Low immunogenicity of LNP allows repeated administrations of CRISPR-Cas9 mRNA into skeletal muscle in mice.

Genome editing therapy for Duchenne muscular dystrophy (DMD) holds great promise, however, one major obstacle is delivery of the CRISPR-Cas9/sgRNA system to skeletal muscle tissues. In general, AAV vectors are used for in vivo delivery, but AAV injections cannot be repeated because of neutralization antibodies. Here we report a chemically defined lipid nanoparticle (LNP) system which is able to deliver Cas9 mRNA and sgRNA into skeletal muscle by repeated intramuscular injections. Although the expressions of Cas9 protein and sgRNA were transient, our LNP system could induce stable genomic exon skipping and restore dystrophin protein in a DMD mouse model that harbors a humanized exon sequence. Furthermore, administration of our LNP via limb perfusion method enables to target multiple muscle groups. The repeated administration and low immunogenicity of our LNP system are promising features for a delivery vehicle of CRISPR-Cas9 to treat skeletal muscle disorders.

Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping.

Prolonged expression of the CRISPR-Cas9 nuclease and gRNA from viral vectors may cause off-target mutagenesis and immunogenicity. Thus, a transient delivery system is needed for therapeutic genome editing applications. Here, we develop an extracellular nanovesicle-based ribonucleoprotein delivery system named NanoMEDIC by utilizing two distinct homing mechanisms. Chemical induced dimerization recruits Cas9 protein into extracellular nanovesicles, and then a viral RNA packaging signal and two self-cleaving riboswitches tether and release sgRNA into nanovesicles. We demonstrate efficient genome editing in various hard-to-transfect cell types, including human induced pluripotent stem (iPS) cells, neurons, and myoblasts. NanoMEDIC also achieves over 90% exon skipping efficiencies in skeletal muscle cells derived from Duchenne muscular dystrophy (DMD) patient iPS cells. Finally, single intramuscular injection of NanoMEDIC induces permanent genomic exon skipping in a luciferase reporter mouse and in mdx mice, indicating its utility for in vivo genome editing therapy of DMD and beyond.

A Highly Bioactive Lys-Deficient IFN Leads to a Site-Specific Di-PEGylated IFN with Equivalent Bioactivity to That of Unmodified IFN-α2b.

Although conjugation with polyethylene glycol (PEGylation) improves the pharmacokinetics of therapeutic proteins, it drastically decreases their bioactivity. Site-specific PEGylation counters the reduction in bioactivity, but developing PEGylated proteins with equivalent bioactivity to that of their unmodified counterparts remains challenging. This study aimed to generate PEGylated proteins with equivalent bioactivity to that of unmodified counterparts. Using interferon (IFN) as a model protein, a highly bioactive Lys-deficient protein variant generated using our unique directed evolution methods enables the design of a site-specific di-PEGylated protein. Antiviral activity of our di-PEGylated IFN was similar to that of unmodified IFN-α2b. The di-PEGylated IFN exhibited 3.0-fold greater antiviral activity than that of a commercial PEGylated IFN. Moreover, our di-PEGylated IFN showed higher in vitro and in vivo stability than those of unmodified IFN-α2b. Hence, we propose that highly bioactive Lys-deficient proteins solve the limitation of conventional PEGylation with respect to the reduction in bioactivity of PEGylated proteins.

A convenient technique for live-cell observation on the surface of polytetrafluoroethylene with a phase-contrast microscope.

Phase-contrast microscopy is a convenient technique for live-cell observation on the surface of materials with high optical transmittance. Here, we demonstrate a novel technique to observe living cells on the surface of materials with low optical transmittance, such as polytetrafluoroethylene (PTFE), which are widely used in biomaterials for blood-contacting devices. The surface of a cover glass was coated with a thin PTFE layer with sufficient transmittance, thereby enabling the observation of living cells on the PTFE surface with a phase-contrast microscope.

Nitric oxide production and its contribution to hepatocyte proliferation in normal juvenile rats.

Nitric oxide (NO) has been reported as a key mediator in enhancing hepatocyte proliferation during liver regeneration. Juvenile hepatocytes have a strong ability to proliferate while still in their undifferentiated state but the mechanism of NO production and its contribution to hepatocyte proliferation are not yet fully understood. The present study was designed to investigate NO production in the normal liver and its contribution to hepatocyte proliferation in juvenile rats. Endogenous NO production was evaluated quantitatively using a spin trap followed by electron paramagnetic resonance spectroscopy with the Fe-N, N-diethyldithiocarbamate complex as an NO-trapping reagent in the rat liver. NO production in the liver significantly peaked at 3 weeks after birth, but NO synthase (NOS) 3 expression did not change between 2 to 5 weeks after birth, while NOS 1 and NOS 2 mRNA were not detected. Hepatocyte proliferation, measured by the incorporation of 5-bromo-2'-deoxyuridine into the DNA, was found to decline significantly when endogenous NO production was inhibited by the administration of the NOS inhibitor N(G)-nitro- (L)-arginine methyl ester. These findings indicate that endogenous NO production peaked at 3 weeks after birth and hepatocyte proliferation declined significantly when NO production was inhibited. Thus, this study provides a novel insight into the contribution of NO to hepatic growth and liver maturation in juveniles.

