Twin prime editing mediated exon skipping/reinsertion for restored collagen VII expression in recessive dystrophic epidermolysis bullosa.

Gene editing nucleases, base editors, and prime editors are potential locus specific genetic treatment strategies for recessive dystrophic epidermolysis bullosa (RDEB); however, many RDEB COL7A1 mutations are unique, making the development of personalized editing reagents challenging. 270 of the ∼320 COL7A1 EB mutations reside in exons that can be skipped, and antisense oligonucleotides (ASO) and gene editing nucleases have been used to create in-frame deletions. ASOs are transient and nucleases generate deleterious double stranded DNA breaks (DSB) and uncontrolled mixtures of allele products. We developed a twin prime editing (twinPE) strategy using the PEmax and recently evolved PE6 prime editors and dual prime editing guide RNAs flanking COL7A1 exon five. Prime editing-mediated deletion of exon 5 with a homozygous premature stop codon was achieved in RDEB fibroblasts, keratinocytes, and iPSC with minimal DSBs, and collagen type VII (C7) protein was restored. TwinPE can replace the target exon with recombinase attachment sequences, and we exploited this to re-insert a normal copy of exon 5 using the Bxb1 recombinase. These findings demonstrate that twinPE can facilitate locus-specific, predictable, in-frame deletions and sequence replacement with few DSBs as a strategy that may enable a single therapeutic agent to treat multiple RDEB patient cohorts.

Detection of Increased Relative Expression Units of Bacteroides and Prevotella, and Decreased Clostridium leptum in Stool Samples from Brazilian Rheumatoid Arthritis Patients: A Pilot Study.

Interactions between gut microbes and disease modifying antirheumatic drugs (DMARDs) have been proposed. The aim of the present study was to evaluate the presence of some specific bacteria in stool samples from Brazilian RA patients receiving DMARDs and correlate these data with diet, clinical parameters, and cytokines. Stool samples were used for gut bacteria evalutation by qPCR. Serum samples were used to quantify IL-4 and IL-10 by flow cytometer. Statistics were performed by Pearson chi-square, Mann-Whitney U test, and Spearman's correlation. The study included 20 RA patients and 30 healthy controls. There were no significant differences (P > 0.05) in dietary habits between RA patients and controls. Concerning gut bacteria, we observed an increase in relative expression units (REU) of Bacteroides and Prevotella species in stool samples from patients, and a decrease in REU of Clostridium leptum when compared with healthy controls. Positive correlation between Prevotella and rheumatoid factor was detected. The IL-4 and IL-10 concentrations were increased in patients when compared with controls. We concluded that gut bacteria are different between RA patients receiving DMARDs and healthy controls. Further studies are necessary to determine the real role of gut microbes and their metabolities in clinical response to different DMARDs in RA patients.