CRISPR/Cas9 in the era of nanomedicine and synthetic biology.

The CRISPR/Cas system was first discovered as a defense mechanism in bacteria and is now used as a tool for precise gene-editing applications. Rapidly evolving, it is increasingly applied in therapeutics. However, concerns about safety, specificity, and delivery still limit its potential. In this context, we introduce the concept of nanogenetics and speculate how the rational engineering of the CRISPR/Cas machinery could advance the biomedical field. In nanogenetics, the advantages of traditional approaches of synthetic biology could be expanded by nanotechnology approaches, enabling the design of a new generation of intrinsically safe and specific genome-editing platforms.

SP8 Transcriptional Regulation of Cyclin D1 During Mouse Early Corticogenesis.

Multiple signals control the balance between proliferation and differentiation of neural progenitor cells during corticogenesis. A key point of this regulation is the control of G1 phase length, which is regulated by the Cyclin/Cdks complexes. Using genome-wide chromatin immunoprecipitation assay and mouse genetics, we have explored the transcriptional regulation of Cyclin D1 (Ccnd1) during the early developmental stages of the mouse cerebral cortex. We found evidence that SP8 binds to the Ccnd1 locus on exon regions. In vitro experiments show SP8 binding activity on Ccnd1 gene 3'-end, and point to a putative role for SP8 in modulating PAX6-mediated repression of Ccnd1 along the dorso-ventral axis of the developing pallium, creating a medialLow-lateralHigh gradient of neuronal differentiation. Activation of Ccnd1 through the promoter/5'-end of the gene does not depend on SP8, but on ßcatenin (CTNNB1). Importantly, alteration of the Sp8 level of expression in vivo affects Ccnd1 expression during early corticogenesis. Our results indicate that Ccnd1 regulation is the result of multiple signals and that SP8 is a player in this regulation, revealing an unexpected and potentially novel mechanism of transcriptional activation.

Varicella-zoster virus glycoprotein E is a critical determinant of virulence in the SCID mouse-human model of neuropathogenesis.

Varicella-zoster virus (VZV) is a neurotropic alphaherpesvirus. VZV infection of human dorsal root ganglion (DRG) xenografts in immunodeficient mice models the infection of sensory ganglia. We examined DRG infection with recombinant VZV (recombinant Oka [rOka]) and the following gE mutants: gEΔ27-90, gEΔCys, gE-AYRV, and gE-SSTT. gEΔ27-90, which lacks the gE domain that interacts with a putative receptor insulin-degrading enzyme (IDE), replicated as extensively as rOka, producing infectious virions and significant cytopathic effects within 14 days of inoculation. Since neural cells express IDE, the gE/IDE interaction was dispensable for VZV neurotropism. In contrast, gEΔCys, which lacks gE/gI heterodimer formation, was significantly impaired at early times postinfection; viral genome copy numbers increased slowly, and infectious virus production was not detected until day 28. Delayed replication was associated with impaired cell-cell spread in ganglia, similar to the phenotype of a gI deletion mutant (rOkaΔgI). However, at later time points, infection of satellite cells and other supportive nonneuronal cells resulted in extensive DRG tissue damage and cell loss such that cytopathic changes observed at day 70 were more severe than those for rOka-infected DRG. The replication of gE-AYRV, which is impaired for trans-Golgi network (TGN) localization, and the replication of gE-SSTT, which contains mutations in an acidic cluster, were equivalent to that of rOka, causing significant cytopathic effects and infectious virus production by day 14; genome copy numbers were equivalent to those of rOka. These experiments suggest that the gE interaction with cellular IDE, gE targeting to TGN sites of virion envelopment, and phosphorylation at SSTT are dispensable for VZV DRG infection, whereas the gE/gI interaction is critical for VZV neurovirulence.

Analysis of the functions of glycoproteins E and I and their promoters during VZV replication in vitro and in skin and T-cell xenografts in the SCID mouse model of VZV pathogenesis.

