Angelman syndrome is a devastating neurogenetic disorder for which there is currently no effective treatment. It is caused by mutations or epimutations affecting the expression or function of the maternally inherited allele of the ubiquitin-protein ligase E3A ( UBE3A ) gene. The paternal UBE3A allele is imprinted in neurons of the central nervous system (CNS) by the UBE3A antisense ( UBE3A-AS ) transcript, which represents the distal end of the small nucleolar host gene 14 ( SNHG14 ) transcription unit. Reactivating the expression of the paternal UBE3A allele in the CNS has long been pursued as a therapeutic option for Angelman syndrome. Here, we described the development of an antisense oligonucleotide (ASO) therapy for Angelman syndrome that targets an evolutionarily conserved region demarcating the start of the UBE3A-AS transcript. We designed and chemically optimized gapmer ASOs targeting specific sequences at the start of the human UBE3A-AS transcript. We showed that ASOs targeting this region precisely and efficiently repress the transcription of UBE3A-AS , reactivating the expression of the paternal UBE3A allele in neurotypical and Angelman syndrome induced pluripotent stem cell–derived neurons. We further showed that human-targeted ASOs administered to the CNS of cynomolgus macaques by lumbar intrathecal injection repress UBE3A-AS and reactivate the expression of the paternal UBE3A allele throughout the CNS. These findings support the advancement of this investigational molecular therapy for Angelman syndrome into clinical development ( ClinicalTrials.gov , NCT04259281).

Streptococcus gallolyticussubspeciesgallolyticus(Sgg) has a strong clinical association with colorectal cancer (CRC) and actively promotes the development of colon tumors. However, the molecular determinants involved inSggpathogenicity in the gut are unknown. Bacterial type VII secretion systems (T7SS) mediate pathogen interactions with their host and are important for virulence in pathogenic mycobacteria andStaphylococcus aureus. Through genome analysis, we identified a locus inSggstrain TX20005 that encodes a putative type VII secretion system (designated asSggT7SS^T05). We showed that core genes within theSggT7SS^T05locus are expressedin vitroand in the colon of mice. Western blot analysis showed thatSggEsxA, a protein predicted to be a T7SS secretion substrate, is detected in the bacterial culture supernatant, indicating that thisSggT7SS^T05is functional. Deletion ofSggT7SS^T05(TX20005Δesx) resulted in impaired bacterial adherence to HT29 cells and abolished the ability ofSggto stimulate HT29 cell proliferation. Analysis of bacterial culture supernatants suggest thatSggT7SS^T05-secreted factors are responsible for the pro-proliferative activity ofSgg, whereasSggadherence to host cells requires bothSggT7SS^T05-secreted and bacterial surface-associated factors. In a murine gut colonization model, TX20005Δesxshowed significantly reduced colonization compared to the parent strain. Furthermore, in a mouse model of CRC, mice exposed to TX20005 had a significantly higher tumor burden compared to saline-treated mice, whereas those exposed to TX20005Δesxdid not. Examination of theSggload in the colon in the CRC model suggests thatSggT7SS^T05-mediated activities are directly involved in the promotion of colon tumors. Taken together, these results revealSggT7SS^T05as a previously unrecognized pathogenicity determinant forSggcolonization of the colon and promotion of colon tumors.

Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating disease of the central nervous system. Dysregulation of STAT3, a transcription factor pivotal to various cellular processes including Th17 cell differentiation, has been implicated in MS. Here, we report that STAT3 is activated in infiltrating monocytic cells near active MS lesions and that activation of STAT3 in myeloid cells is essential for leukocyte infiltration, neuroinflammation, and demyelination in experimental autoimmune encephalomyelitis (EAE). Genetic disruption ofStat3in peripheral myeloid lineage cells abrogated EAE, which was associated with decreased antigen-specific T helper cell responses. Myeloid cells from immunizedStat3mutant mice exhibited impaired antigen-presenting functions and were ineffective in driving encephalitogenic T cell differentiation. Single-cell transcriptome analyses of myeloid lineage cells from preclinical wild-type and mutant mice revealed that loss of myeloid STAT3 signaling disrupted antigen-dependent cross-activation of myeloid cells and T helper cells. This study identifies a previously unrecognized requisite for myeloid cell STAT3 in the activation of myelin-reactive T cells and suggests myeloid STAT3 as a potential therapeutic target for autoimmune demyelinating disease.

Prenatal alcohol exposure (PAE) results in cerebral cortical dysgenesis. Single-cell RNA sequencing was performed on murine fetal cerebral cortical cells from six timed pregnancies, to decipher persistent cell- and sex-specific effects of an episode of PAE during early neurogenesis. We found, in an analysis of 38 distinct neural subpopulations across 8 lineage subtypes, that PAE altered neural maturation and cell cycle and disrupted gene co-expression networks. Whereas most differentially regulated genes were inhibited, particularly in females, PAE also induced sex-independent neural expression of fetal hemoglobin, a presumptive epigenetic stress adaptation. PAE inhibited Bcl11a, Htt, Ctnnb1, and other upstream regulators of differentially expressed genes and inhibited several autism-linked genes, suggesting that neurodevelopmental disorders share underlying mechanisms. PAE females exhibited neural loss of X-inactivation, with correlated activation of autosomal genes and evidence for spliceosome dysfunction. Thus, episodic PAE persistently alters the developing neural transcriptome, contributing to sex- and cell-type-specific teratology.

Mus musculus is the only known species from which embryonic stem cells (ESC) can be isolated under conditions requiring only leukemia inhibitory factor (LIF). Other species are non-permissive in LIF media, and form developmentally primed epiblast stem cells (EpiSC) similar to cells derived from post-implantation, egg cylinders. To evaluate whether non-permissiveness extends to induced pluripotent stem cells (iPSC), we derived iPSC from the eight founder strains of the mouse Collaborative Cross. Two strains, NOD/ShiLtJ and the WSB/EiJ, were non-permissive, consistent with the previous classification of NOD/ShiLtJ as non-permissive to ESC derivation. We determined non-permissiveness is recessive, and that non-permissive genomes do not compliment. We overcame iPSC non-permissiveness by using GSK3B and MEK inhibitors with serum, a technique we termed 2iS reprogramming. Although used for ESC derivation, GSK3B and MEK inhibitors have not been used during iPSC reprogramming because they inhibit survival of progenitor differentiated cells. iPSC derived in 2iS are more transcriptionally similar to ESC than EpiSC, indicating that 2iS reprogramming acts to overcome genetic background constraints. Finally, of species tested for ESC or iPSC derivation, only some M. musculus strains are permissive under LIF culture conditions suggesting that this is an evolutionarily derived characteristic in the M. musculus lineage.