Mirus Bio (United States)

To enable simple and effective high titer recombinant lentivirus production, we examined key parameters for the generation of lentivirus including: transfection optimization, media change, incubation time and DNA vector selection. These results illustrate the importance of optimizing transfection processes for high titer recombinant lentivirus production.

Abstract Transfection is an enabling technique for cellular and molecular biologists to study expression and knockdown of gene expression. Over the past decade, many researchers have utilized liposomal systems to deliver nucleic acids; however, this method has many caveats including low transfection efficiency and high cellular toxicity in some cell types. To address these shortcomings, Mirus Bio has developed a novel non-liposomal technology called the TransIT-X2™ Dynamic Delivery System. This unique polymeric delivery system is able to effectively condense negatively charged nucleic acids (plasmid DNA and/or siRNA), enter cells and break out of the endosomal compartment, a critical barrier for transfection. The plasmid and siRNA can then be delivered to their respective functional locations, the nucleus and the cytoplasm. In this study, we demonstrate functional transfection of labeled plasmid DNA co-delivered with labeled siRNA illustrating the dual abilities of the TransIT-X2 Dynamic Delivery System. Additionally, extensive head-to-head comparisons with a popular liposomal reagent, Lipofectamine® 2000 were performed using a luciferase expression assay in 30 cell types; TransIT-X2 demonstrated superior performance in 20 cell types with 11 showing greater than 2-fold higher expression compared to Lipofectamine 2000. Transfection efficiency of green fluorescent protein (GFP) was measured using flow cytometry and visualized by microscopy; most cell types had greater than 50% GFP efficiency. Lastly, gene knockdown after delivery of siRNA or miRNA was assessed. Two endogenous genes, GAPDH and AHA1, were targeted for siRNA knockdown and mRNA levels were assessed by qRT-PCR. miRNA function was assessed by the decrease in the miR-1 target mRNA PTK9. The TransIT-X2 Dynamic Delivery System shows effective knockdown via siRNA or miRNA. The TransIT-X2 Dynamic Delivery System offers researchers exceptional broad-spectrum transfection of plasmid DNA and siRNA. This cutting edge, non-liposomal polymeric delivery method enhances endocytic release of the transfection complexes allowing researchers to obtain very high transfection efficiencies. Citation Format: James Ludtke, Anthony Lauer, Austin Storck, Nicholas Rossi, Karen Neder, Anatoly Pinchuk, Laura Juckem. A novel dynamic delivery system enabling high efficiency transfection of cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 479. doi:10.1158/1538-7445.AM2014-479

We investigated the voluntary contraction direction dependence of motor unit number index (MUNIX) for multifunctional muscles in patients with amyotrophic lateral sclerosis (ALS). The MUNIX technique was applied in nine first dorsal interosseous muscles of eight ALS subjects, using surface electromyography (EMG) signals from index finger abduction and flexion, respectively. In seven examined muscles, the MUNIX derived from the index finger abduction mode was smaller than that from the flexion mode. For the remaining two muscles, one had the same MUNIX; the other showed an abduction mode MUNIX much higher than the flexion mode MNUIX. Across all muscles, the median MUNIX was 96 for the index finger abduction mode and 161 for the flexion mode. The findings reveal the dependence of multifunctional muscle MUNIX on voluntary contraction directions in ALS patients. Based on this analysis, we further explored the concept of "multidimensional MUNIX" for an appropriate performance or interpretation of MUNIX in multifunctional muscles of ALS patients. An effort towards such a development was presented using both abduction and flexion mode surface EMG for MUNIX calculation.

