Optimizing the lyophilization of Lumbricus terrestris erythrocruorin.

Haemorrhagic shock is a leading cause of death worldwide. Blood transfusions can be used to treat patients suffering severe blood loss but donated red blood cells (RBCs) have several limitations that limit their availability and use. To solve the problems associated with donated RBCs, several acellular haemoglobin-based oxygen carriers (HBOCs) have been developed to restore the most important function of blood: oxygen transport. One promising HBOC is the naturally extracellular haemoglobin (i.e. erythrocruorin) of Lumbricus terrestris (LtEc). The goal of this study was to maximise the portability of LtEc by lyophilising it and then testing its stability at elevated temperatures. To prevent oxidation, several cryoprotectants were screened to determine the optimum formulation for lyophilisation that could minimise oxidation of the haem iron and maximise recovery. Furthermore, samples were also deoxygenated prior to storage to decrease auto-oxidation, while resuspension in a solution containing ascorbic acid was shown to partially reduce LtEc that had oxidised during storage (e.g. from 42% Fe3+ to 11% Fe3+). Analysis of the oxygen equilibria and size of the resuspended LtEc showed that the lyophilisation, storage, and resuspension processes did not affect the oxygen transport properties or the structure of the LtEc, even after 6 months of storage at 40 °C. Altogether, these efforts have yielded a shelf-stable LtEc powder that can be stored for long periods at high temperatures, but future animal studies will be necessary to prove that the resuspended product is a safe and effective oxygen transporter in vivo.

Sequencing of the Lumbricus terrestris genome reveals degeneracy in its erythrocruorin genes.

The erythrocruorin of Lumbricus terrestris (LtEc) is a relatively large macromolecular assembly that consists of at least four different hemoglobin subunits (A, B, C, and D) and four linker subunits (L1, L2, L3, and L4). The complexity and stability of this large structure make LtEc an attractive hemoglobin-based oxygen carrier that could potentially be used as a substitute for donated red blood cells. However, the sequences of the LtEc subunit sequences must be determined before a scalable recombinant expression platform can be developed. The goal of this study was to sequence the L. terrestris genome to identify the complete sequences of the LtEc subunit genes. Our results revealed multiple homologous genes for each subunit (e.g., two homologous A globin genes; A1 and A2), with the exception of the L4 linker. Some of the homologous genes encoded identical peptide sequences (C1 and C2, L1a and L1b), while cDNA and mass spectrometry experiments revealed that some of the homologs are not expressed (e.g., A2). In contrast, multiple sequences for the B, D, L2, and L4 subunits were detected in LtEc samples. These observations reveal novel degeneracy in LtEc and other annelids, along with some new revisions to its previously published peptide sequences.

The artificial oxygen carrier erythrocruorin-characteristics and potential significance in medicine.

The diminishing supply and increasing costs of donated blood have motivated research into novel hemoglobin-based oxygen carriers (HBOCs) that can serve as red blood cell (RBC) substitutes. HBOCs are versatile agents that can be used in the treatment of hemorrhagic shock. However, many of the RBC substitutes that are based on mammalian hemoglobins have presented key limitations such as instability and toxicity. In contrast, erythrocruorins (Ecs) are other types of HBOCs that may not suffer these disadvantages. Ecs are giant metalloproteins found in annelids, crustaceans, and some other invertebrates. Thus far, the Ecs of Lumbricus terrestris (LtEc) and Arenicola marina (AmEc) are the most thoroughly studied. Based on data from preclinical transfusion studies, it was found that these compounds not only efficiently transport oxygen and have anti-inflammatory properties, but also can be modified to further increase their effectiveness. This literature review focuses on the structure, properties, and application of Ecs, as well as their advantages over other HBOCs. Development of methods for both the stabilization and purification of erythrocruorin could confer to enhanced access to artificial blood resources.

Transcriptomic analysis of the innate immune response to in vitro transfection of plasmid DNA.

