Arming Immune Cell Therapeutics with Polymeric Prodrugs.

Engineered immune cells are an exciting therapeutic modality, which survey and attack tumors. Backpacking strategies exploit cell targeting capabilities for delivery of drugs to combat tumors and their immune-suppressive environments. Here, a new platform for arming cell therapeutics through dual receptor and polymeric prodrug engineering is developed. Macrophage and T cell therapeutics are engineered to express a bioorthogonal single chain variable fragment receptor. The receptor binds a fluorescein ligand that directs cell loading with ligand-tagged polymeric prodrugs, termed "drugamers." The fluorescein ligand facilitates stable binding of drugamer to engineered macrophages over 10 days with 80% surface retention. Drugamers also incorporate prodrug monomers of the phosphoinositide-3-kinase inhibitor, PI-103. The extended release of PI-103 from the drugamer sustains antiproliferative activity against a glioblastoma cell line compared to the parent drug. The versatility and modularity of this cell arming system is demonstrated by loading T cells with a second fluorescein-drugamer. This drugamer incorporates a small molecule estrogen analog, CMP8, which stabilizes a degron-tagged transgene to provide temporal regulation of protein activity in engineered T cells. These results demonstrate that this bioorthogonal receptor and drugamer system can be used to arm multiple immune cell classes with both antitumor and transgene-activating small molecule prodrugs.

BRF1 mutations alter RNA polymerase III-dependent transcription and cause neurodevelopmental anomalies.

RNA polymerase III (Pol III) synthesizes tRNAs and other small noncoding RNAs to regulate protein synthesis. Dysregulation of Pol III transcription has been linked to cancer, and germline mutations in genes encoding Pol III subunits or tRNA processing factors cause neurogenetic disorders in humans, such as hypomyelinating leukodystrophies and pontocerebellar hypoplasia. Here we describe an autosomal recessive disorder characterized by cerebellar hypoplasia and intellectual disability, as well as facial dysmorphic features, short stature, microcephaly, and dental anomalies. Whole-exome sequencing revealed biallelic missense alterations of BRF1 in three families. In support of the pathogenic potential of the discovered alleles, suppression or CRISPR-mediated deletion of brf1 in zebrafish embryos recapitulated key neurodevelopmental phenotypes; in vivo complementation showed all four candidate mutations to be pathogenic in an apparent isoform-specific context. BRF1 associates with BDP1 and TBP to form the transcription factor IIIB (TFIIIB), which recruits Pol III to target genes. We show that disease-causing mutations reduce Brf1 occupancy at tRNA target genes in Saccharomyces cerevisiae and impair cell growth. Moreover, BRF1 mutations reduce Pol III-related transcription activity in vitro. Taken together, our data show that BRF1 mutations that reduce protein activity cause neurodevelopmental anomalies, suggesting that BRF1-mediated Pol III transcription is required for normal cerebellar and cognitive development.

CHD8 regulates neurodevelopmental pathways associated with autism spectrum disorder in neural progenitors.

Truncating mutations of chromodomain helicase DNA-binding protein 8 (CHD8), and of many other genes with diverse functions, are strong-effect risk factors for autism spectrum disorder (ASD), suggesting multiple mechanisms of pathogenesis. We explored the transcriptional networks that CHD8 regulates in neural progenitor cells (NPCs) by reducing its expression and then integrating transcriptome sequencing (RNA sequencing) with genome-wide CHD8 binding (ChIP sequencing). Suppressing CHD8 to levels comparable with the loss of a single allele caused altered expression of 1,756 genes, 64.9% of which were up-regulated. CHD8 showed widespread binding to chromatin, with 7,324 replicated sites that marked 5,658 genes. Integration of these data suggests that a limited array of direct regulatory effects of CHD8 produced a much larger network of secondary expression changes. Genes indirectly down-regulated (i.e., without CHD8-binding sites) reflect pathways involved in brain development, including synapse formation, neuron differentiation, cell adhesion, and axon guidance, whereas CHD8-bound genes are strongly associated with chromatin modification and transcriptional regulation. Genes associated with ASD were strongly enriched among indirectly down-regulated loci (P < 10(-8)) and CHD8-bound genes (P = 0.0043), which align with previously identified coexpression modules during fetal development. We also find an intriguing enrichment of cancer-related gene sets among CHD8-bound genes (P < 10(-10)). In vivo suppression of chd8 in zebrafish produced macrocephaly comparable to that of humans with inactivating mutations. These data indicate that heterozygous disruption of CHD8 precipitates a network of gene-expression changes involved in neurodevelopmental pathways in which many ASD-associated genes may converge on shared mechanisms of pathogenesis.

