A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing.

The development of clinically viable delivery methods presents one of the greatest challenges in the therapeutic application of CRISPR/Cas9 mediated genome editing. Here, we report the development of a lipid nanoparticle (LNP)-mediated delivery system that, with a single administration, enabled significant editing of the mouse transthyretin (Ttr) gene in the liver, with a >97% reduction in serum protein levels that persisted for at least 12 months. These results were achieved with an LNP delivery system that was biodegradable and well tolerated. The LNP delivery system was combined with a sgRNA having a chemical modification pattern that was important for high levels of in vivo activity. The formulation was similarly effective in a rat model. Our work demonstrates that this LNP system can deliver CRISPR/Cas9 components to achieve clinically relevant levels of in vivo genome editing with a concomitant reduction of TTR serum protein, highlighting the potential of this system as an effective genome editing platform.

Genomic Scars Generated by Polymerase Theta Reveal the Versatile Mechanism of Alternative End-Joining.

For more than half a century, genotoxic agents have been used to induce mutations in the genome of model organisms to establish genotype-phenotype relationships. While inaccurate replication across damaged bases can explain the formation of single nucleotide variants, it remained unknown how DNA damage induces more severe genomic alterations. Here, we demonstrate for two of the most widely used mutagens, i.e. ethyl methanesulfonate (EMS) and photo-activated trimethylpsoralen (UV/TMP), that deletion mutagenesis is the result of polymerase Theta (POLQ)-mediated end joining (TMEJ) of double strand breaks (DSBs). This discovery allowed us to survey many thousands of available C. elegans deletion alleles to address the biology of this alternative end-joining repair mechanism. Analysis of ~7,000 deletion breakpoints and their cognate junctions reveals a distinct order of events. We found that nascent strands blocked at sites of DNA damage can engage in one or more cycles of primer extension using a more downstream located break end as a template. Resolution is accomplished when 3' overhangs have matching ends. Our study provides a step-wise and versatile model for the in vivo mechanism of POLQ action, which explains the molecular nature of mutagen-induced deletion alleles.

FANCJ promotes DNA synthesis through G-quadruplex structures.

Our genome contains many G-rich sequences, which have the propensity to fold into stable secondary DNA structures called G4 or G-quadruplex structures. These structures have been implicated in cellular processes such as gene regulation and telomere maintenance. However, G4 sequences are prone to mutations particularly upon replication stress or in the absence of specific helicases. To investigate how G-quadruplex structures are resolved during DNA replication, we developed a model system using ssDNA templates and Xenopus egg extracts that recapitulates eukaryotic G4 replication. Here, we show that G-quadruplex structures form a barrier for DNA replication. Nascent strand synthesis is blocked at one or two nucleotides from the G4. After transient stalling, G-quadruplexes are efficiently unwound and replicated. In contrast, depletion of the FANCJ/BRIP1 helicase causes persistent replication stalling at G-quadruplex structures, demonstrating a vital role for this helicase in resolving these structures. FANCJ performs this function independently of the classical Fanconi anemia pathway. These data provide evidence that the G4 sequence instability in FANCJ(-/-) cells and Fancj/dog1 deficient C. elegans is caused by replication stalling at G-quadruplexes.

A Polymerase Theta-dependent repair pathway suppresses extensive genomic instability at endogenous G4 DNA sites.

Genomes contain many sequences that are intrinsically difficult to replicate. Tracts of tandem guanines, for instance, have the potential to adopt stable G-quadruplex structures, which are prone to cause genome alterations. Here we describe G4 DNA-induced mutagenesis in Caenorhabditis elegans and identify a non-canonical DNA break repair mechanism that generates deletions characterized by an extremely narrow size distribution, minimal homology of exactly one nucleotide at the junctions, and by the occasional presence of templated insertions. This typical mutation profile is fully dependent on the A-family polymerase Theta, the absence of which leads to profound loss of sequences surrounding G4 motifs. Theta-mediated end-joining prevails over non-homologous end joining and homologous recombination and prevents genomic havoc at replication fork barriers at the expense of small deletions. G4 DNA-induced deletions also manifest in the genomes of wild isolates of C. elegans, indicating a protective role for this pathway during evolution.

Nonpeptidergic allosteric antagonists differentially bind to the CXCR2 chemokine receptor.