The innate immune response to cytosolic DNA is intended to protect the host from viral infections, but it can also inhibit the delivery and expression of therapeutic transgenes in gene and cell therapies. The goal of this work was to use mRNA sequencing to identify genes that may influence transfection efficiency in four different cell types (PC-3, Jurkat, HEK-293T, and primary T cells). The highest transfection efficiency was observed in HEK-293T cells, which upregulated only 142 genes with no known antiviral functions after transfection with lipofectamine. Lipofection upregulated 1,057 cytokine-stimulated genes (CSGs) in PC-3 cells, which exhibited a significantly lower transfection efficiency. However, when PC-3 cells were transfected in serum-containing media or electroporated, the observed transfection efficiencies were significantly higher while the expression levels of cytokines and CSGs decreased. In contrast, lipofection of Jurkat and primary T cells only upregulated a few genes, but several of the antiviral CSGs that were absent in HEK-293T cells and upregulated in PC-3 cells were observed to be constitutively expressed in T cells, which may explain the relatively low Lipofection efficiencies observed with T cells (8%-21% GFP+). Indeed, overexpression of one CSG (IFI16) significantly decreased transfection efficiency in HEK-293T cells.

Nonviral gene delivery to T cells with Lipofectamine LTX.

Retroviral gene delivery is widely used in T cell therapies for hematological cancers. However, viral vectors are expensive to manufacture, integrate genes in semirandom patterns, and their transduction efficiency varies between patients. In this study, several nonviral gene delivery vehicles, promoters, and additional variables were compared to optimize nonviral transgene delivery and expression in both Jurkat and primary T cells. Transfection of Jurkat cells was maximized to a high efficiency (63.0% ± 10.9% EGFP+  cells) by transfecting cells with Lipofectamine LTX in X-VIVO 15 media. However, the same method yielded a much lower transfection efficiency in primary T cells (8.1% ± 0.8% EGFP+ ). Subsequent confocal microscopy revealed that a majority of the lipoplexes did not enter the primary T cells, which might be due to relatively low expression levels of heparan sulfate proteoglycans detected via messenger RNA-sequencing. Pyrin and HIN (PYHIN) DNA sensors (e.g., AIM2 and IFI16) that can induce apoptosis or repress transcription after binding cytoplasmic DNA were also detected at high levels in primary T cells. Therefore, transfection of primary T cells appears to be limited at the level of cellular uptake or DNA sensing in the cytoplasm. Both of these factors should be considered in the development of future viral and nonviral T cell gene delivery methods.

Enhancement of transgene expression by the ß-catenin inhibitor iCRT14.

The innate immune response is an essential defense mechanism that allows cells to detect pathogen-associated molecular patterns (PAMPs) like endotoxin or cytosolic DNA and then induce the expression of defensive genes that restrict the replication of viruses and other pathogens. However, the therapeutic DNA used in some gene therapy treatments can also trigger the innate immune response, which activates host cell genes that may inhibit transgene expression. The goal of this study was to enhance transgene expression by inhibiting key components of the innate immune response with small molecule inhibitors (iCRT14, curcumin, Amlexanox, H-151, SC-514, & VX-702). Most of the inhibitors significantly increased transgene (luciferase) expression at least 2-fold, but the ß-catenin/TCF4 inhibitor iCRT14 showed the highest enhancement (16 to 35-fold) in multiple cell lines (PC-3, MCF7, & MB49) without significantly decreasing cellular proliferation. Alternatively, cloning a ß-catenin/TCF4 binding motif (TCAAAG) into the EF1α promoter also enhanced transgene expression up to 8-fold. To further investigate the role of ß-catenin/TCF4 in transgene expression, mRNA-sequencing experiments were conducted to identify host cell genes that were upregulated following transfection with PEI but down-regulated after the addition of iCRT14. As expected, transfection with plasmid DNA activated the innate immune response and upregulated hundreds (687) of defensive genes, but only 7 of those genes were down-regulated in the presence of iCRT14 (e.g., PTGS2 & PLA1A). Altogether, these results show that transgene expression can be enhanced by inhibiting the innate immune response with SMIs like iCRT14, which inhibits ß-catenin/TCF4 to prevent the expression of specific host cell genes.