The two VZV glycoproteins, gE and gI, are encoded by genes that are designated open reading frames, ORF67 and ORF68, located in the short unique region of the VZV genome. These proteins have homologs in the other alphaherpesviruses. Like their homologues, VZV gE and gI exhibit prominent co-localization in infected cells and form heterodimers. However, VZV gE is much larger than its homologues because it has a unique N-terminal domain, consisting of 188 amino acids that are not present in these other gene products. VZV gE also differs from the related gE proteins, in that it is essential for viral replication. Targeted mutations of gE that are compatible with VZV replication in cultured cells have varying phenotypes in skin and T-cell xenografts in the SCID mouse model of VZV pathogenesis in vivo. While gI is dispensable for growth in cultured cells in vitro, this glycoprotein is essential for VZV infection of differentiated human skin and T cells in vivo. The promoter regions of gE and gI are regulated by the cellular transactivator, specificity protein factor 1 (Sp1) in combination with the major VZV transactivator in reporter construct experiments and some Sp1 promoter elements are important for VZV virulence in vivo. Further analysis of VZV gE and gI functions and their interactions with other viral and host cell proteins are important areas for studies of VZV replication and pathogenesis.

Functions of the unique N-terminal region of glycoprotein E in the pathogenesis of varicella-zoster virus infection.

Varicella-zoster virus (VZV) is an alphaherpesvirus that infects skin, lymphocytes, and sensory ganglia. VZV glycoprotein E (gE) has a unique N-terminal region (aa1-188), which is required for replication and includes domains involved in secondary envelopment, efficient cell-cell spread, and skin infection in vivo. The nonconserved N-terminal region also mediates binding to the insulin-degrading enzyme (IDE), which is proposed to be a VZV receptor. Using viral mutagenesis to make the recombinant rOka-DeltaP27-G90, we showed that amino acids in this region are required for gE/IDE binding in infected cells; this deletion reduced cell-cell spread in vitro and skin infection in vivo. However, a gE point mutation, linker insertions, and partial deletions in the aa27-90 region, and deletion of a large portion of the unique N-terminal region, aa52-187, had similar or more severe effects on VZV replication in vitro and in vivo without disrupting the gE/IDE interaction. VZV replication in T cells in vivo was not impaired by deletion of gE aa27-90, suggesting that these gE residues are not essential for VZV T cell tropism. However, the rOka-DeltaY51-P187 mutant failed to replicate in T cell xenografts as well as skin in vivo. VZV tropism for T cells and skin, which is necessary for its life cycle in the human host, requires this nonconserved region of the N-terminal region of VZV gE.

Deletion of the first cysteine-rich region of the varicella-zoster virus glycoprotein E ectodomain abolishes the gE and gI interaction and differentially affects cell-cell spread and viral entry.

Varicella-zoster virus (VZV) glycoprotein E (gE) is the most abundant glycoprotein in infected cells and, in contrast to those of other alphaherpesviruses, is essential for viral replication. The gE ectodomain contains a unique N-terminal region required for viral replication, cell-cell spread, and secondary envelopment; this region also binds to the insulin-degrading enzyme (IDE), a proposed VZV receptor. To identify new functional domains of the gE ectodomain, the effect of mutagenesis of the first cysteine-rich region of the gE ectodomain (amino acids 208 to 236) was assessed using VZV cosmids. Deletion of this region was compatible with VZV replication in vitro, but cell-cell spread of the rOka-DeltaCys mutant was reduced significantly. Deletion of the cysteine-rich region abolished the binding of the mutant gE to gI but not to IDE. Preventing gE binding to gI altered the pattern of gE expression at the plasma membrane of infected cells and the posttranslational maturation of gI and its incorporation into viral particles. In contrast, deletion of the first cysteine-rich region did not affect viral entry into human tonsil T cells in vitro or into melanoma cells infected with cell-free VZV. These experiments demonstrate that gE/gI heterodimer formation is essential for efficient cell-cell spread and incorporation of gI into viral particles but that it is dispensable for infectious varicella-zoster virion formation and entry into target cells. Blocking gE binding to gI resulted in severe impairment of VZV infection of human skin xenografts in SCIDhu mice in vivo, documenting the importance of cell fusion mediated by this complex for VZV virulence in skin.

