Fig. 5: Design and validation of housekeeping-like sLCRs for gene therapy compatible with AAV-vectors size constraints.

a Schematic generation and validation of sLCRs based on broadly expressed genes and transcription factors in humans and rodents (partially assembled using BioRender.com). b Bar plot of the sequence lengths of housekeeping-like sLCRs (HKGTs) and well-known broadly expressed promoters. Note all HKGTs (housekeeping genetic tracing sLCRs) to be shorter than well established UBC, EF1A, CMV and hPGK promoters. c Box plot of normalized mCherry fluorescence intensities for EFS or HK sLCRs transduced stably in human, mouse, and hamster cell lines. Data distribution is shown, with box indicating the interquartile range and inner line indicating the median. Whiskers extend to represent the data range, including outliers. Small dots denote individual datapoints for triplicate measurements of cell lines of human, mouse, and hamster origin (n = 6), genetically engineered with each of the nine constructs. Statistical significance values were calculated by two-sided unpaired t-test. d Heatmap of the normalized mCherry fluorescence intensities of EFS or HK sLCRs transduced stably into human, mouse, and hamster cell lines. e Scatter plot with varying dot size indicating mean log10-transformed intensities and interquartile range of HKs LCR-driven and EFS-driven mCherry. Dot size represents standard deviation, color-coded for corresponding identities. Note that EFS is the strongest promoter, and HKGT4 is the strongest promoter with lowest variability across all the lines tested. Source data are provided in the Source Data file.