Optimization of electroporation and other non-viral gene delivery strategies for T cells.

CAR-T therapy is a particularly effective treatment for some types of cancer that uses retroviruses to deliver the gene for a chimeric antigen receptor (CAR) to a patient's T cells ex vivo. The CAR enables the T cells to bind and eradicate cells with a specific surface marker (e.g., CD19+ B cells) after they are transfused back into the patient. This treatment was proven to be particularly effective in treating non-Hodgkin's lymphoma (NHL) and acute lymphoblastic leukemia (ALL), but the current CAR-T cell manufacturing process has a few significant drawbacks. For example, while lentiviral and gammaretroviral transduction are both relatively effective, the process of producing viral vectors is time-consuming and costly. Additionally, patients must undergo follow up appointments for several years to monitor them for any unanticipated side effects associated with the virus. Therefore, several studies have endeavored to find alternative non-viral gene delivery methods that are less expensive, more precise, simple, and safe. This review focuses on the current state of the most promising non-viral gene delivery techniques, including electroporation and transfection with cationic polymers or lipids.

Purification of Lumbricus terrestris erythrocruorin (LtEc) with anion exchange chromatography.

The naturally extracellular hemoglobin (erythrocruorin) of the Canadian nightcrawler, Lumbricus terrestris (LtEc), is a unique oxygen transport protein that may be an effective substitute for donated human blood. Indeed, this ultra-high molecular weight (~3.6 MDa) hemoglobin has already been shown to avoid the side effects associated with previous hemoglobin-based oxygen carriers and its high thermal stability (Tm = 56°C) and resistance to heme oxidation (kox = 0.04 hr-1 × 103 at 20°C) allow it to be stored for long periods of time without refrigeration. However, before it can be tested in human clinical trials, an effective and scalable purification process for LtEc must be developed. We have previously purified LtEc for animal studies with tangential flow filtration (TFF), which allows rapid and scalable purification of LtEc based on its relatively large size, but that type of size-based purification may not be able to specifically remove some impurities and high MW (>500 kDa) contaminants like endotoxin (MW = ~1-4 MDa). Anion exchange (AEX) and immobilized metal affinity chromatography (IMAC) are two purification methods that have been previously used to purify mammalian hemoglobins, but they have not yet been used to purify large invertebrate hemoglobins like LtEc. Therefore, the goal of this study was to determine if AEX and IMAC resins could successfully purify LtEc from crude earthworm homogenate, while also preserving its macromolecular structure and function. Both processes were able to produce purified LtEc with low levels of endotoxin, but IMAC purification induced significantly higher levels of heme oxidation and subunit dissociation than AEX. In addition, the IMAC process required an additional desalting step to enable LtEc binding. In contrast, AEX produced highly pure LtEc that was not dissociated. LtEc purified by AEX also exhibits similar oxygen binding characteristics (P50 = 27.33 ± 1.82 mm Hg, n = 1.58 ± 0.17) to TFF-purified LtEc (P50 = 28.84 ± 0.40 mm Hg, n = 1.93 ± 0.02). Therefore, AEX appears to be the optimal method for LtEc purification.

Components from the Human c-myb Transcriptional Regulation System Reactivate Epigenetically Repressed Transgenes.

