Dual DNA Transfection Using 1,6-Hexanedithiol-Conjugated Maleimide-Functionalized PU-PEI600 For Gene Correction in a Patient iPSC-Derived Fabry Cardiomyopathy Model

Non-viral gene delivery holds promises for treating inherited diseases. However, the limited cloning capacity of plasmids may hinder the co-delivery of distinct genes to the transfected cells. Previously, the conjugation of maleimide-functionalized polyurethane grafted with small molecular weight polyethylenimine (PU-PEI600-Mal) using 1,6-hexanedithiol (HDT) could promote the co-delivery and extensive co-expression of two different plasmids in target cells. Herein, we designed HDT-conjugated PU-PEI600-Mal for the simultaneous delivery of CRISPR/Cas9 components to achieve efficient gene correction in the induced pluripotent stem cell (iPSC)-derived model of Fabry cardiomyopathy (FC) harboring GLA IVS4 + 919 G > A mutation. This FC in vitro model recapitulated several clinical FC features, including cardiomyocyte hypertrophy and lysosomal globotriaosylceramide (Gb3) deposition. As evidenced by the expression of two reporter genes, GFP and mCherry, the addition of HDT conjugated two distinct PU-PEI600-Mal/DNA complexes and promoted the co-delivery of sgRNA/Cas9 and homology-directed repair DNA template into target cells to achieve an effective gene correction of IVS4 + 919 G > A mutation. PU-PEI600-Mal/DNA with or without HDT-mediated conjugation consistently showed neither the cytotoxicity nor an adverse effect on cardiac induction of transfected FC-iPSCs. After the gene correction and cardiac induction, disease features, including cardiomyocyte hypertrophy, the mis-regulated gene expressions, and Gb3 deposition, were remarkably rescued in the FC-iPSC-differentiated cardiomyocytes. Collectively, HDT-conjugated PU-PEI600-Mal-mediated dual DNA transfection system can be an ideal approach to improve the concurrent transfection of non-viral-based gene editing system in inherited diseases with specific mutations.

Transfected plasmid DNA is incorporated into the nucleus via nuclear envelope reformation at telophase

DNA transfection is an important technology in the life sciences, wherein nuclear entry of DNA is necessary to achieve expression of the exogenous DNA. Non-viral vectors and their transfection reagents are useful as safe tools for transfection. However, they have no effects for transfection of non-proliferating cells, the reason for which remains unknown. This study aimed to elucidate the mechanism by which transfected DNA enters the nucleus for gene expression. To monitor the intracellular behavior of transfected DNA, we introduced a plasmid bearing lacO repeats and RFP-coding sequences into cells expressing GFP-LacI and observed plasmid behavior and RFP expression. RFP expression appeared only after mitosis. Electron microscopy showed that plasmids were wrapped with nuclear envelope (NE)-like membranes or associated with chromosomes at telophase. Depletion of BAF, which is involved in NE reformation, delayed plasmid RFP expression. These results suggest that transfected DNA is incorporated into the nucleus via NE reformation at telophase.

Microtubule inhibitors enhance DNA transfection efficiency through autophagy receptor p62/SQSTM1

Ectopic gene expression is an indispensable tool in biology and medicine. However, it is often limited by the low efficiency of DNA transfection. It is known that depletion of p62/SQSTM1 enhances DNA transfection efficiency by preventing the degradation of transfected DNA. Therefore, p62 is a potential target of drugs to increase transfection efficiency. To identify drugs that enhance transfection efficiency, a non-biased high-throughput screening was applied to over 4,000 compounds from the Osaka University compound library, and their p62-dependency was evaluated. The top-scoring drugs were mostly microtubule inhibitors, such as colchicine and vinblastine, and all of them showed positive effects only in the presence of p62. To understand the mechanisms, the time of p62-dependent ubiquitination was examined using polystyrene beads that were introduced into cells as materials that mimicked transfected DNA. The microtubule inhibitors caused a delay in ubiquitination. Furthermore, the level of phosphorylated p62 at S405, which is required for ubiquitination during autophagosome formation, markedly decreased in the drug-treated cells. These results suggest that microtubule inhibitors inhibit p62-dependent autophagosome formation. Our findings provide new insights into the mechanisms of DNA transfection and also provide a solution to increase DNA transfection efficiency.

