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Glia Maturation Factor Gene Editing Improves Neurocognitive Function in an Alzheimer's Disease Mouse Model
Author(s) -
Raikwar Sudhanshu P,
Ahmed Mohammad E,
Dubova Iuliia,
Mentor Shireen,
Saeed Daniyal,
Zahoor Haris,
Premkumar Keerthivaas,
Thangavel Ramasamy,
Selvakumar Govindhasamy P.,
Kempuraj Duraisamy,
Zaheer Smita,
Iyer Shankar S.,
Zaheer Asgar
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.620.10
Subject(s) - genome editing , neuroinflammation , cas9 , crispr , neurodegeneration , biology , genetic enhancement , mcherry , neuroscience , viral vector , microglia , disease , gene , immunology , medicine , genetics , green fluorescent protein , recombinant dna , pathology , inflammation
Alzheimer's Disease (AD) is a progressive neurodegenerative disorder that causes an irreversible cognitive decline in an estimated 5.5 million Americans. If the present trend continues, by 2050 the number of AD patients will surpass 16 million with ~$1.1 trillion in healthcare costs thereby necessitating the development of novel therapeutic strategies to effectively treat AD. Towards fulfillment of the unmet need for an effective AD therapy, our ultimate goal is to develop a robust AD patient‐specific personalized precision‐guided targeted gene editing and stem cell therapy. Glia maturation factor (GMF), a brain specific pro‐inflammatory molecule discovered in our laboratory plays a crucial role in neuroinflammation and AD pathophysiology. We hypothesized that CRISPR/Cas9‐mediated GMF gene editing in microglia is a novel approach to reduce neuroinflammation, neurodegeneration, AD pathophysiology and improve cognitive function. To achieve GMF gene editing, we developed recombinant adeno‐associated viral (AAV) and lentiviral vectors (LVs) expressing CRISPR/Cas9 and GMF‐specific sgRNAs. Confocal microscopy of BV2 cells transduced with an AAV simultaneously co‐expressing Staphylococcus aureus CRISPR/Cas9 (SaCas9) and a GMF specific sgRNA revealed a small subset of BV2 cells expressing SaCas9 while completely lacking GMF expression, thereby confirming successful biallelic GMF gene editing. To further improve GMF gene editing efficiency, we generated LVs coexpressing either 1) Streptococcus pyogenes CRISPR/Cas9 (SpCas9), eGFP and Neo r or 2) GMF‐specific individual sgRNAs1‐3, mCherry and Puro r . BV2 cells were sequentially transduced with LV‐EF1α‐SpCas9 and LV‐GMF‐sgRNAs1‐3 to generate stable clones. Confocal microscopy revealed reduced GMF expression in GMF‐edited BV2 cells as compared to non‐edited BV2 cells. DNA sequencing of GMF edited clones revealed indels in the exons 2–3 of the GMF coding sequence thereby conclusively proving SpCas9‐mediated GMF gene editing. Treatment of wild type non‐edited and GMF‐edited BV2 cells with LPS revealed differences in basal p38 MAPK as well as LPS‐induced phosphorylation of p38 MAPK. In wild type non‐edited BV2 cells LPS treatment induced significant upregulation of pp38 MAPK, however in GMF‐edited BV2 cells pp38 MAPK levels were significantly lower, thereby indicating that GMF gene editing leads to attenuation of microglial activation. Further, GMF gene editing in BV2 cells leads to regulation of mitochondrial dynamics by attenuation of NRF2/HO‐1 dependent ferritin activation. Our in vivo GMF gene editing data in the 5XFAD mouse model of AD revealed improved neurocognitive function. Overall, our data suggest that GMF gene editing represents a novel approach to develop precision‐targeted AD patient‐specific therapy. Support or Funding Information VA Merit Award I01BX002477 and NIH Grants AG048205 and NS073670 to AZ This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .