Efficient ssODN-Mediated Targeting by Avoiding Cellular Inhibitory RNAs through Precomplexed CRISPR-Cas9/sgRNA Ribonucleoprotein.

Combined with CRISPR-Cas9 technology and single-stranded oligodeoxynucleotides (ssODNs), specific single-nucleotide alterations can be introduced into a targeted genomic locus in induced pluripotent stem cells (iPSCs); however, ssODN knockin frequency is low compared with deletion induction. Although several Cas9 transduction methods have been reported, the biochemical behavior of CRISPR-Cas9 nuclease in mammalian cells is yet to be explored. Here, we investigated intrinsic cellular factors that affect Cas9 cleavage activity in vitro. We found that intracellular RNA, but not DNA or protein fractions, inhibits Cas9 from binding to single guide RNA (sgRNA) and reduces the enzymatic activity. To prevent this, precomplexing Cas9 and sgRNA before delivery into cells can lead to higher genome editing activity compared with Cas9 overexpression approaches. By optimizing electroporation parameters of precomplexed ribonucleoprotein and ssODN, we achieved efficiencies of single-nucleotide correction as high as 70% and loxP insertion up to 40%. Finally, we could replace the HLA-C1 allele with the C2 allele to generate histocompatibility leukocyte antigen custom-edited iPSCs.

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.

PACAP-PAC1 Receptor Activation Is Necessary for the Sympathetic Response to Acute Intermittent Hypoxia.

Repetitive hypoxia is a key feature of obstructive sleep apnoea (OSA), a condition characterized by intermittent airways obstruction. Patients with OSA present with persistent increases in sympathetic activity and commonly develop hypertension. The objectives of this study were to determine if the persistent increases in sympathetic nerve activity, known to be induced by acute intermittent hypoxia (AIH), are mediated through activation of the pituitary adenylate cyclase activating polypeptide (PACAP) signaling system. Here, we show that the excitatory neuropeptide PACAP, acting in the spinal cord, is important for generating the sympathetic response seen following AIH. Using PACAP receptor knockout mice, and pharmacological agents in Sprague Dawley rats, we measured blood pressure, heart rate, pH, PaCO2, and splanchnic sympathetic nerve activity, under anaesthesia, to demonstrate that the sympathetic response to AIH is mediated via the PAC1 receptor, in a cAMP-dependent manner. We also report that both intermittent microinjection of glutamate into the rostroventrolateral medulla (RVLM) and intermittent infusion of a sub-threshold dose of PACAP into the subarachnoid space can mimic the sympathetic response to AIH. All the sympathetic responses are independent of blood pressure, pH or PaCO2 changes. Our results show that in AIH, PACAP signaling in the spinal cord helps drive persistent increases in sympathetic nerve activity. This mechanism may be a precursor to the development of hypertension in conditions of chronic intermittent hypoxia, such as OSA.

Efficient mRNA delivery system utilizing chimeric VSVG-L7Ae virus-like particles.

The delivery of mRNA is advantageous over DNA delivery as it is transient and does not carry the risk of genomic DNA integration. However, there are currently few efficient mRNA delivery options available, especially for hard-to-transfect cell types, and thus new delivery methods are needed. To this end, we have established a novel mRNA delivery system utilizing chimeric virus-like particles (VLPs). We generated a novel VLP by fusing protein G of Vesicular stomatitis virus (VSV-G) with a ribosomal protein L7Ae of Archeoglobus fulgidus. This system allowed the efficient delivery of EGFP mRNA which was independent from the presence of BoxC/D motif in the mRNA sequence. Our VSVG-L7Ae VLP system demonstrated high transduction efficacy in hard-to-transfect cell lines, such as human induced pluripotent stem cells (iPS cells) and monocytes. In summary, this platform may serve as an efficient and transient transgene delivery tool for an mRNA of interest.

Site-directed mutagenesis of human papillomavirus 18 promoter elements and tissue-specific expression in cervical carcinoma cells.

In this study, the effect of mutations in transcription factor-binding elements was investigated in the human papillomavirus (HPV) 18 P(105) promoter. Site-directed mutagenesis activities, in the AP1/YY1-, KRF-1-, GRE/YY1-, Sp1- and the double mutation (AP1/YY1- and GRE/YY1)-binding sites were assessed in five human cell lines: HeLa (HPV18-positive cervical carcinoma), SiHa (HPV16-positive cervical carcinoma), C33A (HPV-negative cervical carcinoma), H1299 (non-small cell lung carcinoma) and MRC-5 (foetal lung fibroblast). The results indicated that the GRE/YY1 mutation increased the HPV18 P(105) promoter activity in the cervical cell lines by 53-135%. In HeLa and SiHa cells, mutations in the AP1/YY1, KRF-1 and Sp1 transcription factor-binding sites resulted in reduced promoter activity. For C33A, mutations in KRF-1 and Sp1 reduced the promoter activity, while the GRE/YY1 mutation increased the activity. The double mutation, AP1/YY1 and GRE/YY1, appeared to display an additive effect of the two individual mutations in cervical cells. Compared with HeLa cells, HPV18 P(105) promoter activity was more than 80-fold lower in H1299 cells and more than 500-fold lower in MRC-5 cells. Hence in this study, a comprehensive site-directed mutagenesis analysis, of important transcription factor-binding elements, in the HPV18 P(105) promoter was accomplished in a range of human cell lines. In particular, we concluded that HPV-induced factors were extremely important in the transcriptional activity of the HPV18 P(105) promoter.

The use of a human papillomavirus 18 promoter for tissue-specific expression in cervical carcinoma cells.

