIM3-), exhausted CD8+ T cells (PD1 +TIM3+) 3 cases, a total of 6 samples (see Table 1 for specific information from 3 patients):

[0067] Table 1 Sample Information 1

[0068]

[0069] Table 2 Sample Information 2

[0070]

[0071]

[0072] Then through high-throughput RNA sequencing, a group of differentially expressed genes were found in CD8+ T cells with different degrees of depletion, and the invasiveness of liver cancer ti...

Embodiment 2

[0091] Design, synthesis, connection, transformation, electrotransfection of embodiment 2 sgRNA

[0092] 1) Design of SH2D1A-sgRNA

[0093] According to the gene sequence of SH2D1A on NCBI Genbank (its sequence is shown in SEQ ID NO.1), combined with the principle of CRISPR / Cas9 gene editing, according to the PAM sequence (NGG) positioning, according to its exon 3 (its sequence is shown in SEQ ID NO. 2), select 5 sites located on SH2D1A exon3, and design sgRNA sequences 1-5, the nucleotide sequences of which are shown in sequence as SEQ ID NO.3-SEQ ID NO.7.

[0094] 2) Synthesis of SH2D1A-sgRNA

[0095] Combining pGL3-U6-sgRNA-PGK-puromycin carrier plasmid (the carrier structure is as follows figure 1 Shown) (Carleton, J.B., et al. (2017). "Multiplex Enhancer Interference RevealsCollaborative Control of Gene Regulation by Estrogen Receptor alpha-BoundEnhancers." Cell Syst 5(4):333-344e335.) and restriction endonuclease BsaI (NEB, R3535S), based on the sgRNA1-5 designed in ...

Embodiment 3

[0155] Example 3 Identification of the effect of target-specific knockout of SH2D1A in CD8+ T cells

[0156] 1) RT-PCR method to detect the expression level of SH2D1AmRNA in CD8+ T cells

[0157] a) Total RNA extraction

[0158] The SH2D1A-knockout exhausted CD8+ T cells 1-5 in Example 2 were lysed by TRIzol (Invitrogen), and then the total RNA 1-5 was extracted using chloroform, isopropanol and 75% ethanol. b) Preparation of cDNA1-5: prepare cDNA by reverse transcription reagent PrimeScript RT Master Mix (Takara) Reaction conditions: 37°C for 15min, 85°C for 5sec, 4°C∞

[0159] The reaction system is as follows:

[0160]

[0161] c) qPCR detection of SH2D1A mRNA expression in exhausted and non-exhausted CD8+ T cells

[0162] According to SH2D1A mRNA sequence situation design specific primer, sequence (SEQ ID NO.18 and SEQ ID NO.19)

[0163] Fluorescent quantitative PCR program: pre-denaturation at 95°C for 30s, followed by 40 cycles: 95°C for 5s, 60°C for 32s.

[0164...

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Abstract

The invention discloses an SH2D1A gene and application of sgRNA to preparation of medicine for T cell depletion recovery or medicine for resisting to a tumor, and relates to the field of T cell molecular biology. By finding, detecting and applying genes with most remarkable expression difference in CD8+T cells with different depletion degrees, through high-throughput RNA sequencing, SH2D1A has animportant adjusting effect in genesis and development of CD8+T cell depletion, and SH2D1A expression is closely related to the genesis and development of CD8+T cell depletion; then, according to the coding sequence of SH2D1A, sgRNA is designed and synthesized, through combination with a plasmid vector and conversion and based on a CRISPR / Cas9 gene editing technology, SH2D1A is knocked down in theCD8+T cells, genesis of CR8+T cell depletion can be reduced remarkably, the anti-tumor capacity of the depleted CD8+T cells is restored remarkably, and sgRNA can be applied to preparation of anti-tumor medicine.

Description

technical field [0001] The present invention belongs to the field of T cell molecular biology, specifically, the present invention relates to SH2D1A gene and its sgRNA and its application. Background technique [0002] Primary liver cancer is one of the most common malignant tumors in my country. Its incidence rate ranks fifth among malignant tumors in the world, and its fatality rate ranks third. According to pathological classification, it can be divided into hepatocyte type, cholangiocyte type and mixed type. Three types, among which hepatocellular carcinoma (HCC) is the most common, accounting for more than 90% of primary liver cancer (Chen W, etal. Cancer statistics in China, 2015. CA Cancer J Clin2016; 66:115-132.) . [0003] The main cause of HCC is chronic active hepatitis, including viral hepatitis caused by hepatitis B virus (HBV) and hepatitis C virus (HCV), alcoholic hepatitis and nonalcoholic fatty liver hepatitis. HBV infection is the main route of common hepat...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): C12N15/113C12N15/90C12N9/22C12N5/10A61K31/7105A61K48/00A61P35/00
Inventor孙倍成何其峰王晓琛黄新立夏安亮
OwnerNANJING DRUM TOWER HOSPITAL