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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.

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.

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 ox...

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&rsquo, 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.

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.

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 (T(m) = 56 °C) and resistance to heme oxidation (k(ox) = 0.04 hr(−1) × 10(3) 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 (P(50) = 27.33 ± 1.82 mm Hg, n = 1.58 ± 0.17) to TFF-purified LtEc (P(50) = 28.84 ± 0.40 mm Hg, n = 1.93 ± 0.02). Therefore, AEX appears to be the optimal method for LtEc purification.

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.

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.

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.

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.

Background The hemoglobin of the Earthworm Lumbricus terrestris (also known as erythrocruorin, or LtEc) is a naturally occurring high molecular weight protein assembly (3.6 MDa) that is extremely stable, resistant to oxidation, and transports oxygen similarly to human whole blood. Therefore, LtEc may serve as an alternative to donated human red blood cells, however, a suitable purification process must be developed to produce highly pure LtEc on a large scale that can be evaluated in an animal model to determine the safety and efficacy of LtEc.

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.

Hemoglobin (Hb) that is purified from red blood cells (RBCs) is commonly subjected to harsh processing conditions, such as high temperatures and extensive column separation, which may damage the Hb by altering the heme prosthetic group and/or the Hb protein structure. In this study, bovine and human Hb purified by tangential flow filtration (TFF) was compared to commercial preparations of human Hb (Hemosol, Inc., Toronto, Canada) and bovine Hb (Biopure, Inc., Cambridge, MA). Purified Hbs were characterized by measuring their overall purity (SDS-PAGE, SEC, and ESI-MS), susceptibility to oxidation (k(ox)), responses to physiological conditions (pH, [Cl(-)], [IHP], and T), and ligand binding kinetics (O(2), NO, and CO). All Hbs evaluated possessed comparable biophysical properties, however, a small amount of catalase was detected in the TFF-purified Hbs that reduced the rate of autoxidation. Mass changes observed by mass spectrometry suggest that structural alterations may be introduced into Hbs that are purified by extensive chromatographic separations. These results demonstrate that TFF is a suitable process for the purification of Hb from RBCs with a quality equivalent to that of commercial Hb preparations that employ more extensive purification strategies. This work also shows that TFF can yield highly pure Hb which can be used for Hb-based O(2) carrier synthesis.