Natural IgM Abs play an important role in clearing pathogens and preventing autoimmunity. The molecular mechanisms that mediate these processes are understood only to a limited degree. This shortcoming is largely due to the fact that isolated natural IgM Abs commonly recognize a variety of unrelated antigens (Ags). Such polyspecificity is generally believed to be an inherent property of natural Abs. However, there is increasing evidence that it may be induced by non‐physiological binding conditions. In this study, we compared the specificity of natural IgM Abs in conventional buffers and undiluted sera deficient in immunoglobulins. All tested Abs were found to lose their polyspecificity in the serum environment. When in serum, the Abs no longer reacted with conventional screening Ags, including hapten‐BSA conjugates, ssDNA, thyroglobulin and myosin, but fully retained their reactivity with specific peptide Ags selected from a phage display library. The acquisition by IgM of narrow binding specificity was also observed when muscle tissue sections were used as a source of endogenous Ags. The loss of polyspecificity by different Abs was dependent on the presence of different serum constituents. The results from this study suggest that the seemingly inherent polyspecificity of natural IgM Abs is largely an in vitro phenomenon related to the lack of normal blood components in the medium.

BackgroundDespite a prodigious investment of funds, we cannot treat or prevent arteriosclerosis and restenosis, particularly its major pathology, arterial intimal hyperplasia. A cornerstone question lies behind all approaches to the disease: what causes the pathology?HypothesisI argue that the question itself is misplaced because it implies that intimal hyperplasia is a novel pathological phenomenon caused by new mechanisms. A simple inquiry into arterial morphology shows the opposite is true. The normal multi-layer cellular organization of the tunica intima is identical to that of diseased hyperplasia; it is the standard arterial system design in all placentals at least as large as rabbits, including humans. Formed initially as one-layer endothelium lining, this phenotype can either be maintained or differentiate into a normal multi-layer cellular lining, so striking in its resemblance to diseased hyperplasia that we have to name it "benign intimal hyperplasia". However, normal or "benign" intimal hyperplasia, although microscopically identical to pathology, is a controllable phenotype that rarely compromises blood supply. It is remarkable that each human heart has coronary arteries in which a single-layer endothelium differentiates early in life to form a multi-layer intimal hyperplasia and then continues to self-renew in a controlled manner throughout life, relatively rarely compromising the blood supply to the heart, causing complications requiring intervention only in a small fraction of the population, while all humans are carriers of benign hyperplasia. Unfortunately, this fundamental fact has not been widely appreciated in arteriosclerosis research and medical education, which continue to operate on the assumption that the normal arterial intima is always an "ideal" single-layer endothelium. As a result, the disease is perceived and studied as a new pathological event caused by new mechanisms. The discovery that normal coronary arteries are morphologically indistinguishable from deadly coronary arteriosclerosis continues to elicit surprise.ConclusionTwo questions should inform the priorities of our research: (1) what controls switch the single cell-layer intimal phenotype into normal hyperplasia? (2) how is normal (benign) hyperplasia maintained? We would be hard-pressed to gain practical insights without scrutinizing our premises.

Over the last few years, hydrodynamic tail vein delivery has established itself as a simple, yet very effective method for gene transfer into small rodents. Hydrodynamic delivery of plasmid DNA expression vectors or small interfering RNA allows for a broad range of in vivo experiments, including the testing of regulatory elements, antibody generation, evaluation of gene therapy approaches, basic biology and disease model creation (non-heritable transgenics). The recent development of the hydrodynamic limb vein procedure provides a safe nucleic acid delivery technique with equally high efficiency in small and large research animals and, importantly, the prospects for clinical translation.

RNA interference is a powerful tool for target-specific knockdown of gene expression. The triggers for this process are duplex small interfering RNAs (siRNAs) of 21-25 nt with 2-bp 3' overhangs produced in cells by the RNase III family member Dicer. We have observed that short RNAs that are long enough to serve as Dicer substrates (D-siRNA) can often evoke more potent RNA interference than the corresponding 21-nt siRNAs; this is probably a consequence of the physical handoff of the Dicer-produced siRNAs to the RNA-induced silencing complex. Here we describe the design parameters for D-siRNAs and a protocol for in vitro and in vivo intraperitoneal delivery of D-siRNAs and siRNAs to macrophages. siRNA delivery and transfection and analysis of macrophages in vivo can be accomplished within 36 h.