Commercially Supplied Amine-Modified siRNAs May Require Ultrafiltration prior to Conjugation with Amine-Reactive Compounds

Conjugation of siRNA to macromolecules such as serum albumin has multiple potential benefits, including enhanced extravasation via albumin-mediated transcytosis across endothelial cells and reduced renal clearance. In attempting to conjugate siRNA to albumin, we used commercially sourced amine-modified siRNA and reacted it with the heterobifunctional linker succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) to introduce a maleimide group suitable for conjugation to the thiol group of the surface-exposed cysteine residue (Cys 34) within albumin. We found the conjugation of the SMCC-treated siRNA to bovine serum albumin (BSA) to be very inefficient and investigated the cause of the low yield of conjugate. Ultrafiltration with phosphate-buffered saline prior to activation with SMCC dramatically increased the yield of siRNA-albumin conjugate (~15-fold). Communication with the commercial supplier revealed that ammonium acetate buffer was used in a desalting step as part of the siRNA purification process prior to supply, likely resulting in ammonium counterions to the siRNA polyanion, which would interfere with conjugation by consuming the SMCC. After ultrafiltration, a greatly reduced amount of SMCC could be used to affect conjugation, without significant reduction in yield. These data indicate that amine-modified siRNA sourced commercially may require ultrafiltration or dialysis prior to use in conjugation reactions

A CX3CRI Reporter hESC Line Facilitates Integrative Analysis of In-Vitro-Derived Microglia and Improved Microglia Identity upon Neuron-Glia Co-culture

Multiple protocols have been published for generation of iMGLs from hESCs/iPSCs. To date, there are no guides to assist researchers to determine the most appropriate methodology for microglial studies. To establish a framework to facilitate future microglial studies, we first performed a comparative transcriptional analysis between iMGLs derived using three published datasets, which allowed us to establish the baseline protocol that is most representative of bona fide human microglia. Secondly, using CRISPR to tag the classic microglial marker CX3CR1 with nanoluciferase and tdTomato, we generated and functionally validated a reporter ESC line. Finally, using this cell line, we demonstrated that co-culture of iMGL precursors with human glia and neurons enhanced transcriptional resemblance of iMGLs to ex vivo microglia. Together, our comprehensive molecular analysis and reporter cell line are a useful resource for neurobiologists seeking to use iMGLs for disease modeling and drug screening studies.Peer reviewe

Oral and maxillofacial surgery cases

The obvious motor symptoms of Parkinson's disease result from a loss of dopaminergic neurons from the substantia nigra. Embryonic stem cell-derived neural progenitor or precursor cells, adult neurons and fetal midbrain tissue have all been used to replace dying dopaminergic neurons. Transplanted cell survival is compromised by factors relating to the new environment, for example; hypoxia, mechanical trauma and excitatory amino acid toxicity. In this study we investigate, using live-cell fluorescence Ca(2+) and Cl(-) imaging, the functional properties of catecholaminergic neurons as they mature. We also investigate whether GABA has the capacity to act as a neurotoxin early in the development of these neurons. From day 13 to day 21 of differentiation [Cl(-)](i) progressively dropped in tyrosine hydroxylase positive (TH(+)) neurons from 56.0 (95% confidence interval, 55.1, 56.9) mM to 6.9 (6.8, 7.1) mM. At days 13 and 15 TH(+) neurons responded to GABA (30 µM) with reductions in intracellular Cl(-) ([Cl(-)](i)); from day 21 the majority of neurons responded to GABA (30 µM) with elevations of [Cl(-)](i). As [Cl(-)](i) reduced, the ability of GABA (30 µM) to elevate intracellular Ca(2+) ([Ca(2+)](i)) did also. At day 13 of differentiation a three hour exposure to GABA (30 µM) or L-glutamate (30 µM) increased the number of midbrain dopaminergic (TH(+) and Pitx3(+)) neurons labeled with the membrane-impermeable nuclear dye TOPRO-3. By day 23 cultures were resistant to the effects of both GABA and L-glutamate. We believe that neuronal susceptibility to amino acid excitotoxicity is dependent upon neuronal maturity, and this should be considered when isolating cells for transplantation studies

Directed expression of Gata2, Mash1, and Foxa2 synergize to induce the serotonergic neuron phenotype during in vitro differentiation of embryonic stem cells

