Megakaryocytic microparticles-mediated nucleic acid delivery for gene therapy

Cell-derived microparticles (MPs) are 0.1 to 1 micron extracellular vesicles (EVs), budding off cellular plasma membranes under stress or activation. MPs play an important role in cell-to-cell communication by transferring cargo from parent to target cells. Among circulating MPs, megakaryocyte-derived MPs (MkMPs) are the most abundant MPs in circulation (1). We have demonstrated that, in vitro, MkMPs specifically targeted and were taken up by human hematopoietic stem & progenitor cells (HSPCs) via fusion or endocytosis following specific receptor recognition (2). MkMPs transfer cargo to HSPCs and induce potent Mk differentiation of HSPCs in the absence of thrombopoietin (3). Here, we explored the capability of human MkMPs to transfer DNA and siRNA to HSPCs, and developed MkMP-based strategies for gene therapy. From our current protocol, we were able to achieve loading of plasmid DNA (pGFPns: encoding eGFP) into over 80% of MPs and functional delivery to HSPCs though co-culture to perform eGFP expression. DNA delivery efficiency were increased by optimized co-culture methods, chemically via fusogens to enhance membrane fusion, and physically to enhance contact between MPs and HSPCs. As a result, we were able to make possible that more than 20% of HSPCs express GFP. Functional RNA delivery was also studied by examining the impact of siR-MYB mediated c-myb silencing in enhancing Mk differentiation of CD34+ cells. Our data demonstrate that MkMP-based delivery of siR-MYB to HSPCs enabled c-myb silencing that resulted in enhanced Mk differentiation beyond that of unloaded MkMPs, as assessed by a 29% increase of CD41 expression (an Mk marker), indicating functional siRNA delivery. To sum, functional pDNA and siRNA delivery to HSPCs via MkMPs demonstrate the potential of this delivery system for targeting the stem-cell compartment, suggesting that MkMPs constitute a potentially useful therapeutic delivery system for gene therapy, with applications in regenerative and transfusion medicine. References: 1. Flaumenhaft, R., Dilks, J. R., Richardson, J., Alden, E., Patel-Hett, S. R., Battinelli, E., Klement, G. L., Sola-Visner, M., and Italiano, J. E., Jr. (2009) Megakaryocyte-derived microparticles: direct visualization and distinction from platelet-derived microparticles. Blood 113, 1112-1121 2. Jiang, J., Kao, C., and Papoutsakis, E. T. (2017) How do megakaryocytic microparticles target and deliver cargo to alter the fate of hematopoietic stem cells? Journal of Controlled Release 247, 1-18. doi:10.1016/j.jconrel.2016.1012.1021 3. Jiang, J., Woulfe, D. S., and Papoutsakis, E. T. (2014) Shear enhances thrombopoiesis and formation of microparticles that induce megakaryocytic differentiation of stem cells. Blood 124, 2094-210

Synthetic methylotrophy: Engineering methanol metabolism in a nonnative host

Methylotrophy is the ability of microorganisms to utilize one carbon compounds such as methane and methanol for growth and energy generation. The recent discovery of abundant natural gas reserves has prompted considerable interest in utilizing these compounds as substrates or co-substrates in industrial fermentations of chemicals and fuels. Increased biomass and product yields are expected from these compounds since both are more reduced than lignocellulosic sugars. Native methylotrophic microorganisms are poor industrial host organisms since many are strict aerobes, produce few metabolites and lack genetic engineering tools. Therefore, the development of synthetic methylotrophy in nonnative host organisms is of considerable interest. Here, the development of Escherichia coli as a platform microorganism for methanol metabolism is presented. Incorporation of native methylotrophic enzymes confers methylotrophic properties to E. coli, allowing the nonnative metabolism of methanol into biomass and metabolites. For example, as shown in Figure 1, methanol supplementation provides a 50% improvement in biomass yield on yeast extract in engineered E. coli. Further discussion will be given on how to overcome the challenges involved in synthetic methylotrophy, including unfavorable methanol oxidation, methanol toxicity, carbon conservation and regulatory limitations. Specifically, a high-throughput fluorescence activated cell sorting assay was developed to identify engineered methanol dehydrogenase mutants exhibiting improved methanol oxidation characteristics. Separately, chemical mutagenesis and directed evolution led to the isolation of a mutant methylotrophic E. coli strain exhibiting improved methanol tolerance. The importance of the pentose phosphate pathway during nonnative methanol metabolism in E. coli and its regulatory tuning for optimal methanol assimilation will also be discussed. Finally, data demonstrating the use of methanol as a substrate for cell growth, energy generation and metabolite production in E. coli will be presented. This work was supported by the US DOE ARPA-E agency through contract no. DE-AR0000432. Figure Please click Additional Files below to see the full abstract

