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Method for knocking out PD-L1 gene in keratinocytes by CRISPR/Cas9 gene editing method

A PD-L1 and gene editing technology, applied to other methods of inserting foreign genetic material, cells modified by introducing foreign genetic material, animal cells, etc., can solve the problems of psoriasis patients who are difficult to continue to adhere to treatment and adverse reactions.

Pending Publication Date: 2019-09-20
SHANGHAI CHANGHAI HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the past few years, PD-L1 antibodies (such as durvalumab, avelumab, atezolizumab) have achieved promising results in cancer treatment, but they are often accompanied by skin-related adverse reactions. Among these adverse reactions, the onset and exacerbation of psoriasis Make it difficult for patients to continue to adhere to treatment (Fattore D, Annunziata MC, Panariello L, Marasca C, Fabbrocini G. Successful treatment of psoriasis induced by immune checkpoint inhibitors with apremilast. European journal of cancer. 2019,110:107-9.)

Method used

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  • Method for knocking out PD-L1 gene in keratinocytes by CRISPR/Cas9 gene editing method
  • Method for knocking out PD-L1 gene in keratinocytes by CRISPR/Cas9 gene editing method
  • Method for knocking out PD-L1 gene in keratinocytes by CRISPR/Cas9 gene editing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Expression of PD-L1 in keratinocytes of skin lesions of patients with psoriasis

[0045] 1. Materials and methods

[0046] (1) Subjects and samples

[0047] A total of 27 subjects participated in this study, including 18 in the disease group and 9 in the healthy control group. The skin samples of the disease group were provided by patients with psoriasis in the Dermatology Department of Shanghai Changhai Hospital, and the skin samples of the healthy control group were provided by volunteers who underwent plastic surgery at the Department of Plastic Surgery of Shanghai Changhai Hospital. This clinical study was approved by the Ethics Committee of Shanghai Changhai Hospital.

[0048] Subject inclusion and exclusion criteria are as follows:

[0049] 1. Inclusion criteria

[0050] Psoriasis group: (1) Patients with vulgaris psoriasis diagnosed by two or more dermatologists; (2) without blood and severe cardiovascular diseases; (3) without other autoimmune dis...

Embodiment 2

[0075] Example 2: Using CRISPR / Cas9 technology to construct a PD-L1 knockout keratinocyte model

[0076] In humans, the full length of the PD-L1 gene contains 7 exons and is located at the p24.1 site on chromosome 9. PD-L1 protein is a type I transmembrane protein, consisting of 290 amino acids, including IgV-like and IgC-like hydrophobic transmembrane domains. As the third member of the B7 protein family, it has 15%-20% homology with B7.1 and B7.2 proteins (Kythreotou A, Siddique A, Mauri FA, Bower M, Pinato DJ.Pd-L1.Journal of clinical pathology. 2018,71(3):189-94.). Through previous studies, it is known that PD-L1 plays an important role in psoriasis, and immunohistochemical results show that epidermal keratinocytes of healthy people express a large amount of PD-L1, while PD-L1 is expressed in keratinocytes of skin lesions of patients with psoriasis. The expression of -L1 was significantly downregulated. Kim et al. (Kim J, Bissonnette R, Lee J, Correa da Rosa J, Suarez-F...

Embodiment 3

[0150] Example 3: Effects of PD-L1 Knockout on Cell Regulatory Factors Secreted by Keratinocytes

[0151] PD-L1 mainly plays its role by binding to its specific receptor PD-1. PD-L1 knockout mice or PD-L1 antibody blocking mice have been widely used in the research of autoimmune diseases and autologous transplantation diseases. However, PD-L1 is widely distributed, and it is not very good to distinguish whether the intervening PD-L1 plays a role in tissues or lymphocytes. Keratinocytes can regulate the proliferation and activity of T cells through PD-L1 (Mazanet MM, Hughes CCW. B7-H1 Is Expressed by Human Endothelial Cells and Suppresses T Cell Cytokine Synthesis. The Journal of Immunology. 2002, 169 (7): 3581- 8.). (Ritprajak P, Hashiguchi M, Tsushima F, Chalermsarp N, Azuma M.Keratinocyte-associated B7-H1 directly regulates cutaneous effector CD8+T cell responses.Journal of immunology.2010,184(9):4918-25.) by Ritprajak et al. The study of K14-PD-L1 transgenic mice found tha...

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Abstract

The invention relates to the field of cell model construction, and concretely relates to a method for knocking out a PD-L1 gene in keratinocytes by a CRISPR / Cas9 gene editing method, and a keratinocyte model constructed by the method and an application thereof. The method has the advantages that the PD-L1 knockout keratinocyte stable-transfection strain constructed by the invention can be used not only for the research of psoriasis, but also for the research of other skin diseases with low expression of PD-L1. The construction of the PD-L1 knockout cell strain is helpful for exploring the role of PD-L1 in skin-related diseases such as psoriasis, pityriasis rosea, and allergic contact dermatitis, and further explores the function of PD-L1 in keratinocytes.

Description

technical field [0001] The invention relates to the field of cell model construction, specifically, a CRISPR / Cas9 gene editing method for knocking out the PD-L1 gene in keratinocytes, and the keratinocyte model constructed by the method and its application. Background technique [0002] Psoriasis is a genetically related, immune-mediated chronic inflammatory disease, usually with skin and joint lesions. The clinical manifestations of psoriasis are red plaques with clear boundaries on the skin surface, accompanied by silvery white dry scales, often accompanied by pruritus symptoms, and easy to relapse, which brings great physiological, psychological and economic burdens to patients. [0003] Skin consists of epidermis (including stratum corneum, granular layer, spinous layer and basal layer), dermis (papillary layer and reticular layer) and subcutaneous tissue. The main component of the epidermis is keratinocytes, which play an important role in the skin's immune response. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/90C12N15/867C12N5/10
CPCC12N15/113C12N15/907C12N15/86C12N5/0629C07K14/70532C12N2310/10C12N2310/20C12N2740/15043C12N2800/107C12N2510/00
Inventor 胡晋红薛潇春阳凌燕兰芬唐晓萌施林林
Owner SHANGHAI CHANGHAI HOSPITAL
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