Investigation of serotonergic neuronal activity and its relationship to disease has been limited by a lack of physiologically relevant in vitro cell models. Serotonergic neurons derived from embryonic stem cells (ESCs) could provide a platform for such studies and provide models for use in drug discovery. Here, we report enhancement of serotonergic differentiation using a genetic approach. Expression of Gata2 increased the yield of serotonergic neurons. Enhancement was only achieved when Gata2 was expressed under the control of the tissue-specific promoter of the transcription factor Nkx6.1. High levels of Gata2 expression in ESCs compromised pluripotency and induced non-neuronal differentiation. Combined directed expression of Gata2, pro-neural gene Mash1, and forkhead transcription factor Foxa2 further enhanced serotonergic neural differentiation, resulting in a 10-fold increase in serotonin content. These neurons were also capable of depolarization (KCl, 30 mM)-induced elevations of intracellular Ca(2+). The presence of sonic hedgehog during differentiation produced a further modest increase in numbers (1.5-fold). Transgene expression did not influence the number of tyrosine hydroxylase positive neurons in the cultures after 20 days, implying that Gata2, Mash1, and Foxa2 modulate in vitro differentiation at a time beyond the decision-point for dopaminergic or nondopaminergic commitment. This study demonstrates that the directed expression of specific transcription factors enhances serotonergic neuron differentiation in vitro and highlights the importance of transgene expression at the right stage of ESC differentiation to effect the generation of a desired neural subtype

Self-Crosslinking Lipopeptide/DNA/PEGylated Particles: A New Platform for DNA Vaccination Designed for Assembly in Aqueous Solution

Delivery of plasmids for gene expression in vivo is an inefficient process that requires improvement and optimization to unlock the clinical potential of DNA vaccines. With ease of manufacture and biocompatibility in mind, we explored condensation of DNA in aqueous solution with a self-crosslinking, endosome-escaping lipopeptide (LP), stearoyl-Cys-His-His-Lys-Lys-Lys-amide (stearoyl-CH2K3), to produce cationic LP/DNA complexes. To test whether poly(ethylene glycol) (PEG)-ylation of these cationic complexes to neutralize the surface charge would improve the distribution, gene expression, and immune responses poly(ethylene glycol), these LP/DNA complexes were combined with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000). Fluorescence imaging illustrated that the cationic complexes exhibited the highest degree of localization and lowest degree of dispersion throughout the injected muscle, suggesting impaired mobility of cationic particles upon administration. Nanoluciferase reporter assays over a 90-day period demonstrated that gene expression levels in muscle were highest for PEGylated particles, with over a 200-fold higher level of expression than the cationic particles observed at 30 days. Humoral and cell-mediated immune responses were evaluated in vivo after injection of an ovalbumin expression plasmid. PEGylation improved both immune responses to the DNA complexes in mice. Overall, this suggests that PEGylation of cationic lipopeptide complexes can significantly improve both the transgene expression and immunogenicity of intramuscular DNA vaccines. Keywords: DNA vaccine, PEGylation, lipopeptide, intramuscular, non-viral gene delivery, gene expression, humoral and cell-mediated immunit

Tissue-specific Calibration of Real-time PCR Facilitates Absolute Quantification of Plasmid DNA in Biodistribution Studies

Analysis of the tissue distribution of plasmid DNA after administration of nonviral gene delivery systems is best accomplished using quantitative real-time polymerase chain reaction (qPCR), although published strategies do not allow determination of the absolute mass of plasmid delivered to different tissues. Generally, data is expressed as the mass of plasmid relative to the mass of genomic DNA (gDNA) in the sample. This strategy is adequate for comparisons of efficiency of delivery to a single site but it does not allow direct comparison of delivery to multiple tissues, as the mass of gDNA extracted per unit mass of each tissue is different. We show here that by constructing qPCR standard curves for each tissue it is possible to determine the dose of intact plasmid remaining in each tissue, which is a more useful parameter when comparing the fates of different formulations of DNA. We exemplify the use of this tissue-specific qPCR method by comparing the delivery of naked DNA, cationic DNA complexes, and neutral PEGylated DNA complexes after intramuscular injection. Generally, larger masses of intact plasmid were present 24 hours after injection of DNA complexes, and neutral complexes resulted in delivery of a larger mass of intact plasmid to the spleen