A persistent challenge for mammalian cell engineering is the undesirable epigenetic silencing of transgenes. Foreign DNA can be incorporated into closed chromatin before and after it has been integrated into a host cell's genome. To identify elements that mitigate epigenetic silencing, we tested components from the c-myb and NF-kB transcriptional regulation systems in transiently transfected DNA and at chromosomally integrated transgenes in PC-3 and HEK 293 cells. DNA binding sites for MYB (c-myb) placed upstream of a minimal promoter enhanced expression from transiently transfected plasmid DNA. We targeted p65 and MYB fusion proteins to a chromosomal transgene, UAS-Tk-luciferase, that was silenced by ectopic Polycomb chromatin complexes. Transient expression of Gal4-MYB induced an activated state that resisted complete re-silencing. We used custom guide RNAs and dCas9-MYB to target MYB to different positions relative to the promoter and observed that transgene activation within ectopic Polycomb chromatin required proximity of dCas9-MYB to the transcriptional start site. Our report demonstrates the use of MYB in the context of the CRISPR-activation system, showing that DNA elements and fusion proteins derived from c-myb can mitigate epigenetic silencing to improve transgene expression in engineered cell lines.

Enhancement of transgene expression by nuclear transcription factor Y and CCCTC-binding factor.

If a transgene is effectively delivered to a cell, its expression may still be limited by epigenetic mechanisms that silence the transgene. Indeed, once the transgene reaches the nucleus, it may be bound by histone proteins and condensed into heterochromatin or associated with repressor proteins that block transcription. In this study, we sought to enhance transgene expression by adding binding motifs for several different epigenetic enzymes either upstream or downstream of two promoters (CMV and EF1α). Screening these plasmids revealed that luciferase expression was enhanced 10-fold (10.4 ± 5.8) by the addition of a CCAAT box just upstream of the EF1α promoter to recruit nuclear transcription factor Y (NF-Y), while inserting a CCCTC-binding factor (CTCF) motif downstream of the EF1α promoter enhanced expression at least 14-fold (14.03 ± 6.54). ChIP assays confirmed that NF-Y and CTCF bound to the motifs that were added to each plasmid, but the presence of NF-Y and CTCF did not significantly affect the levels of histone acetylation (H3K9ac) or methylation (H3K9me3). Overall, these results show that transgene expression from the EF1α promoter can be significantly increased with motifs that recruit NF-Y or CTCF. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1581-1588, 2018.

Synthesis of Cationic Polymer Libraries for Gene Delivery Using Diglycidyl Ethers.

Gene therapy has the potential to cure many different genetic diseases, if safe and effective gene delivery vectors can be developed. This chapter describes protocols for the synthesis of novel polymers using diglycidyl ether and diamine or polyamine monomers for transgene delivery and expression. The resulting poly (amino ethers) are able to transfect a higher number of cells, with lower cytotoxicity than other commercially available polymers (e.g., Polyethyleneimine, PEI).

Increasing the stability of Lumbricus terrestris erythrocruorin via poly(acrylic acid) conjugation.

Since donated red blood cells must be constantly refrigerated, they are often unavailable in remote areas and battlefields. The goal of this study was to synthesize a highly stable blood substitute that does not require refrigeration. Specifically, the extracellular haemoglobin (a.k.a. erythrocruorin, Ec) of the earthworm Lumbricus terrestris erythrocruororin (LtEc) was cross-linked with poly(acrylic acid) (PAA) and ethylene diamine (EDA). PAGE analysis of the LtEc nanoparticles reveals cross-linking between subunits, while dynamic light scattering and scanning electron microscopy show that cross-linking significantly increases the size of the LtEc nanoparticles (164 ± 13.9 nm). Cross-linking also significantly increased the thermal stability of the LtEc nanoparticles by 10 °C (Tm = 72 ± 0.84 °C) relative to native LtEc (Tm = 62 ± 0.6 °C). In addition, while native LtEc rapidly dissociates at pH 9, the LtEc nanoparticles resist subunit dissociation up to pH 10. The oxygen affinity of the LtEc nanoparticles (P50 = 6.85 ± 0.13 mm Hg) is much higher than native LtEc (P50 = 26.67 ± 0.4 mm Hg), but the cooperativity (n = 2.43 ± 0.12) is not affected. Altogether, these results show that cross-linking LtEc with PAA and EDA provides a potential blood substitute with increased stability and oxygen affinity.

