Use of CRISPR gene editing to investigate novel therapeutic strategies to counteract the effects of Hypoxia-Inducible Factor-1alpha single nucleotide polymorphisms

Secondary Supervisor(s): Dr Hannah Bridgewater (Warwick)

University of Registration: Coventry University

BBSRC Research Themes: Understanding the Rules of Life (Immunology)

Project Outline

This project will use cutting-edge CRISPR gene editing techniques to introduce single nucleotide polymorphisms (SNPs) in monocyte cell lines. These cell lines will be used to elucidate the molecular mechanisms by which Hypoxia-inducible factor-1 (HIF-1) SNPs affect macrophage phenotypes relevant to progression of a range of diseases, and to enable testing of novel siRNA therapeutics.

HIF-1 is a transcription factor which is constitutively expressed but is only stable in low oxygen conditions and therefore accumulates to facilitate adaptation of cells to hypoxia. HIF-1 is present at high levels in a variety of diseases including arthritis, cardiovascular conditions, preeclampsia, and solid tumours, and is linked to poor outcomes. HIF-1 is extremely important for the activity of human macrophages, phagocytic white blood cells which play vital roles in maintaining health and in a range of diseases^(1,2).The research team has significant experience in macrophage biology^(1,3).

It is known that SNPs within the oxygen-regulated alpha subunit (HIF-1α) can affect expression and activity of HIF-1, and negatively influence disease outcomes⁽⁴⁾. The majority of studies aimed at understanding the role of these SNPs have focused on coding regions of the gene. Disease-linked SNPs often occur within the Oxygen-Dependent Degradation domain of HIF-1a, which is responsible for its regulation by hypoxia, causing aberrant upregulation of the protein in normoxia.

However, there is also evidence that genetic variations in the 3’ untranslated region (3’UTR) lead to changes in gene expression, by creating new miRNA binding sites or by inactivating existing sites. Hence, 3’UTR variation in this gene has the potential to affect risk and susceptibility to a wide variety of diseases, as well as affecting outcomes and responses to therapies, although functional studies are lacking. One particular SNP (rs2057482) in the HIF1A 3’UTR has been linked to increased levels of HIF-1α protein in cardiovascular diseases, in femoral osteonecrosis, and in tumour tissues. The rs2057482 SNP was shown to reduce the ability of microRNAs to bind to the HIF1A 3’UTR to repress HIF-1α mRNA levels, resulting in increased expression of HIF-1 protein, disease progression, and poor outcomes⁽⁵⁾. CRISPR-edited cell lines containing HIF1A SNPs will be created to test their role in the modulation of monocyte phenotype in a hypoxic cell culture model.

Project aims

• Generate CRISPR-edited macrophage cell lines containing either HIF1A 3’ UTR SNPs or the corresponding normal WT sequence.

• Investigate the mechanisms by which 3’UTR SNPs (starting initially with the rs2057482 SNP) affect HIF1A expression and cell phenotype under different conditions (hypoxia and normoxia) by carrying out functional assays on the CRISPR-edited macrophages.

• Determine the therapeutic potential of siRNA designed to specifically target these 3’UTR mutated mRNAs.

Methodology

1. All-in-one plasmid vectors targeted to the 3’ UTR HIF1A (pCas-Guide-EF1a-GFP), alongside a DNA template (including the 3’ UTR SNP sequence) will be designed and cloned.

2. Plasmid and DNA template will be transfected into the THP-1 human monocyte cell line.

3. Clones from step 3 will be expanded and sequenced to confirm the correct genome edit.

4. HIF-1 protein expression will be determined by Western blot in the WT and CRISPR-edited THP-1 cell lines in hypoxia and normoxia.

5. Effects of anti-HIF-1 SNP tailored siRNA on HIF-1 levels and macrophage phenotypes will be determined.

This research evaluates the potential of cutting-edge CRISPR gene editing technologies to validate functionally and clinically relevant “genetic drug” targets and provide proof of concept for future 3’UTR SNP therapeutic studies in many human diseases.

References

1. Burke et al. doi: 10.1002/path.1029.

2. Qiu et al. doi: 10.1016/j.heliyon.2023.e17167.

3. Feehan et al. 10.1038/s41467-024-48138-y.

4. Gladek et al. doi: 10.1002/gcc.22449.

5. Wang et al. doi: 10.18632/oncotarget.7263.