Role of human transcription elongation factor DSIF in the suppression of senescence and apoptosis.

DSIF is an evolutionarily conserved, ubiquitously expressed, heterodimeric transcription elongation factor composed of two subunits, Spt4 and Spt5. Previous biochemical studies have shown that DSIF positively and negatively regulates RNA polymerase II elongation in collaboration with other protein factors. While several data suggest that DSIF is a 'general' elongation factor, there is also evidence that DSIF exerts a tissue- and gene-specific function. Here we sought to address the question of whether physiological functions of DSIF are general or specific, by using a sophisticated knockdown approach and gene expression microarray analysis. We found that Spt5 is essential for cell growth of various human cell lines and that Spt5 knockdown causes senescence and apoptosis. However, Spt5 knockdown affects a surprisingly small number of genes. In Spt5 knockdown cells, the p53 signaling pathway is activated and mediates part of the knockdown-induced transcriptional change, but apoptotic cell death occurs in the absence of p53. Structure-function analysis of Spt5 shows that the C-terminal approximately 300 amino acid residues are not required to support cell proliferation. These results suggest that one of the functions of Spt5 is to suppress senescence and apoptosis, and that this function is exerted through its association with Spt4 and Pol II.

P-TEFb-mediated phosphorylation of hSpt5 C-terminal repeats is critical for processive transcription elongation.

Human DSIF, a heterodimer composed of hSpt4 and hSpt5, plays opposing roles in transcription elongation by RNA polymerase II (RNA Pol II). Here, we describe an evolutionarily conserved repetitive heptapeptide motif (consensus = G-S-R/Q-T-P) in the C-terminal region (CTR) of hSpt5, which, like the C-terminal domain (CTD) of RNA Pol II, is highly phosphorylated by P-TEFb. Thr-4 residues of the CTR repeats are functionally important phosphorylation sites. In vitro, Thr-4 phosphorylation is critical for the elongation activation activity of DSIF, but not to its elongation repression activity. In vivo, Thr-4 phosphorylation is critical for epidermal growth factor (EGF)-inducible transcription of c-fos and for efficient progression of RNA Pol II along the gene. We consider this phosphorylation to be a switch that converts DSIF from a repressor to an activator. We propose the "mini-CTD" hypothesis, in which phosphorylated CTR is thought to function in a manner analogous to phosphorylated CTD, serving as an additional code for active elongation complexes.

Human Spt6 stimulates transcription elongation by RNA polymerase II in vitro.

Recent studies have suggested that Spt6 participates in the regulation of transcription by RNA polymerase II (RNAPII). However, its underlying mechanism remains largely unknown. One possibility, which is supported by genetic and biochemical studies of Saccharomyces cerevisiae, is that Spt6 affects chromatin structure. Alternatively, Spt6 directly controls transcription by binding to the transcription machinery. In this study, we establish that human Spt6 (hSpt6) is a classic transcription elongation factor that enhances the rate of RNAPII elongation. hSpt6 is capable of stimulating transcription elongation both individually and in concert with DRB sensitivity-inducing factor (DSIF), comprising human Spt5 and human Spt4. We also provide evidence showing that hSpt6 interacts with RNAPII and DSIF in human cells. Thus, in vivo, hSpt6 may regulate multiple steps of mRNA synthesis through its interaction with histones, elongating RNAPII, and possibly other components of the transcription machinery.

A novel hydrogen peroxide-induced phosphorylation and ubiquitination pathway leading to RNA polymerase II proteolysis.

DNA damage-induced ubiquitination of the largest subunit of RNA polymerase II, Rpb1, has been implicated in transcription-coupled repair for years. The studies so far, however, have been limited to the use of bulky helix-distorting DNA damages caused by UV light and cisplatin, which are corrected by the nucleotide excision repair pathway. Non-bulky, non-helix-distorting damages are caused at high frequency by reactive oxygen species in cells and corrected by the base excision repair pathway. Contrary to a classic view, we recently found that the second type of DNA lesions also causes RNA polymerase II stalling in vitro. In this paper, we show that hydrogen peroxide (H(2)O(2)) causes significant ubiquitination and proteasomal degradation of Rpb1 by mechanisms that are distinct from those employed after UV irradiation. UV irradiation and H(2)O(2) treatment cause characteristic changes in protein kinases phosphorylating the carboxyl-terminal domain at Ser-2 and -5. The H(2)O(2)-induced ubiquitination is likely dependent on unusual Ser-5 phosphorylation by ERK1/2. Moreover, the H(2)O(2)-induced ubiquitination occurs on transcriptionally engaged polymerases without the help of Cockayne syndrome A and B proteins and von Hippel-Lindau tumor suppressor proteins, which are all required for the UV-induced ubiquitination. These results suggest that stalled polymerases are recognized and ubiquitinated differentially depending on the types of DNA lesions. Our findings may have general implications in the basic mechanism of transcription-coupled nucleotide excision repair and base excision repair.