Prolonging the shelf life of Lumbricus terrestris erythrocruorin for use as a novel blood substitute.

Limitations associated with the storage of red blood cells have motivated the development of novel blood substitutes that are able to withstand long-term storage at elevated temperatures. The hemoglobin of the earthworm Lumbricus terrestris (LtEc) is an attractive blood substitute candidate, since it is resistant to oxidation and aggregation during storage. Several factors were investigated to optimize the thermal and oxidative stability of LtEc during storage, including pH, antioxidant supplements, and deoxygenation. A strategy for the reduction of fully oxidized LtEc with antioxidants was also developed. Overall, LtEc was shown to have the highest thermal stability in Ringer's Modified Lactate solution with 10 mM HEPES at pH 7.0. Deoxygenation of the LtEc was also shown to significantly reduce oxidation of the ferrous heme iron (e.g., %Fe2+ after 7 d at 37 °C = 75.7%). However, even in cases where oxidation does occur, the addition of 1.8 mM ascorbic acid (AA) was found to reduce 98.3% of the oxidized LtEc (37 µM heme). Most importantly, the oxygen transport properties of LtEc were unaffected by storage at high temperatures or oxidation followed by reduction with AA. These results show that LtEc can be stored at high temperatures (37 °C) without any significant loss of function.

Glutaraldehyde cross-linking increases the stability of Lumbricus terrestris erythrocruorin.

Since donated red blood cells must be constantly refrigerated, they are not available in remote areas and battlefields. We have previously shown that the hemoglobin of the earthworm Lumbricus terrestris (LtEc) is an effective and safe substitute for donated blood that is stable enough to be stored for long periods at the relatively high temperatures that may be encountered in remote areas. The goal of this study was to further increase the thermal stability of LtEc by covalently cross-linking LtEc with glutaraldehyde (gLtEc). Our results show that the melting temperatures of the gLtEc samples steadily increase as the molar ratio of glutaraldehyde to heme increases (from Tm = 57°C for native LtEc up to Tm = 68°C at a ratio of 128:1). In addition, while native LtEc is susceptible to subunit dissociation at alkaline pH (8-10), cross-linking with glutaraldehyde completely prevents dissociation of gLtEc at pH 10. Increasing the molar ratio of glutaraldehyde:heme also significantly increased the oxygen affinity of gLtEc, but this effect was decreased by cross-linking gLtEc in the deoxygenated T state. Finally, while gLtEc samples cross-linked at low G:H ratios (e.g., 2:1) exhibited slight increases in oxidation rate in Tris buffer, no significant difference in oxidation rate was observed between native LtEc and the gLtEc samples in Ringer's Solution, which contains antioxidants. Overall, cross-linking LtEc with glutaraldehyde significantly increases its thermal and structural stability without any loss of function, making gLtEc an attractive blood substitute for deployment in remote areas and battlefields. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:521-528, 2018.

Direct comparison of oligochaete erythrocruorins as potential blood substitutes.

While many blood substitutes are based on mammalian hemoglobins (e.g., human hemoglobin, HbA), the naturally extracellular hemoglobins of invertebrates (a.k.a. erythrocruorins, Ecs) are intriguing alternative oxygen carriers. Specifically, the erythrocruorin of Lumbricus terrestris has been shown to effectively deliver oxygen in mice and rats without the negative side effects observed with HbA. In this study, the properties of six oligochaete Ecs (Lumbricus terrestris, Eisenia hortensis, Eisenia fetida, Eisenia veneta, Eudrilus eugeniae, and Amynthas gracilis) were compared in vitro to identify the most promising blood substitute candidate(s). Several metrics were used to compare the Ecs, including their oxidation rates, dissociation at physiological pH, thermal stability, and oxygen transport characteristics. Overall, the Ecs of Lumbricus terrestris (LtEc) and Eisenia fetida (EfEc) were identified as promising candidates, since they demonstrated high thermal and oligomeric stability, while also exhibiting relatively low oxidation rates. Interestingly, the O2 affinity of LtEc (P50 = 26.25 mmHg at 37 °C) was also observed to be uniquely lower than EfEc and all of the other Ecs (P50 = 9.29-13.62 mmHg). Subsequent alignment of the primary sequences of LtEc and EfEc revealed several significant amino acid substitutions within the D subunit interfaces that may be responsible for this significant change in O2 affinity. Nonetheless, these results show that LtEc and EfEc are promising potential blood substitutes that are resistant to oxidation and denaturation, but additional experiments will need to be conducted to determine their safety, efficacy, and the effects of their disparate oxygen affinities in vivo.

