Inactive Cas9 blocks vitreous-induced expression of Mdm2 and proliferation and survival of retinal pigment epithelial cells

Na Chen; Zhengping Hu; Yanhui Yang; Haote Han; Hetian Lei

doi:10.1016/j.exer.2019.107716

Inactive Cas9 blocks vitreous-induced expression of Mdm2 and proliferation and survival of retinal pigment epithelial cells

Exp Eye Res. 2019 Sep:186:107716. doi: 10.1016/j.exer.2019.107716. Epub 2019 Jul 3.

Authors

Na Chen¹, Zhengping Hu², Yanhui Yang³, Haote Han², Hetian Lei⁴

Affiliations

¹ Schepens Eye Research Institute of Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, MA, USA; Department of Ophthalmology, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, 200127, PR China.
² Schepens Eye Research Institute of Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, MA, USA.
³ Schepens Eye Research Institute of Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, MA, USA; School of Basic Medical Sciences, Ningxia Medical University, Yinchuan, Ningxia, China.
⁴ Schepens Eye Research Institute of Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, MA, USA. Electronic address: hetian_lei@meei.harvard.edu.

PMID: 31278903
DOI: 10.1016/j.exer.2019.107716

Abstract

Mouse double minute (MDM)2 single nucleotide polymorphism (SNP) 309G allele in the second promoter of MDM2 enhances vitreous-induced expression of Mdm2 and degradation of the tumor suppressor protein p53. This MDM2^SNP309G contributes to certain cancer development and experimental proliferative vitreoretinopathy. The goal of this study is to discover a novel strategy to only block vitreous-induced expression of Mdm2 for preventing vitreous-induced cell proliferation and survival and thus find a potential novel strategy to treat proliferation-related diseases. We created two mutations (D10A and H840A) in Streptococcus pyogenes (Sp)Cas9 within the nuclease domains (RuvC1 and HNH, respectively) to render this SpCas9 nuclease dead named as dCas9 in a lentiCRISPR v2 vector. Then an MDM2-sgRNA targeting the second promoter of human MDM2 gene was cloned into this vector for producing lentivirus to infect human retinal pigment epithelial (RPE) cells with, which carry a heterozygous genotype of MDM2^{SNP309 T/G}. lacZ-sgRNA was used as a control. As a result, we discovered that vitreous from experimental rabbits induced a 1.9 ± 0.2 fold increase in Mdm2 and a 2.0 ± 0.2 fold decrease in p53 in the RPE cells with dCas9/lacZ-sgRNA compared to those with dCas9/MDM2-sgRNA, suggesting that dCas9 under the guidance of the MDM2-sgRNA prevented RV-stimulated increase in Mdm2. In addition, we found that the rabbit vitreous significantly enhanced cell proliferation (1.5 ± 0.2 fold), survival against apoptosis (2.2 ± 0.2 fold), migration (10 ± 1.5%) and contraction (112.7 ± 14.1 mm²) of the cells with dCas9/lacZ-sgRNA compared with those with dCas9/MDM2-sgRNA. These results indicated that application of the dCas9 targeted to the P2 of MDM2 is a potential therapeutic approach to diseases due to the P2-driven aberrant expression of Mdm2 - such as proliferative vitreoretinopathy.

Keywords: CRISPR; Mdm2; Proliferation and survival; Vitreous; dSpCas9.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Animals
CRISPR-Cas Systems / physiology*
Cell Proliferation / physiology
Cell Survival / physiology
Cells, Cultured
Epithelial Cells / physiology*
Humans
Mice
Molecular Targeted Therapy / methods
Polymorphism, Single Nucleotide
Proto-Oncogene Proteins c-mdm2 / metabolism*
Retinal Pigment Epithelium / physiology*
Tumor Suppressor Protein p53 / metabolism
Vitreoretinopathy, Proliferative / physiopathology
Vitreous Body / metabolism*

Substances

Tumor Suppressor Protein p53
Proto-Oncogene Proteins c-mdm2