Epidermal Growth Factor Mediated Healing in Stem Cell-derived Vocal Fold Mucosa

Background: The goal of vocal fold wound healing is the reconstitution of functional tissue, including a structurally and functionally intact epithelium. Mechanisms underlying reepithelialization in vocal folds are not known, although it is suspected that healing involves the interplay between several growth factors. We used a three-dimensional human embryonic stem cell-derived model of vocal fold mucosa to examine the effects of one growth factor, exogenous epidermal growth factor (EGF), on wound healing. Materials and methods: A scratch wound was created in the in vitro model. Rate of wound healing, epidermal growth factor receptor (EGFR) activation, and cell proliferation after injury were analyzed with and without application of both exogenous EGF and an EGFR inhibitor, gefitinib. Results: Wound repair after injury was significantly hastened by application of exogenous EGF (13.3 μm/h, ±2.63) compared with absence of exogenous EGF (7.1 μm/h ±2.84), but inhibited with concurrent addition of Gefitinib (5.2 μm/h, ±2.23), indicating that EGF mediates wound healing in an EGFR-dependent manner. Immunohistochemistry revealed that EGFR activation occurred only in the presence of exogenous EGF. Although not statistically significant, increased density of Ki67 staining in the epithelium adjacent to the scratch wound was observed after treatment with EGF, suggesting a tendency for exogenous EGF to increase epithelial cell proliferation. Conclusions: Exogenous EGF increases the rate of wound healing in an EGFR-dependent manner in a three-dimensional stem cell-derived model of vocal fold mucosa. This model of wound healing can be used to gain insight into the mechanisms that regulate vocal fold epithelial repair after injury

Virus-Free CRISPR CAR T cells induce solid tumor regression

Chimeric antigen receptor (CAR) T cell therapy has shown promising efficacy in treating hematologic malignancies and has led to the FDA-approval of three CAR T cell products. However, there has been little success in treating solid tumors, as clinical trials to date have yielded little to no responses and no improvement in survival. Current methods of CAR T cell production typically involve the use of viral vectors which can give rise to complications such as insertional mutagenesis, leading to gene silencing or oncogene activation. In addition, GMP-grade viral vector manufacturing can be expensive with lengthy wait times for new batches. Here we have developed a virus-free strategy in primary T cells that has eliminated the use of viral vectors through the use of CRISPR-Cas9 to precisely edit the chimeric antigen receptor into the TRAC gene1. Our method of virus free production begins through the generation of a double stranded DNA (dsDNA) template produced by polymerase chain reaction (PCR). This template is then combined with a SpCas9-single guide RNA to create a ribonucleoprotein (RNP) complex. Isolated human primary T cells from adult healthy donors are then nucleofected with the RNP and dsDNA template on day 2 of ex vivo expansion. Flow cytometry is then utilized to immunophenotype the cell product and analyze the percent of efficiency of CAR gene transfer. Within the cell product, the editing efficiencies are \u3e95% TCR knockout and 35% CAR+. Transcriptional profiling indicates that the virus-free CART cells have a favorable memory-like phenotype. In addition to our in vitro work, in vivo mice studies with anti-GD2 CART products demonstrate regression of GD2+ solid tumors upon virus-free CART treatment, showing similar potency and survival to viral-produced CAR T cells. The production of virus-free CAR T cells has high potential to enable the rapid and flexible manufacturing of highly defined and highly potent CAR T cell products for the treatment of solid tumors. 1 Mueller, K. et al. CRISPR-mediated insertion of a chimeric antigen receptor produces nonviral T cell products capable of inducing solid tumor regression. bioRxiv preprint doi: https://doi.org/10.1101/2021.08.06.455489 (2021)

Virus-Free CRISPR CAR T cells induce solid tumor regression

Chimeric antigen receptor (CAR) T cell therapy has shown promising efficacy in treating hematologic malignancies and has led to the FDA-approval of three CAR T cell products. However, there has been little success in treating solid tumors, as clinical trials to date have yielded little to no responses and no improvement in survival. Current methods of CAR T cell production typically involve the use of viral vectors which can give rise to complications such as insertional mutagenesis, leading to gene silencing or oncogene activation. In addition, GMP-grade viral vector manufacturing can be expensive with lengthy wait times for new batches. Here we have developed a virus-free strategy in primary T cells that has eliminated the use of viral vectors through the use of CRISPR-Cas9 to precisely edit the chimeric antigen receptor into the TRAC gene1. Our method of virus free production begins through the generation of a double stranded DNA (dsDNA) template produced by polymerase chain reaction (PCR). This template is then combined with a SpCas9-single guide RNA to create a ribonucleoprotein (RNP) complex. Isolated human primary T cells from adult healthy donors are then nucleofected with the RNP and dsDNA template on day 2 of ex vivo expansion. Flow cytometry is then utilized to immunophenotype the cell product and analyze the percent of efficiency of CAR gene transfer. Within the cell product, the editing efficiencies are \u3e95% TCR knockout and 35% CAR+. Transcriptional profiling indicates that the virus-free CART cells have a favorable memory-like phenotype. In addition to our in vitro work, in vivo mice studies with anti-GD2 CART products demonstrate regression of GD2+ solid tumors upon virus-free CART treatment, showing similar potency and survival to viral-produced CAR T cells. The production of virus-free CAR T cells has high potential to enable the rapid and flexible manufacturing of highly defined and highly potent CAR T cell products for the treatment of solid tumors. 1 Mueller, K. et al. CRISPR-mediated insertion of a chimeric antigen receptor produces nonviral T cell products capable of inducing solid tumor regression. bioRxiv preprint doi: https://doi.org/10.1101/2021.08.06.455489 (2021)