The histone deacetylase inhibitor Entinostat enhances polymer-mediated transgene expression in cancer cell lines.

Eukaryotic cells maintain an immense amount of genetic information by tightly wrapping their DNA around positively charged histones. While this strategy allows human cells to maintain more than 25,000 genes, histone binding can also block gene expression. Consequently, cells express histone acetyl transferases (HATs) to acetylate histone lysines and release DNA for transcription. Conversely, histone deacetylases (HDACs) are employed for restoring the positive charge on the histones, thereby silencing gene expression by increasing histone-DNA binding. It has previously been shown that histones bind and silence viral DNA, while hyperacetylation of histones via HDAC inhibition restores viral gene expression. In this study, we demonstrate that treatment with Entinostat, an HDAC inhibitor, enhances transgene (luciferase) expression by up to 25-fold in human prostate and murine bladder cancer cell lines when used with cationic polymers for plasmid DNA delivery. Entinostat treatment altered cell cycle progression, resulting in a significant increase in the fraction of cells present in the G0/G1 phase at low micromolar concentrations. While this moderate G0/G1 arrest disappeared at higher concentrations, a modest increase in the fraction of apoptotic cells and a decrease in cell proliferation were observed, consistent with the known anticancer effects of the drug. DNase accessibility studies revealed no significant change in plasmid transcriptional availability with Entinostat treatment. However, quantitative PCR studies indicated that Entinostat treatment, at the optimal dose for enhancing transgene expression, led to an increase in the amount of plasmid present in the nucleus in two cancer cell lines. Taken together, our results show that Entinostat enhances polymer- mediated transgene expression and can be useful in applications related to transient protein expression in mammalian cells. Biotechnol. Bioeng. 2016;113: 1345-1356. © 2015 Wiley Periodicals, Inc.

Purification of diverse hemoglobins by metal salt precipitation.

Although donated blood is the preferred material for transfusion, its limited availability and stringent storage requirements have motivated the development of blood substitutes. The giant extracellular hemoglobin (aka erythrocruorin) of the earthworm Lumbricus terrestris (LtEc) has shown promise as a blood substitute, but an efficient purification method for LtEc must be developed to meet the potential large demand for blood substitutes. In this work, an optimized purification process that uses divalent and trivalent metal salts to selectively precipitate human, earthworm, and bloodworm hemoglobin (HbA, LtEc, and GdHb, respectively) from crude solutions was developed. Although several metal ions were able to selectively precipitate LtEc, Zn(2+) and Ni(2+) provided the lowest heme oxidation and highest overall yield of LtEc. In contrast, Zn(2+) was the only metal ion that completely precipitated HbA and GdHb. Polyacrylamide gel electrophoresis (PAGE) analysis shows that metal precipitation removes several impurities to provide highly pure hemoglobin samples. Heme oxidation levels were relatively low for Zn(2+)-purified HbA and LtEc (2.4±1.3% and 5.3±2.1%, respectively), but slightly higher for Ni(2+)-purified LtEc (8.4±1.2%). The oxygen affinity and cooperativity of the precipitated samples are also identical to samples purified with tangential flow filtration (TFF) alone, indicating the metal precipitation does not significantly affect the function of the hemoglobins. Overall, these results show that hemoglobins from several different species can be highly purified using a combination of metal (Zn(2+)) precipitation and tangential flow filtration.

