Prime Editor 3 Mediated Beta-Thalassemia Mutations of the <i>HBB</i> Gene in Human Erythroid Progenitor Cells

Recently developed Prime Editor 3 (PE3) has been implemented to induce genome editing in various cell types but has not been proven in human hematopoietic stem and progenitor cells. Using PE3, we successfully installed the beta-thalassemia (beta-thal) mutations in the HBB gene in the erythroid progenitor cell line HUDEP-2. We inserted the mCherry reporter gene cassette into editing plasmids, each including the prime editing guide RNA (pegRNA) and nick sgRNA. The plasmids were electroporated into HUDEP-2 cells, and the PE3 modified cells were identified by mCherry expression and collected using fluorescence-activated cell sorting (FACS). Sanger sequencing of the positive cells confirmed that PE3 induced precise beta-thal mutations with editing ratios from 4.55 to 100%. Furthermore, an off-target analysis showed no unintentional edits occurred in the cells. The editing ratios and parameters of pegRNA and nick sgRNA were also analyzed and summarized and will contribute to enhanced PE3 design in future studies. The characterization of the HUDEP-2 beta-thal cells showed typical thalassemia phenotypes, involving ineffective erythropoiesis, abnormal erythroid differentiation, high apoptosis rate, defective alpha-globin colocalization, cell viability deterioration, and ROS resisting deficiency. These HUDEP-2 beta-thal cells could provide ideal models for future beta-thal gene therapy studies

Improved gene therapy for MFRP deficiency-mediated retinal degeneration by knocking down endogenous bicistronic Mfrp and Ctrp5 transcript

The membrane frizzled-related protein (Mfrp) and C1-tumor necrosis factor related protein 5 (Ctrp5) genes are transcribed as a bicistronic unit and dysregulation of either gene is associated with retinal degeneration in the retinal pigment epithelium (RPE) cells. However, the mechanisms that regulate the expression of the bicistronic transcript remain controversial. Here, we identified a microRNA-based negative feedback loop that helps maintain a normal expression level of the bicistronic Mfrp and Ctrp5 transcript. Specifically, miR-149-3p, a conserved microRNA, binds to the 3′UTR of the Mfrp gene. In MFRP-deficient rd6 mice, the miR-149-3p levels were compromised compared with those in WT mice, resulting in an increase in the bicistronic transcript. We also report a capsid-modified rAAVDJ-3M vector that is capable of robustly and specifically transducing RPE cells following subretinal delivery. Compared with the parental vector, the modified vector elicited similar levels of serum anti-rAAV antibodies, but recruited fewer microglial infiltrations. Most significantly, we also demonstrate that simultaneous overexpressing of MFRP and knockdown of the bicistronic transcript was more effective in rescuing vision than MFRP overexpression alone. Our findings offer new insights into the function of MFRP and provide a promising therapeutic strategy for the treatment of MFRP-associated ocular diseases