Biomanufacturing of platelet-like cells and cell microparticles for cell therapy applications

Megakaryocytes (Mks) are large polyploid cells derived from hematopoietic stem/progenitor cells (HSPCs) triggered by thrombopoietin (Tpo). During differentiation and maturation from HSPCs, Mks migrate from bone marrow toward blood vessels, and give rise to proplatelets (PPTs) and platelets (PLTs) released into blood circulation. We have shown that Mks also shed megakaryocytic microparticles (MkMPs) (1), which are 0.1 to 1 micron extracellular vesicles (EVs). These MkMPs specifically target HSPCs in vitro and induce them into Mk differentiation in the absence of Tpo by delivery of cargo such as proteins and RNA (1,2). PLTs (collected from donated blood) are an expensive cell product in limited supply due to their short life time (4-5 days at room temperature; freezing is not possible) and large needs in Transfusion Medicine for patients with thrombotic deficiencies. Culture-derived PLTs has been shown to have functional activity as PLTs and hold a great potential for providing abundant PLT supply. In this study, we examined biomanufacturing issues of cells or cell derived particles for potential use in lieu of collected PLTs. First, we examined the possibility that MkMPs may be used in lieu of collected PLTs. To examine this, we tested the hypothesis that human MkMPs (huMkMPs) might interact with murine HSPCs and promote Mk and PLT biogenesis in vivo. If this hypothesis is correct, it would suggest that huMkMPs can be used in Transfusion Medicine in lieu of PLTs, especially because huMkMPs would interact more efficiently with huHSPCs than with muHSPCs and also because huMkMPs can be stored frozen. To test this hypothesis, we investigated the interaction of huMkMPs with huHSPCs, both in vivo and in vitro. Injection of huMkMPs to wild-type mice enhanced PLT levels by up to 49%, while reticulated (newly synthesized) PLTs increased from 11.8 % to 15.9 % (a substantial and statistically significant increase). Furthermore, huMkMPs were able to rescue the PLT levels of antibody-induced thrombocytopenic mice by up to 52% Taken together, these data show that huMkMPs target murine HSPCs to enhance PLT biogenesis in vivo. How would one then optimize Mk, PLT and MkMP biomanufacturing for practical applications at large scale? Difficulties regarding PLTs yield per Mk or per input HSPC and PLT functionality remain unsolved. We have shown that shear forces enhance Mk maturation, and the production and function of PPTs, PLTs and MkMPs (1). To achieve metrics suitable for biomanufacturing of PLTs and MkMPs, we improve at the late stage of Mk culture. Cultures under mixing conditions imposing increased biomechanical forces in different culture vessels were carried out. PPTs, PLPs, and MkMPs production under mixing condition were enhanced by ≥ 4-6 fold. Furthermore, PLTs and MkMPs generated under increased biomechanical forces maintained their biological functionality. These data suggest that biomanufacturing of these PLTs and MkMPs (and other cell types and EVs) produced under optimized culture condition engaging optimal biomechanical forces show great potential for serving as PLTs substitutes in Transfusion Medicine, and, more broadly, as agents for novel cell therapies. 1. Jiang, J., Woulfe, D. S., and Papoutsakis, E. T. (2014) Shear enhances thrombopoiesis and formation of microparticles that induce megakaryocytic differentiation of stem cells. Blood 124, 2094-2103 2. Jiang, J., Kao, C. Y., and Papoutsakis, E. T. (2017) How do megakaryocytic microparticles target and deliver cargo to alter the fate of hematopoietic stem cells? J Control Release 247, 1-1

Modeling the effect of bioreactor pH on Chinese Hamster Ovary (CHO) cell metabolism and site-specific N-linked glycosylation of VRC01

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A model-based optimization framework for the inference of regulatory interactions using time-course DNA microarray expression data