The use of tissue-specific promoter elements in the treatment of cervical cancer has been explored in this paper. The P(105) promoter of human papillomavirus 18 (HPV18) was utilised to direct tissue-specific expression in a number of cell types. Expression was examined in three cervical carcinoma cell lines: HeLa (HPV18 positive), SiHa (HPV16 positive), and C33A cells (HPV negative); the epithelial cell line, H1299; and the foetal fibroblast cell line, MRC5, utilising a luciferase expression vector. Expression was highest in the cervical cell lines by a factor of at least 80. The effect of a number of mutations in the P(105) promoter on expression levels was examined. Three deletion constructs of the long control region (LCR) were investigated: an 800 bp fragment (LCR800), a 400 bp fragment (LCR400), and a 200 bp fragment (LCR200), as well as the full length product LCR of HPV18 (LCR1000). The LCR800 construct of the HPV18 P(105) promoter had the highest level of expression in the cervical cell lines and was also highest in the HPV18-positive HeLa cell line. Site-directed mutagenesis was then employed on the LCR800 construct to create four further constructs that each had inactivating mutations in one of the four E2 binding sites (E2BSs). Overall, this study indicated that the LCR800 construct of the HPV18 P(105) promoter could be utilised as a tissuerestricted promoter in cervical cancer cells.

Substance P, tyrosine hydroxylase and serotonin terminals in the rat caudal nucleus ambiguus.

Substance P (SP), tyrosine hydroxylase (TH) and serotonin inputs onto laryngeal motoneurons (LMNs) are known to exist, but the distribution of their terminals in the caudal nucleus ambiguus (NA), remains unclear. Using immunofluorescence and confocal microscopy, we assessed simultaneously the distribution of SP, TH, serotonin and synaptophysin immunoreactive (ir) terminals in the caudal NA. SP, TH and serotonin-ir varicosities were considered to represent immunoreactive synapses if, using confocal microscopy, they were co-localized with the presynaptic protein, synaptophysin. Relative to the total number of synapses, we found only a modest number of SP, TH or serotonin-ir synaptic terminals in the caudal NA. The density of SP-ir synaptic terminals was higher than that of TH-ir and serotonin-ir synaptic terminals. Our results suggest that SP, TH, and serotonin-ir inputs may play only a modest role in regulating the activity of LMN. We conclude that SP, TH and serotonin are not always co-localized in terminals forming inputs with LMN and that they arise from separate subpopulations of neurons.

Catestatin attenuates the effects of intrathecal nicotine and isoproterenol.

Catestatin (Cts; human chromogranin A(352-372)) is a neuropeptide derived from chromogranin A (ChgA). In the periphery it is released from the terminals of preganglionic neurons. In the adrenal medulla it inhibits catecholamine release by non-competitively antagonizing nicotinic cholinergic receptors. ChgA is present in the central nervous system, but the extent to which it is present within bulbospinal sympathoexcitatory neurons is unknown. We investigated the distribution of ChgA in the brainstem and its relationship to sympathoexcitatory neurons by combining immunofluorescence and in situ hybridization. A possible role for Cts in modulating the effect of other neurotransmitter systems in the spinal cord was examined by intrathecal injection of Cts, in conjunction with nicotine (1 microg-100 microg) and isoproterenol (0.12 microg-2.5 microg), in the anesthetized rat. Cts attenuated the hypotensive effect of isoproterenol on mean arterial pressure (maximum dose, 2.5 microg isoproterenol; -27 mmHg pre-Cts to -18 mmHg post-Cts), splanchnic sympathetic nerve activity (at 2.5 microg isoproterenol; 10.5% pre-Cts to 2.4% post-Cts), HR (at 2.5 microg isoproterenol; 1.1% pre-Cts to -1.6% post-Cts), and the dp/dt max of carotid pulse pressure (at 2.5 microg isoproterenol 17.3% pre-Cts to 9.3% post-Cts). Cts attenuated the hypertensive effect of nicotine on mean arterial pressure (at 10 microg nicotine, 19.3 mmHg pre-Cts to 6.8 mmHg post-Cts), splanchnic sympathetic nerve activity (at 10 microg nicotine, 10.7% pre-Cts to 4.5% post-Cts), and HR (at 10 microg nicotine, 4.1% pre-Cts to 2.0% post-Cts). The results indicate that Cts antagonizes both central nicotinic acetylcholine receptors and beta-adrenoceptors that are involved in cardiovascular regulation in vivo.

Differential regulation of the central neural cardiorespiratory system by metabotropic neurotransmitters.

Central neurons in the brainstem and spinal cord are essential for the maintenance of sympathetic tone, the integration of responses to the activation of reflexes and central commands, and the generation of an appropriate respiratory motor output. Here, we will discuss work that aims to understand the role that metabotropic neurotransmitter systems play in central cardiorespiratory mechanisms. It is well known that blockade of glutamatergic, gamma-aminobutyric acidergic and glycinergic pathways causes major or even complete disruption of cardiorespiratory systems, whereas antagonism of other neurotransmitter systems barely affects circulation or ventilation. Despite the lack of an 'all-or-none' role for metabotropic neurotransmitters, they are nevertheless significant in modulating the effects of central command and peripheral adaptive reflexes. Finally, we propose that a likely explanation for the plethora of neurotransmitters and their receptors on cardiorespiratory neurons is to enable differential regulation of outputs in response to reflex inputs, while at the same time maintaining a tonic level of sympathetic activity that supports those organs that significantly autoregulate their blood supply, such as the heart, brain, retina and kidney. Such an explanation of the data now available enables the generation of many new testable hypotheses.