Agent-based modeling reveals benefits of heterogeneous and stochastic cell populations during cGAS-mediated IFNβ production

Motivation The cGAS pathway is a component of the innate immune system responsible for the detection of pathogenic DNA and upregulation of interferon beta (IFNβ). Experimental evidence shows that IFNβ signaling occurs in highly heterogeneous cells and is stochastic in nature; however, the benefits of these attributes remain unclear. To investigate how stochasticity and heterogeneity affect IFNβ production, an agent-based model is developed to simulate both DNA transfection and viral infection. Results We show that heterogeneity can enhance IFNβ responses during infection. Furthermore, by varying the degree of IFNβ stochasticity, we find that only a percentage of cells (20–30%) need to respond during infection. Going beyond this range provides no additional protection against cell death or reduction of viral load. Overall, these simulations suggest that heterogeneity and stochasticity are important for moderating immune potency while minimizing cell death during infection. Availability and implementation Model repository is available at: https://github.com/ImmuSystems-Lab/AgentBasedModel-cGASPathway. Contact [email protected] Supplementary information Supplementary data are available at Bioinformatics online.

λ Recombineering Used to Engineer the Genome of Phage T7

Bacteriophage T7 and T7-like bacteriophages are valuable genetic models for lytic phage biology that have heretofore been intractable with in vivo genetic engineering methods. This manuscript describes that the presence of λ Red recombination proteins makes in vivo recombineering of T7 possible, so that single base changes and whole gene replacements on the T7 genome can be made. Red recombination functions also increase the efficiency of T7 genome DNA transfection of cells by ~100-fold. Likewise, Red function enables two other T7-like bacteriophages that do not normally propagate in E. coli to be recovered following genome transfection. These results constitute major technical advances in the speed and efficiency of bacteriophage T7 engineering and will aid in the rapid development of new phage variants for a variety of applications.

Optimization of human papillomavirus-based pseudovirus techniques for efficient gene transfer

Human papillomavirus (HPV) L1 and L2 capsid proteins self-assemble into virions capable of efficiently packaging either its 8 kilobase genome or non-viral DNA. The ability of HPV capsids to package non-viral DNA makes these a useful tool for delivering plasmids to study proteins of interest in a variety of cell types. We describe optimization of current methods and present new protocols for using HPV capsids to deliver non-viral DNA thereby providing an alternative to DNA transfection. Using keratinocyte generated extracellular matrices can enhance infection efficiency in keratinocytes, hepatocytes and neuronal cells. Furthermore, we describe a suspension-based efficient technique for infecting different cell types.

Definition of a Novel Plasmid-Based Gene Transfection Protocol of Mammalian Skeletal Muscles by Means of In Vivo Electroporation

We describe an original electroporation protocol for in vivo plasmid DNA transfection. The right hind limbs of C57 mice are exposed to a specifically designed train of permeabilizing electric pulses by transcutaneous application of tailored needle electrodes, immediately after the injection of pEGFP-C1 plasmid encoding GFP (Green Fluorescente Protein). The electroporated rodents show a greater GFP expression than the controls at three different time points (4, 10, and 15 days). The electroporated muscles display only mild interstitial myositis, with a significant increase in inflammatory cell infiltrates. Finally, mild gait abnormalities are registered in electroporated mice only in the first 48 h after the treatment. This protocol has proven to be highly efficient in terms of expression levels of the construct, is easy to apply since it does not require surgical exposure of the muscle and is well tolerated by the animals because it does not cause evident morphological and functional damage to the electroporated muscle.

Electroporation-Based Treatments in Urology

The observation that an application of a pulsed electric field (PEF) resulted in an increased permeability of the cell membrane has led to the discovery of the phenomenon called electroporation (EP). Depending on the parameters of the electric current and cell features, electroporation can be either reversible or irreversible. The irreversible electroporation (IRE) found its use in urology as a non-thermal ablative method of prostate and renal cancer. As its mechanism is based on the permeabilization of cell membrane phospholipids, IRE (as well as other treatments based on EP) provides selectivity sparing extracellular proteins and matrix. Reversible EP enables the transfer of genes, drugs, and small exogenous proteins. In clinical practice, reversible EP can locally increase the uptake of cytotoxic drugs such as cisplatin and bleomycin. This approach is known as electrochemotherapy (ECT). Few in vivo and in vitro trials of ECT have been performed on urological cancers. EP provides the possibility of transmission of genes across the cell membrane. As the protocols of gene electrotransfer (GET) over the last few years have improved, EP has become a well-known technique for non-viral cell transfection. GET involves DNA transfection directly to the cancer or the host skin and muscle tissue. Among urological cancers, the GET of several plasmids encoding prostate cancer antigens has been investigated in clinical trials. This review brings into discussion the underlying mechanism of EP and an overview of the latest progress and development perspectives of EP-based treatments in urology.