Kinome-level screening identifies inhibition of polo-like kinase-1 (PLK1) as a target for enhancing non-viral transgene expression.

Human cells contain hundreds of kinase enzymes that regulate several cellular processes, which likely include transgene delivery and expression. We identified several kinases that influence gene delivery and/or expression by performing a kinome-level screen in which, we identified small-molecule kinase inhibitors that significantly enhanced non-viral (polymer-mediated) transgene (luciferase) expression in cancer cells. The strongest enhancement was observed with several small-molecule inhibitors of Polo-like Kinase 1 (PLK 1) (e.g., HMN-214 and BI 2536), which enhanced luciferase expression up to 30-fold by arresting cells in the G2/M phase of the cell cycle and influencing intracellular trafficking of plasmid DNA. Knockdown of PLK 1 using an shRNA-expressing lentivirus further confirmed the enhancement of polymer-mediated transgene expression. In addition, pairwise and three-way combinations of PLK1 inhibitors with the histone deacetylase-1 (HDAC-1) inhibitor Entinostat and the JAK/STAT inhibitor AG-490 enhanced luciferase expression to levels significantly higher than individual drug treatments acting alone. These findings indicate that inhibition of specific intracellular kinases (e.g., PLK1) can significantly enhance non-viral transgene expression for applications in biotechnology and medicine.

Hemoglobin regulates the migration of glioma cells along poly(ε-caprolactone)-aligned nanofibers.

Aligned fibers have been shown to facilitate cell migration in the direction of fiber alignment while oxygen (O2 )-carrying solutions improve the metabolism of cells in hypoxic culture. Therefore, U251 aggregate migration on poly(ε-caprolactone) (PCL)-aligned fibers was studied in cell culture media supplemented with the O2 storage and transport protein hemoglobin (Hb) obtained from bovine, earthworm and human sources at concentrations ranging from 0 to 5 g/L within a cell culture incubator exposed to O2 tensions ranging from 1 to 19% O2 . Individual cell migration was quantified using a wound healing assay. In addition, U251 cell aggregates were developed and aggregate dispersion/cell migration quantified on PCL-aligned fibers. The results of this work show that the presence of bovine or earthworm Hb improved individual cell viability at 1% O2 , while human Hb adversely affected cell viability at increasing Hb concentrations and decreasing O2 levels. The control data suggests that decreasing the O2 tension in the incubator from 5 to 1% O2 decreased aggregate dispersion on the PCL-aligned fibers. However, the addition of bovine Hb at 5% O2 significantly improved aggregate dispersion. At 19% O2 , Hb did not impact aggregate dispersion. Also at 1% O2 , aggregate dispersion appeared to increase in the presence of earthworm Hb, but only at the latter time points. Taken together, these results show that Hb-based O2 carriers can be utilized to improve O2 availability and the migration of glioma spheroids on nanofibers.

Applying horizontal gene transfer phenomena to enhance non-viral gene therapy.

Horizontal gene transfer (HGT) is widespread amongst prokaryotes, but eukaryotes tend to be far less promiscuous with their genetic information. However, several examples of HGT from pathogens into eukaryotic cells have been discovered and mimicked to improve non-viral gene delivery techniques. For example, several viral proteins and DNA sequences have been used to significantly increase cytoplasmic and nuclear gene delivery. Plant genetic engineering is routinely performed with the pathogenic bacterium Agrobacterium tumefaciens and similar pathogens (e.g. Bartonella henselae) may also be able to transform human cells. Intracellular parasites like Trypanosoma cruzi may also provide new insights into overcoming cellular barriers to gene delivery. Finally, intercellular nucleic acid transfer between host cells will also be briefly discussed. This article will review the unique characteristics of several different viruses and microbes and discuss how their traits have been successfully applied to improve non-viral gene delivery techniques. Consequently, pathogenic traits that originally caused diseases may eventually be used to treat many genetic diseases.