Cellular and viral factors regulate the varicella-zoster virus gE promoter during viral replication.

Varicella-zoster virus (VZV) glycoprotein E (gE) is essential for viral replication and is involved in cell-to-cell spread, secondary envelopment, and entry. We created a set of mutations in the gE promoter to investigate the role of viral and cellular transcriptional factors in regulation of the gE promoter. Deletion or point mutation of the two Sp1 sites in the gE promoter abolished Sp1 binding and IE62-mediated transactivation of the gE promoter in vitro. Incorporation of the deletion or the point mutations disrupting both of the Sp1 binding sites into the VZV genome was not compatible with viral replication. A point mutation altering the atypical Sp1 binding site was lethal, while altering the second site impaired VZV replication significantly, indicating functional differences between the two Sp1 binding sites. Deletions in the gE promoter that abolished putative binding sites for cellular transcriptional factors other than Sp1, identified by bioinformatics analysis, were inserted in the VZV genome. Replication of the viruses with mutations of the gE promoter did not differ from control recombinants in melanoma cells or primary human tonsil T cells in vitro. These deletions did not affect infection of human skin xenografts in SCIDhu mice. These results indicate that Sp1 is required for IE62-mediated transactivation of the gE promoter and that this transcriptional factor appears to be the only cellular factor essential for regulation of the gE promoter.

The ubiquitous cellular transcriptional factor USF targets the varicella-zoster virus open reading frame 10 promoter and determines virulence in human skin xenografts in SCIDhu mice in vivo.

Varicella-zoster virus (VZV) open reading frame 10 (ORF10) is a determinant of virulence in SCIDhu skin xenografts but not in human T cells in vivo. In this analysis of the regulation of ORF10 transcription, we have identified four ORF10-related transcripts, including a major 1.3-kb RNA spanning ORF10 only and three other read-through transcripts. Rapid-amplification-of-cDNA-ends experiments indicated that the 1.3-kb transcript of ORF10 has single initiation and termination sites. In transient expression assays, the ORF10 promoter was strongly stimulated by the major VZV transactivator, IE62. Deletion analyses revealed approximate boundaries for the full ORF10 promoter activity between -75 and -45 and between +5 and -8, relative to the ORF10 transcription start site. The recombinant virus POKA10-Deltapro, with the ORF10 promoter deletion, blocked transcription of ORF10 and also of ORF9A and ORF9 mRNAs, whereas expression of read-through ORF9A/9/10 and ORF9/10 transcripts was increased, compensating for the loss of the monocistronic mRNAs. The cellular factor USF bound specifically to its consensus site within the ORF10 promoter and was required for IE62 transactivation, whereas disrupting the predicted TATA boxes or Oct-1 binding elements had no effect. The USF binding site was disrupted in the recombinant virus, POKA10-proDeltaUSF, and no ORF10 protein was produced. Both ORF10 promoter mutants reduced VZV replication in SCIDhu skin xenografts. These observations provided further evidence of the contribution of the ORF10 protein to VZV pathogenesis in skin and demonstrated that VZV depends upon the cellular transcriptional factor USF to support its virulence in human skin in vivo.

Essential functions of the unique N-terminal region of the varicella-zoster virus glycoprotein E ectodomain in viral replication and in the pathogenesis of skin infection.

Varicella-zoster virus (VZV) glycoprotein E (gE) is a multifunctional protein important for cell-cell spread, envelopment, and possibly entry. In contrast to other alphaherpesviruses, gE is essential for VZV replication. Interestingly, the N-terminal region of gE, comprised of amino acids 1 to 188, was shown not to be conserved in the other alphaherpesviruses by bioinformatics analysis. Mutational analysis was performed to investigate the functions associated with this unique gE N-terminal region. Linker insertions, serine-to-alanine mutations, and deletions were introduced in the gE N-terminal region in the VZV genome, and the effects of these mutations on virus replication and cell-cell spread, gE trafficking and localization, virion formation, and replication in vivo in the skin were analyzed. In summary, mutagenesis of the gE N-terminal region identified a new functional region in the VZV gE ectodomain essential for cell-cell spread and the pathogenesis of VZV skin tropism and demonstrated that different subdomains of the unique N-terminal region had specific roles in viral replication, cell-cell spread, and secondary envelopment.

