Application of CRISPR/Cas9 gene editing to target Neuronal Interleukin-16 a novel protein involved in osteoarthritis progression
Dooley, Claire
Dooley, Claire
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Publication Date
2022-09-07
Type
Thesis
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Abstract
Osteoarthritis (OA) is a debilitating joint disease affecting an increasing proportion of the elderly population worldwide annually. Current treatments for the disease are symptomatic with management of pain and side effects the only options. The field of osteoarthritis research has endeavored to understand the mechanisms of cartilage degeneration and disease progression. This has led to the development of several proposed cell and gene therapies that aim to reverse or slow the progression of OA. The underlying molecular mechanisms involved in OA are complex and remain to be elucidated in order to increase understanding of the disease to address the need to identify novel treatment options. One such novel protein is neuronal Interleukin-16 (nIL-16) previously identified as expressed in late osteoarthritic cartilage. nIL-16 is an IL-16 isoform previously reported in neural tissue only. The other known isoform of IL-16, peripheral IL-16 (pIL-16) has been associated with control of T-cell proliferation. nIl-16 has been reported to function as an anchoring protein via its’ PDZ domains in neural tissues leading to the hypothesis that its role in late-stage OA is to anchor ion channels to the chondrocyte membrane causing an influx of calcium during chondrocyte hypertrophy, eventually leading to inappropriate endochondral ossification. The main focus of this thesis was to determine if CRISPR/Cas9 gene editing applied to the PDZ domains of nIL-16 could contribute to elucidate their role in OA progression. CRISPR/Cas9 gene editing has begun to be widely used to understand the gene function in many different contexts. With the ability to target CRISPR/Cas9 to specific sites of interest, targeted double stranded breaks can create edits in the genomic sequence leading to insertions, deletions or frameshift mutations allowing investigation of the outcomes of these changes on gene and protein function. This thesis investigated the suitability of CRISPR/Cas9 technology as a means to investigate the role of nIL-16 in OA progression. The target sites of interest were the PDZ domains in the nIL-16 isoform. Different CRISPR/Cas formats and non-viral mechanisms of delivery of CRISPR/Cas9 to human MSCs were explored with the aim of identifying the most suitable method to investigate whether editing of these domains was possible in hMSCs. Peptide assisted CRISPR plasmid DNA and electroporation of CRISPR plasmid DNA and the CRISPR ribonucleoprotein were assessed as methods of CRISPR gene editing in hMSCs A comparison of the effects of both formats on hMSC survival, proliferation and growth post electroporation was analyzed through examination of gene expression changes in the NF-ΚB, PI3K/AKT/mTOR, MAPK/ERK signaling pathways, Autophagy, senescence, and the DNA damage repair response to double stranded breaks. Electroporation of both plasmid DNA and RNP successfully delivered CRISPR/Cas9 to hMSCs with the RNP having higher levels of viability and editing efficacy compared to the plasmid DNA format. Autophagy was activated in hMSCs regardless of the format of CRISPR used. However, the hMSCs treated with nIL-16 targeting plasmid DNA were visibly more stressed. This was reflected in the gene expression changes with metabolic stress evident through upregulated expression of signaling transducers such as mTOR, MAPK1 and SMAD4 as well as evidence of growth cycle arrest with upregulated p53 and downregulation of p16ink4a. When hMSCs were electroporated with the RNP complexes, there was an initial acute reaction to the electroporation of the RNP complexes but the hMSCs returned to a state of homeostasis within 24 hours of the electroporation with down regulation of NF-ΚB, PI3K/AKT/mTOR, MAPK/ERK pathways as well as rapid autophagic clearance of cell debris. The work in this thesis has demonstrated that the RNP format of CRISPR/Cas9 can efficiently perform edits of hMSCs. Using two RNP complexes targeting up and down stream of the nIL-16 PDZ domains of interest successfully edited the genomic sequence, and the cells were healthy and proliferating post electroporation. This method can therefore be used to further examine the role of nIL-16 in OA progression. Furthermore, this method can be applied to novel genes of interest in OA progression and beyond to uncover their functions in disease progression and to identify targets for drug or gene therapy development.
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Publisher
NUI Galway
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Attribution-NonCommercial-NoDerivs 3.0 Ireland