All Publications


  • Peptide nucleic acid-dependent artifact can lead to false-positive triplex gene editing signals. Proceedings of the National Academy of Sciences of the United States of America Ho, P. Y., Zhang, Z., Hayes, M. E., Curd, A., Dib, C., Rayburn, M., Tam, S. N., Srivastava, T., Hriniak, B., Li, X., Leonard, S., Wang, L., Tarighat, S., Sim, D. S., Fiandaca, M., Coull, J. M., Ebens, A., Fordyce, M., Czechowicz, A. 2021; 118 (45)

    Abstract

    Triplex gene editing relies on binding a stable peptide nucleic acid (PNA) sequence to a chromosomal target, which alters the helical structure of DNA to stimulate site-specific recombination with a single-strand DNA (ssDNA) donor template and elicits gene correction. Here, we assessed whether the codelivery of PNA and donor template encapsulated in Poly Lactic-co-Glycolic Acid (PLGA)-based nanoparticles can correct sickle cell disease and x-linked severe combined immunodeficiency. However, through this process we have identified a false-positive PCR artifact due to the intrinsic capability of PNAs to aggregate with ssDNA donor templates. Here, we show that the combination of PNA and donor templates but not either agent alone results in different degrees of aggregation that result in varying but highly reproducible levels of false-positive signal. We have identified this phenomenon in vitro and confirmed that the PNA sequences producing the highest supposed correction in vitro are not active in vivo in both disease models, which highlights the importance of interrogating and eliminating carryover of ssDNA donor templates in assessing various gene editing technologies such as PNA-mediated gene editing.

    View details for DOI 10.1073/pnas.2109175118

    View details for PubMedID 34732575

  • Head and neck squamous cancer progression is marked by CLIC4 attenuation in tumor epithelium and reciprocal stromal upregulation of miR-142-3p, a novel post-transcriptional regulator of CLIC4. Oncotarget Carofino, B. L., Dinshaw, K. M., Ho, P. Y., Cataisson, C. n., Michalowski, A. M., Ryscavage, A. n., Alkhas, A. n., Wong, N. W., Koparde, V. n., Yuspa, S. H. 2019; 10 (68): 7251–75

    Abstract

    Chloride intracellular channel 4 (CLIC4) is a tumor suppressor implicated in processes including growth arrest, differentiation, and apoptosis. CLIC4 protein expression is diminished in the tumor parenchyma during progression in squamous cell carcinoma (SCC) and other neoplasms, but the underlying mechanisms have not been identified. Data from The Cancer Genome Atlas suggest this is not driven by genomic alterations. However, screening and functional assays identified miR-142-3p as a regulator of CLIC4. CLIC4 and miR-142-3p expression are inversely correlated in head and neck (HN) SCC and cervical SCC, particularly in advanced stage cancers. In situ localization revealed that stromal immune cells, not tumor cells, are the predominant source of miR-142-3p in HNSCC. Furthermore, HNSCC single-cell expression data demonstrated that CLIC4 is lower in tumor epithelial cells than in stromal fibroblasts and endothelial cells. Tumor-specific downregulation of CLIC4 was confirmed in an SCC xenograft model concurrent with immune cell infiltration and miR-142-3p upregulation. These findings provide the first evidence of CLIC4 regulation by miRNA. Furthermore, the distinct localization of CLIC4 and miR-142-3p within the HNSCC tumor milieu highlight the limitations of bulk tumor analysis and provide critical considerations for both future mechanistic studies and use of miR-142-3p as a HNSCC biomarker.

    View details for DOI 10.18632/oncotarget.27387

    View details for PubMedID 31921386

    View details for PubMedCentralID PMC6944452