The Wnt/beta-catenin pathway regulates Gli-mediated Myf5 expression during somitogenesis.

Canonical Wnt/beta-catenin signaling regulates the activation of the myogenic determination gene Myf5 at the onset of myogenesis, but the underlying molecular mechanism is unknown. Here, we report that the Wnt signal is transduced in muscle progenitor cells by at least two Frizzled (Fz) receptors (Fz1 and/or Fz6), through the canonical beta-catenin pathway, in the epaxial domain of newly formed somites. We show that Myf5 activation is dramatically reduced by blocking the Wnt/beta-catenin pathway in somite progenitor cells, whereas expression of activated beta-catenin is sufficient to activate Myf5 in somites but not in the presomitic mesoderm. In addition, we identified Tcf/Lef sequences immediately 5' to the Myf5 early epaxial enhancer. These sites determine the correct spatiotemporal expression of Myf5 in the epaxial domain of the somite, mediating the synergistic action of the Wnt/beta-catenin and the Shh/Gli pathways. Taken together, these results demonstrate that Myf5 is a direct target of Wnt/beta-catenin, and that its full activation requires a cooperative interaction between the canonical Wnt and the Shh/Gli pathways in muscle progenitor cells.

Chimeric snRNA molecules carrying antisense sequences against the splice junctions of exon 51 of the dystrophin pre-mRNA induce exon skipping and restoration of a dystrophin synthesis in Delta 48-50 DMD cells.

Deletions and point mutations in the dystrophin gene cause either the severe progressive myopathy Duchenne muscular dystrophy (DMD) or the milder Becker muscular dystrophy, depending on whether the translational reading frame is lost or maintained. Because internal in-frame deletions in the protein produce only mild myopathic symptoms, it should be possible, by preventing the inclusion of specific mutated exon(s) in the mature dystrophin mRNA, to restore a partially corrected phenotype. Such control has been previously accomplished by the use of synthetic oligonucleotides; nevertheless, a significant drawback to this approach is caused by the fact that oligonucleotides would require periodic administrations. To circumvent this problem, we have produced several constructs able to express in vivo, in a stable fashion, large amounts of chimeric RNAs containing antisense sequences. In this paper we show that antisense molecules against exon 51 splice junctions are able to direct skipping of this exon in the human DMD deletion 48-50 and to rescue dystrophin synthesis. We also show that the highest skipping activity was found when antisense constructs against the 5' and 3' splice sites are coexpressed in the same cell.

The meso-angioblast: a multipotent, self-renewing cell that originates from the dorsal aorta and differentiates into most mesodermal tissues.

We have previously reported the origin of a class of skeletal myogenic cells from explants of dorsal aorta. This finding disagrees with the known origin of all skeletal muscle from somites and has therefore led us to investigate the in vivo origin of these cells and, moreover, whether their fate is restricted to skeletal muscle, as observed in vitro under the experimental conditions used. To address these issues, we grafted quail or mouse embryonic aorta into host chick embryos. Donor cells, initially incorporated into the host vessels, were later integrated into mesodermal tissues, including blood, cartilage, bone, smooth, skeletal and cardiac muscle. When expanded on a feeder layer of embryonic fibroblasts, the clonal progeny of a single cell from the mouse dorsal aorta acquired unlimited lifespan, expressed hemo-angioblastic markers (CD34, Flk1 and Kit) at both early and late passages, and maintained multipotency in culture or when transplanted into a chick embryo. We conclude that these newly identified vessel-associated stem cells, the meso-angioblasts, participate in postembryonic development of the mesoderm, and we speculate that postnatal mesodermal stem cells may be derived from a vascular developmental origin.