<p>Abstract</p> <p>Background</p> <p>Proteins are the primary regulatory agents of transcription even though mRNA expression data alone, from systems like DNA microarrays, are widely used. In addition, the regulation process in genetic systems is inherently non-linear in nature, and most studies employ a time-course analysis of mRNA expression. These considerations should be taken into account in the development of methods for the inference of regulatory interactions in genetic networks.</p> <p>Results</p> <p>We use an S-system based model for the transcription and translation process. We propose an optimization-based regulatory network inference approach that uses time-varying data from DNA microarray analysis. Currently, this seems to be the only model-based method that can be used for the analysis of time-course "relative" expressions (expression ratios). We perform an analysis of the dynamic behavior of the system when the number of experimental samples available is varied, when there are different levels of noise in the data and when there are genes that are not considered by the experimenter. Our studies show that the principal factor affecting the ability of a method to infer interactions correctly is the similarity in the time profiles of some or all the genes. The less similar the profiles are to each other the easier it is to infer the interactions. We propose a heuristic method for resolving networks and show that it displays reasonable performance on a synthetic network. Finally, we validate our approach using real experimental data for a chosen subset of genes involved in the sporulation cascade of <it>Bacillus anthracis</it>. We show that the method captures most of the important known interactions between the chosen genes.</p> <p>Conclusion</p> <p>The performance of any inference method for regulatory interactions between genes depends on the noise in the data, the existence of unknown genes affecting the network genes, and the similarity in the time profiles of some or all genes. Though subject to these issues, the inference method proposed in this paper would be useful because of its ability to infer important interactions, the fact that it can be used with time-course DNA microarray data and because it is based on a non-linear model of the process that explicitly accounts for the regulatory role of proteins.</p

Small RNAs in the Genus Clostridium

The genus Clostridium includes major human pathogens and species important to cellulose degradation, the carbon cycle, and biotechnology. Small RNAs (sRNAs) are emerging as crucial regulatory molecules in all organisms, but they have not been investigated in clostridia. Research on sRNAs in clostridia is hindered by the absence of a systematic method to identify sRNA candidates, thus delegating clostridial sRNA research to a hit-and-miss process. Thus, we wanted to develop a method to identify potential sRNAs in the Clostridium genus to open up the field of sRNA research in clostridia. Using comparative genomics analyses combined with predictions of rho-independent terminators and promoters, we predicted sRNAs in 21 clostridial genomes: Clostridium acetobutylicum, C. beijerinckii, C. botulinum (eight strains), C. cellulolyticum, C. difficile, C. kluyveri (two strains), C. novyi, C. perfringens (three strains), C. phytofermentans, C. tetani, and C. thermocellum. Although more than one-third of predicted sRNAs have Shine-Dalgarno (SD) sequences, only one-sixth have a start codon downstream of SD sequences; thus, most of the predicted sRNAs are noncoding RNAs. Quantitative reverse transcription-PCR (Q-RT-PCR) and Northern analysis were employed to test the presence of a randomly chosen set of sRNAs in C. acetobutylicum and several C. botulinum strains, leading to the confirmation of a large fraction of the tested sRNAs. We identified a conserved, novel sRNA which, together with the downstream gene coding for an ATP-binding cassette (ABC) transporter gene, responds to the antibiotic clindamycin. The number of predicted sRNAs correlated with the physiological function of the species (high for pathogens, low for cellulolytic, and intermediate for solventogenic), but not with 16S rRNA-based phylogeny

The transcriptional program underlying the physiology of clostridial sporulation

A detailed microarray analysis of transcription during sporulation of the strict anaerobe and endospore former Clostridium acetobutylicum is presented

Synthetic tolerance: three noncoding small RNAs, DsrA, ArcZ and RprA, acting supra-additively against acid stress

ABSTRACT Synthetic acid tolerance, especially during active cell growth, is a desirable phenotype for many biotechnological applications. Natively, acid resistance in Escherichia coli is largely a stationary-phase phenotype attributable to mechanisms mostly under the control of the stationary-phase sigma factor RpoS. We show that simultaneous overexpression of noncoding small RNAs (sRNAs), DsrA, RprA and ArcZ, which are translational RpoS activators, increased acid tolerance (based on a low-pH survival assay) supra-additively up to 8500-fold during active cell growth, and provided protection against carboxylic acid and oxidative stress. Overexpression of rpoS without its regulatory 5 0 -UTR resulted in inferior acid tolerance. The supra-additive effect of overexpressing the three sRNAs results from the impact their expression has on RpoS-protein levels, and the beneficial perturbation of the interconnected RpoS and H-NS networks, thus leading to superior tolerance during active growth. Unlike the overexpression of proteins, overexpression of sRNAs imposes hardly any metabolic burden on cells, and constitutes a more effective strain engineering strategy