Epigenetic studies relied so far on correlations between epigenetic marks and gene expression pattern. Technologies developed for epigenome editing now enable direct study of functional relevance of precise epigenetic modifications and gene regulation. The reversible nature of epigenetic modifications, including DNA methylation, has been already exploited in cancer therapy for remodeling the aberrant epigenetic landscape. However, this was achieved non-selectively using epigenetic inhibitors. Epigenetic editing at specific loci represents a novel approach that might selectively and heritably alter gene expression. Here, we developed a CRISPR-Cas9-based tool for specific DNA methylation consisting of deactivated Cas9 (dCas9) nuclease and catalytic domain of the DNA methyltransferase DNMT3A targeted by co–expression of a guide RNA to any 20 bp DNA sequence followed by the NGG trinucleotide. We demonstrated targeted CpG methylation in a ∼35 bp wide region by the fusion protein. We also showed that multiple guide RNAs could target the dCas9-DNMT3A construct to multiple adjacent sites, which enabled methylation of a larger part of the promoter. DNA methylation activity was specific for the targeted region and heritable across mitotic divisions. Finally, we demonstrated that directed DNA methylation of a wider promoter region of the target loci IL6ST and BACH2 decreased their expression.

Null mutations in the UGT1A1 gene result in Crigler-Najjar syndrome type I (CNSI), characterized by severe hyperbilirubinemia and constant risk of developing neurological damage. Phototherapy treatment lowers plasma bilirubin levels, but its efficacy is limited and liver transplantation is required. To find alternative therapies, we applied AAV liver-specific gene therapy to a lethal mouse model of CNSI. We demonstrated that a single neonatal hUGT1A1 gene transfer was successful and the therapeutic effect lasted up to 17 months postinjection. The therapeutic effect was mediated by the presence of transcriptionally active double-stranded episomes. We also compared the efficacy of two different gene therapy approaches: liver versus skeletal muscle transgene expression. We observed that 5-8% of normal liver expression and activity levels were sufficient to significantly reduce bilirubin levels and maintain lifelong low plasma bilirubin concentration (3.1 -1.5 mg/dl). In contrast, skeletal muscle was not able to efficiently lower bilirubin (6.4 -2.0 mg/dl), despite 20-30% of hUgt1a1 expression levels, compared with normal liver. We propose that this remarkable difference in gene therapy efficacy could be related to the absence of the Mrp2 and Mrp3 transporters of conjugated bilirubin in muscle. Taken together, our data support the concept that liver is the best organ for efficient and long-term CNSI gene therapy, and suggest that the use of extra-hepatic tissues should be coupled to the presence of bilirubin transporters.

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Adeno-associated virus (AAV) -mediated gene therapy is a promising strategy to treat liver-based monogenic diseases. However, two major obstacles limit its success: first, vector dilution in actively dividing cells, such as hepatocytes in neonates/children, due to the non-integrating nature of the vector; second, development of an immune response against the transgene and/or viral vector. Crigler-Najjar Syndrome Type I is a rare monogenic disease with neonatal onset, caused by mutations in the liver-specific UGT1 gene, with toxic accumulation of unconjugated bilirubin in plasma, tissues and brain. To establish an effective and long lasting cure, we applied AAV-mediated liver gene therapy to a relevant mouse model of the disease. Repeated gene transfer to adults by AAV-serotype switching, upon neonatal administration, resulted in lifelong correction of total bilirubin (TB) levels in both genders. In contrast, vector loss over time was observed after a single neonatal administration. Adult administration resulted in lifelong TB levels correction in male, but not female Ugt1 mice. Our findings demonstrate that neonatal AAV-mediated gene transfer to the liver supports a second transfer of the therapeutic vector, by preventing the induction of an immune response and supporting the possibility to improve AAV-therapeutic efficacy by repeated administration.