The chemokine receptor CXCR2 is involved in different inflammatory diseases, like chronic obstructive pulmonary disease, psoriasis, rheumatoid arthritis, and ulcerative colitis; therefore, it is considered an attractive drug target. Different classes of small CXCR2 antagonists have been developed. In this study, we selected seven CXCR2 antagonists from the diarylurea, imidazolylpyrimide, and thiazolopyrimidine class and studied their mechanisms of action at human CXCR2. All compounds are able to displace (125)I-CXCL8 and inhibit CXCL8-induced beta-arrestin2 recruitment. Detailed studies with representatives of each class showed that these compounds displace and antagonize CXCL8, most probably via a noncompetitive, allosteric mechanism. In addition, we radiolabeled the high-affinity CXCR2 antagonist SB265610 [1-(2-bromophenyl)-3-(4-cyano-1H-benzo[d] [1,2,3]-triazol-7-yl)urea] and subjected [(3)H]SB265610 to a detailed analysis. The binding of this radioligand was saturable and reversible. Using [(3)H]SB265610, we found that compounds of the different chemical classes bind to distinct binding sites. Hence, the use of a radiolabeled low-molecular weight CXCR2 antagonist serves as a tool to investigate the different binding sites of CXCR2 antagonists in more detail.

Astrocytes produce interferon-alpha and CXCL10, but not IL-6 or CXCL8, in Aicardi-Goutières syndrome.

Aicardi-Goutières syndrome (AGS) presents as a severe autosomal recessively inherited neurological brain disease. Clinical and neurological manifestations closely resemble those of congenital viral infection and are generally attributed to a perturbation of innate immunity including a long lasting lymphocytosis and production of interferon-alpha (IFNalpha) in the central nervous system. To clarify the innate immune response evoked in these diseases, we used a 30-mer multiplexed luminex system to measure multiple cytokines and growth factors in the cerebrospinal fluid and serum of patients with AGS and viral meningitis or encephalitis, and febrile controls in whom infection could not be substantiated. In addition to the previously described IFNalpha, both AGS and viral diseases were characterized by expression of CXCL10 and CCL2. In contrast to AGS, viral infection resulted in high levels of IL-6 and CXCL8 in the CNS. Postmortem immunohistochemical staining of brain sections showed that in both AGS and viral CNS infection, astrocytes were responsible for the production of cytokines and not the infiltrating leukocytes. In summary, our data indicate that astrocytes are the predominant cell type responsible for the production of IFNalpha and CXCL10 in AGS. Whereas IFNalpha is assumed to be involved in the neurodegeneration, calcifications and seizures in AGS, CXCL10 may act as the chemoattractant responsible for the influx of activated lymphocytes into the brain. The lack of the inflammatory cytokines IL-6 and CXCL8 in AGS suggest that the neuroinflammatory reaction in this disease is distinct from viral disease.

Identification of the first nonpeptidergic inverse agonist for a constitutively active viral-encoded G protein-coupled receptor.

Human cytomegalovirus (HCMV) encodes a G protein-coupled receptor (GPCR), named US28, which shows homology to chemokine receptors and binds several chemokines with high affinity. US28 induces migration of smooth muscle cells, a feature essential for the development of atherosclerosis, and may serve as a co-receptor for human immunodeficiency virus-type 1 entry into cells. Previously, we have shown that HCMV-encoded US28 displays constitutive activity, whereas its mammalian homologs do not. In this study we have identified a small nonpeptidergic molecule (VUF2274) that inhibits US28-mediated phospholipase C activation in transiently transfected COS-7 cells and in HCMV-infected fibroblasts. Moreover, VUF2274 inhibits US28-mediated HIV entry into cells. In addition, VUF2274 fully displaces radiolabeled RANTES (regulated on activation normal T cell expressed and secreted) binding at US28, apparently with a noncompetitive behavior. Different analogues of VUF2274 have been synthesized and pharmacologically characterized, to understand which features are important for its inverse agonistic activity. Finally, by means of mutational analysis of US28, we have identified a glutamic acid in transmembrane 7 (TM 7), which is highly conserved among chemokine receptors, as a critical residue for VUF2274 binding to US28. The identification of a full inverse agonist provides an important tool to investigate the relevance of US28 constitutive activity in viral pathogenesis.