A recent theory suggests that endocytosis is involved in uptake and intracellular transport of electrotransfected plasmid DNA (pDNA). The goal of the current study was to understand if approaches used previously to improve endocytosis of gene delivery vectors could be applied to enhancing electrotransfection efficiency (eTE). Results from the study showed that photochemically induced endosomal escape, which could increase poly-L-lysine (PLL)-mediated gene delivery, decreased eTE. The decrease could not be blocked by treatment of cells with endonuclease inhibitors (aurintricarboxylic acid and zinc ion) or antioxidants (L-glutamine and ascorbic acid). Chemical treatment of cells with an endosomal trafficking inhibitor that blocks endosome progression, bafilomycin A1, resulted in a significant decrease in eTE. However, treatment of cells with lysosomotropic agents (chloroquine and ammonium chloride) had little effects on eTE. These data suggested that endosomes played important roles in protecting and intracellular trafficking of electrotransfected pDNA.

Electrotransfection (ET) is a nonviral method for delivery of various types of molecules into cells both in vitro and in vivo. Close to 90 clinical trials that involve the use of ET have been performed, and approximately half of them are related to cancer treatment. Particularly, ET is an attractive technique for cancer immunogene therapy because treatment of cells with electric pulses alone can induce immune responses to solid tumors, and the responses can be further enhanced by ET of plasmid DNA (pDNA) encoding therapeutic genes. Compared to other gene delivery methods, ET has several unique advantages. It is relatively inexpensive, flexible, and safe in clinical applications, and introduces only naked pDNA into cells without the use of additional chemicals or viruses. However, the efficiency of ET is still low, partly because biological mechanisms of ET in cells remain elusive. In previous studies, it was believed that pDNA entered the cells through transient pores created by electric pulses. As a result, the technique is commonly referred to as electroporation. However, recent discoveries have suggested that endocytosis plays an important role in cellular uptake and intracellular transport of electrotransfected pDNA. This review will discuss current progresses in the study of biological mechanisms underlying ET and future directions of research in this area. Understanding the mechanisms of pDNA transport in cells is critical for the development of new strategies for improving the efficiency of gene delivery in tumors.

In this cohort treated with cART, pain was less prevalent and less likely to be associated with HIV disease progression or treatment than indicated by studies conducted prior to the widespread use of cART.

The nuclear envelope is a physiological barrier to electrogene transfer. To understand different mechanisms of the nuclear entry for electrotransfected plasmid DNA (pDNA), the current study investigated how manipulation of the mechanisms could affect electrotransfection efficiency (eTE), transgene expression level (EL), and cell viability. In the investigation, cells were first synchronized at G2-M phase prior to electrotransfection so that the nuclear envelope breakdown (NEBD) occurred before pDNA entered the cells. The NEBD significantly increased the eTE and the EL while the cell viability was not compromised. In the second experiment, the cells were treated with a nuclear pore dilating agent (i.e., trans-1,2-cyclohexanediol). The treatment could increase the EL, but had only minor effects on eTE. Furthermore, the treatment was more cytotoxic, compared with the cell synchronization. In the third experiment, a nuclear targeting sequence (i.e., SV40) was incorporated into the pDNA prior to electrotransfection. The incorporation was more effective than the cell synchronization for enhancing the EL, but not the eTE, and the effectiveness was cell type dependent. Taken together, the data described above suggested that synchronization of the NEBD could be a practical approach to improving electrogene transfer in all dividing cells.

or genome editing, such as modification, augmentation, or depletion. The requirement can be achieved through delivery of molecular cargo (DNA, RNA, or protein) into cells, including zinc finger nucleases (ZFNs), [2] transcription activatorlike effector nucleases (TALENs), [3] and CRISPR and associated nucleases such as Cas9. [4] Thus, improving cargo delivery is critical for development of powerful tools used in cell engineering. Molecular cargo can be delivered into cells using either viral or nonviral methods. [5] Compared to nonviral ones, viral methods are in general more efficient. However, they are immunogenic, expensive, and potentially unsafe for clinical use. [6] In addition, viral vehicles have limited delivery capacity. [7] Nonviral methods such as electrotransfer (or electroporation) are increasingly being used in cell engineering. [8] Nonviral delivery is more cost-effective, multiplexable, and applicable to a wide variety of cargo than viral delivery, but currently considered to be inefficient in clinical applications. The low efficiency is often caused by enzymatic degradation of cargo in lysosomes before reaching the target site. Strategies to avoid degradation include facilitating endosomal escape, blocking vesicular transport to lysosomes, and inactivating lysosomal enzymes. Here we propose a new strategy called Pretreatment for Redirection of Endocytic and Autophagic Traffic (pTREAT) that increases the half-life of molecular cargo in cells. The increase is achieved by priming cells with a family of sugar molecules that are disaccharides or oligosaccharides (such as sucrose, trehalose, and raffinose) and cannot be broken down or degraded in mammalian cells. Treatment of cells with the nondegradable sugars (NDSs) can enlarge lysosomes and induce the formation of large nonacidic vesicles called amphisome-like bodies (ALBs), which hinder vesicular trafficking to lysosomes and redirect transport to ALBs. Both changes are reversible and lead to reduction in cargo degradation. To demonstrate the capabilities of the pTREAT method, we applied it to improving the efficiency of electrotransfer of several types of cargo-plasmid DNA, mRNA, Sleeping Beauty transposon, and the CRISPR/Cas9 system-into various cell types including human primary T cells, without compromising cell viability. Cell engineering relies heavily on viral vectors for the delivery of molecular cargo into cells due to their superior efficiency compared to nonviral ones. However, viruses are immunogenic and expensive to manufacture, and have limited delivery capacity. Nonviral delivery approaches avoid these limitations but are currently inefficient for clinical applications. This work demonstrates that the efficiency of nonviral delivery of plasmid DNA, mRNA, Sleeping Beauty transposon, and ribonucleoprotein can be significantly enhanced through pretreatment of cells with the nondegradable sugars (NDS), such as sucrose, trehalose, and raffinose. The enhancement is mediated by the incorporation of the NDS into cell membranes, causing enlar...