Structure-function analysis of human Spt4: evidence that hSpt4 and hSpt5 exert their roles in transcriptional elongation as parts of the DSIF complex.

BACKGROUND: The human Spt4/Spt5 complex, termed DRB-sensitivity inducing factor (DSIF) is a dual regulator of transcription that stimulates, or, when cooperating with negative elongation factor (NELF), represses RNA polymerase II (RNAPII) elongation. Spt4 and Spt5 are also thought to be involved in mRNA capping, homologous DNA recombination, and transcription-coupled DNA repair. As a first step to understanding how these proteins regulate diverse cellular processes, we investigated the structure and function of hSpt4 in vitro. RESULTS: Immunodepletion of hSpt5 from HeLa nuclear extracts resulted in the efficient co-depletion of hSpt4. Using DSIF-depleted nuclear extracts and a series of Spt4 mutants, we examined the amino acid sequence of hSpt4 which was important for hSpt5 binding and for transcriptional repression and activation by DSIF. Unexpectedly, the zinc finger of hSpt4, which is critical for the yeast counterpart to function in vivo, was dispensable for hSpt5 binding and for transcriptional regulation in vitro. CONCLUSION: These and other results suggest: (i) that the central region of hSpt4 is necessary and sufficient for its function in vitro and (ii) that there is no free hSpt4 or hSpt5 in cells. We propose that hSpt4 and hSpt5 exert their roles in transcriptional regulation, and possibly in other nuclear processes, as parts of the DSIF complex.

Human transcription elongation factor NELF: identification of novel subunits and reconstitution of the functionally active complex.

The multisubunit transcription elongation factor NELF (for negative elongation factor) acts together with DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) sensitivity-inducing factor (DSIF)/human Spt4-Spt5 to cause transcriptional pausing of RNA polymerase II (RNAPII). NELF activity is associated with five polypeptides, A to E. NELF-A has sequence similarity to hepatitis delta antigen (HDAg), the viral protein that binds to and activates RNAPII, whereas NELF-E is an RNA-binding protein whose RNA-binding activity is critical for NELF function. To understand the interactions of DSIF, NELF, and RNAPII at a molecular level, we identified the B, C, and D proteins of human NELF. NELF-B is identical to COBRA1, recently reported to associate with the product of breast cancer susceptibility gene BRCA1. NELF-C and NELF-D are highly related or identical to the protein called TH1, of unknown function. NELF-B and NELF-C or NELF-D are integral subunits that bring NELF-A and NELF-E together, and coexpression of these four proteins in insect cells resulted in the reconstitution of functionally active NELF. Detailed analyses using mutated recombinant complexes indicated that the small region of NELF-A with similarity to HDAg is critical for RNAPII binding and for transcriptional pausing. This study defines several important protein-protein interactions and opens the way for understanding the mechanism of DSIF- and NELF-induced transcriptional pausing.

Evidence that negative elongation factor represses transcription elongation through binding to a DRB sensitivity-inducing factor/RNA polymerase II complex and RNA.

Negative elongation factor (NELF) is a human transcription factor complex that cooperates with DRB sensitivity-inducing factor (DSIF)/hSpt4-hSpt5 to repress elongation by RNA polymerase II (RNAPII). NELF activity is associated with five polypeptides, including NELF-A, a candidate gene product for Wolf-Hirschhorn syndrome, and NELF-E, a putative RNA-binding protein with arginine-aspartic acid (RD) dipeptide repeats. Here we report several important findings regarding the DSIF/NELF-dependent elongation control. First, we have established an effective method for purifying the active NELF complex using an epitope-tagging technique. Second, the five polypeptides each are important and together are sufficient for its function in vitro. Third, NELF does not bind to either DSIF or RNAPII alone but does bind to the preformed DSIF/RNAPII complex. Fourth, NELF-E has a functional RNA-binding domain, whose mutations impair transcription repression without affecting known protein-protein interactions. Taken together, we propose that NELF causes RNAPII pausing through binding to the DSIF/RNAPII complex and to nascent transcripts. These results also have implications for how DSIF and NELF are regulated in a gene-specific manner in vivo.