An official website of the United States government
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.
more »
« less
Nonviral gene delivery to T cells with Lipofectamine LTX
Abstract 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.
more »
« less
- PAR ID:
- 10235916
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Biotechnology and Bioengineering
- Volume:
- 118
- Issue:
- 4
- ISSN:
- 0006-3592
- Format(s):
- Medium: X Size: p. 1674-1687
- Size(s):
- p. 1674-1687
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
null (Ed.)Abstract Background Human mesenchymal stem cells (hMSCs) are intensely researched for applications in cell therapeutics due to their unique properties, however, intrinsic therapeutic properties of hMSCs could be enhanced by genetic modification. Viral transduction is efficient, but suffers from safety issues. Conversely, nonviral gene delivery, while safer compared to viral, suffers from inefficiency and cytotoxicity, especially in hMSCs. To address the shortcomings of nonviral gene delivery to hMSCs, our lab has previously demonstrated that pharmacological ‘priming’ of hMSCs with the glucocorticoid dexamethasone can significantly increase transfection in hMSCs by modulating transfection-induced cytotoxicity. This work seeks to establish a library of transfection priming compounds for hMSCs by screening 707 FDA-approved drugs, belonging to diverse drug classes, from the NIH Clinical Collection at four concentrations for their ability to modulate nonviral gene delivery to adipose-derived hMSCs from two human donors. Results Microscope images of cells transfected with a fluorescent transgene were analyzed in order to identify compounds that significantly affected hMSC transfection without significant toxicity. Compound classes that increased transfection across both donors included glucocorticoids, antibiotics, and antihypertensives. Notably, clobetasol propionate, a glucocorticoid, increased transgene production 18-fold over unprimed transfection. Furthermore, compound classes that decreased transfection across both donors included flavonoids, antibiotics, and antihypertensives, with the flavonoid epigallocatechin gallate decreasing transgene production − 41-fold compared to unprimed transfection. Conclusions Our screen of the NCC is the first high-throughput and drug-repurposing approach to identify nonviral gene delivery priming compounds in two donors of hMSCs. Priming compounds and classes identified in this screen suggest that modulation of proliferation, mitochondrial function, and apoptosis is vital for enhancing nonviral gene delivery to hMSCs.more » « less
-
Abstract A high‐throughput non‐viral intracellular delivery platform is introduced for the transfection of large cargos with dosage‐control. This platform, termed Acoustic‐Electric Shear Orbiting Poration (AESOP), optimizes the delivery of intended cargo sizes with poration of the cell membranes via mechanical shear followed by the modulated expansion of these nanopores via electric field. Furthermore, AESOP utilizes acoustic microstreaming vortices wherein up to millions of cells are trapped and mixed uniformly with exogenous cargos, enabling the delivery of cargos into cells with targeted dosages. Intracellular delivery of a wide range of molecule sizes (<1 kDa to 2 MDa) with high efficiency (>90%), cell viability (>80%), and uniform dosages (<60% coefficient of variation (CV)) simultaneously into 1 million cells min−1per single chip is demonstrated. AESOP is successfully applied to two gene editing applications that require the delivery of large plasmids: i) enhanced green fluorescent protein (eGFP) plasmid (6.1 kbp) transfection, and ii) clustered regularly interspaced short palindromic repeats (CRISPR)‐Cas9‐mediated gene knockout using a 9.3 kbp plasmid DNA encoding Cas9 protein and single guide RNA (sgRNA). Compared to alternative platforms, this platform offers dosage‐controlled intracellular delivery of large plasmids simultaneously to large populations of cells while maintaining cell viability at comparable delivery efficiencies.more » « less
-
null (Ed.)Human immunodeficiency virus (HIV) preferentially infects T-lymphocytes by integrating into host DNA and forming a latent transcriptionally silent provirus. As previously shown, HIV-1 alters migration modes of T-lymphocytes by co-regulating viral gene expression with human C-X-C chemokine receptor-4 (CXCR4). Here, we show that motility of infected T-lymphocytes is cell size dependent. In cell migration assays, migrating cells are consistently larger than non-migrating cells. This effect is drug-treatment independent. The cell size dependent motility observed in a previously generated Jurkat latency model correlates with the motility of primary human CD4+ T-cells containing a modified HIV-1 full-length construct JLatd2GFP. In addition, large migrating T-cells, latently infected with HIV, show a slightly decreased rate of reactivation from latency. these results demonstrate that HIV reactivation is cell migration-dependent, where host cell size acts as a catalyst for altered migration velocity. We believe that host cell size controlled migration uncovers an additional mechanism of cellular controlled viral fate determination important for virus dissemination and reactivation from latency. This observation may provide more insights into viral-host interactions regulating cell migration and reactivation from latency and helps in the design and implementation of novel therapeutic strategies.more » « less
-
Abstract CRISPR/Cas9 gene editing is effective in manipulating genetic loci in mammalian cell cultures and whole fish but efficient platforms applicable to fish cell lines are currently limited. Our initial attempts to employ this technology in fish cell lines using heterologous promoters or a ribonucleoprotein approach failed to indicate genomic alteration at targeted sites in a tilapia brain cell line (OmB). For potential use in a DNA vector approach, endogenous tilapia beta Actin (OmBAct), EF1 alpha (OmEF1a), and U6 (TU6) promoters were isolated. The strongest candidate promoter determined by EGFP reporter assay, OmEF1a, was used to drive constitutive Cas9 expression in a modified OmB cell line (Cas9-OmB1). Cas9-OmB1 cell transfection with vectors expressing gRNAs driven by the TU6 promoter achieved mutational efficiencies as high as 81% following hygromycin selection. Mutations were not detected using human and zebrafish U6 promoters demonstrating the phylogenetic proximity of U6 promoters as critical when used for gRNA expression. Sequence alteration to TU6 improved mutation rate and cloning efficiency. In conclusion, we report new tools for ectopic expression and a highly efficient, economical system for manipulation of genomic loci and evaluation of their causal relationship with adaptive cellular phenotypes by CRISPR/Cas9 gene editing in fish cells.more » « less
