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 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.

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.

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.

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.