Complex reorganization and predominant non-homologous repair following chromosomal breakage in karyotypically balanced germline rearrangements and transgenic integration.

We defined the genetic landscape of balanced chromosomal rearrangements at nucleotide resolution by sequencing 141 breakpoints from cytogenetically interpreted translocations and inversions. We confirm that the recently described phenomenon of 'chromothripsis' (massive chromosomal shattering and reorganization) is not unique to cancer cells but also occurs in the germline, where it can resolve to a relatively balanced state with frequent inversions. We detected a high incidence of complex rearrangements (19.2%) and substantially less reliance on microhomology (31%) than previously observed in benign copy-number variants (CNVs). We compared these results to experimentally generated DNA breakage-repair by sequencing seven transgenic animals, revealing extensive rearrangement of the transgene and host genome with similar complexity to human germline alterations. Inversion was the most common rearrangement, suggesting that a combined mechanism involving template switching and non-homologous repair mediates the formation of balanced complex rearrangements that are viable, stably replicated and transmitted unaltered to subsequent generations.

Type 1 T helper cells induce the accumulation of myeloid-derived suppressor cells in the inflamed Tgfb1 knockout mouse liver.

UNLABELLED: Immune-mediated liver injury in hepatitis is due to activated T cells producing interferon-γ (IFN-γ). It is important to identify negative feedback immune mechanisms that can regulate T cell activity. In this study, we demonstrate that liver inflammation mediated by type 1 T helper (Th1) cells can induce the accumulation of myeloid-derived suppressor cells (MDSCs), pleiomorphic cells capable of modulating T cell-mediated immunity, that heretofore have been studied almost exclusively in the context of tumor-associated inflammation. Mice deficient in the gene encoding transforming growth factor-ß1 (Tgfb1(-/-) mice) acutely develop liver necroinflammation caused by IFN-γ-producing clusters of differentiation 4-positive (CD4(+)) T cells. Liver Th1 cell accumulation was accompanied by myeloid cells expressing CD11b and Gr1, phenotypic hallmarks of MDSCs. Isolated Tgfb1(-/-) liver CD11b(+)Gr1(+) cells were functional MDSCs, readily suppressing T cell proliferation in vitro. Pharmacologic inhibitors of inducible nitric oxide (NO) synthase completely eliminated suppressor function. Suppressor function and the production of NO were dependent on cell-cell contact between MDSCs and T cells, and upon IFN-γ, and were specifically associated with the "monocytic" CD11b(+)Ly6G(-) Ly6C(hi) subset of liver Tgfb1(-/-) CD11b(+) cells. The rapid accumulation of CD11b(+)Gr1(+) cells in Tgfb1(-/-) liver was abrogated when mice were either depleted of CD4(+) T cells or rendered unable to produce IFN-γ, showing that Th1 activity induces MDSC accumulation. CONCLUSION: Th1 liver inflammation mobilizes an MDSC response that, through the production of NO, can inhibit T cell proliferation. We propose that MDSCs serve an important negative feedback function in liver immune homeostasis, and that insufficient or inappropriate activity of this cell population may contribute to inflammatory liver pathology.