Palbociclib, ribociclib and abemaciclib were recently approved as chemotherapeutic agents and are currently in the post-marketing surveillance phase. They are used in combination with aromatase inhibitors anastrozole and letrozole or antiestrogen fulvestrant for HR+, HER2− breast cancer treatment. Here, a novel bioanalytical LC-ESI-MS/MS method was developed for the quantitation of these six drugs in human plasma. The samples were prepared by simple protein precipitation followed by solvent evaporation. A Kinetex biphenyl column (150 × 4.6 mm, 2.6 µm) used for chromatographic analysis adequately resolved even the closely eluting aromatase inhibitors’ peaks. The mobile phase consisted of 0.1% formic acid in water and in ACN, in a linear gradient. An additional gradient step was added to eliminate the observed carry-over. The proposed method was fully validated in the relevant linear ranges covering the expected plasma concentrations of all six drugs (correlation coefficients between 0.9996 and 0.9931). The intra-day method precision (CV) ranged from 3.1% to 15%, while intra-day accuracy (%bias) was between −1.5% and 15.0%. The inter-day precision ranged from 1.6% to 14.9%, with accuracy between −14.3% and 14.6%, which is in accordance with the EMA and ICH guidelines on bioanalytical method validation. The method was successfully applied to samples from patients treated for HR+, HER2− breast cancer.

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Anemone sensu lato (including Pulsatilla and Hepatica), tribe Anemoneae (Ranunculaceae), is arranged into two subgenera, Anemone and Anemonidium, with basic chromosome numbers x = 8 and x = 7, respectively. We elucidated the level of divergence of 5S rDNA unit arrays between the subgenera, determined intra‐individual and interspecific sequence variation and tested 5S rDNA phylogenetic signal in revealing the origin of polyploid species. High intra‐individual nucleotide diversity and the presence of 5S rDNA unit array length variants and pseudogenes indicate that weak homogenization forces have shaped 5S rDNA in the investigated species. Our results show that 5S rDNA evolved through two major changes: diversification of 5S rDNA into two lineages, one with long (subgenus Anemone) and one with short 5S rDNA unit arrays (subgenus Anemonidium); and subsequent contraction and expansion of 5S rDNA unit arrays. Phylogenetic analysis based on 5S rDNA supports the hypothesis that A. parviflora could be a parental species and donor of the subgenome D to the allopolyploids A. multifida (BBDD) and A. baldensis (AABBDD). In A. baldensis interlocus exchange possibly occurred, followed by subsequent replacement of the 5S rDNA from subgenome D with those from subgenome B. Here we present evidence that both models, concerted and birth‐and‐death evolution, were probably involved in the evolution of the 5S rDNA multigene family in subgenera Anemone and Anemonidium.

Cas3 nuclease-helicase is part of CRISPR immunity systems in many bacteria and archaea. In type I CRISPR, Cas3 nuclease degrades invader DNA that has been base-paired to crRNA as an R-loop within a "Cascade" complex. An R-loop is a DNA-RNA hybrid that includes a displaced single-strand DNA loop. Purified Cas3 from E. coli and the archaeon M. thermautrophicus can process R-loops without DNA/RNA sequence specificity and without Cascade. This has potential to affect other aspects of microbial biology that involve R-loops. Regulatory RNAs and host cell proteins modulate replication of ColE1 plasmids (e.g., pUC) from R-loop primers. We observed that Cas3 could override endogenous control of a ColE1 replicon, stimulating uncontrolled ("runaway") replication and resulting in much higher plasmid yields. This effect was absent when using helicase-defective Cas3 (Cas3 (K320L) ) or a non-ColE1 plasmid, and was dependent on RNaseHI. Cas3 also promoted formation of plasmid multimers or concatemers, a phenotype consistent with deregulated ColE1 replication and typical of cells lacking RNaseHI. These effects of Cas3 on ColE1 plasmids are inconsistent with it unwinding R-loops in vivo, at least in this assay. We discuss a model of how Cas3 might be able to regulate RNA molecules in vivo, unless it is targeted to CRISPR defense by Cascade, or kept in check by RecG and RNaseHI.