More Like this

1. High-throughput screening of clinically approved drugs that prime nonviral gene delivery to human Mesenchymal stem cells

   https://doi.org/10.1186/s13036-020-00238-1  

   Kozisek, Tyler ; Hamann, Andrew ; Nguyen, Albert ; Miller, Michael ; Plautz, Sarah ; Pannier, Angela K. ( December 2020 , Journal of Biological Engineering)

   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
2. Microfluidic Electroporation Coupling Pulses of Nanoseconds and Milliseconds to Facilitate Rapid Uptake and Enhanced Expression of DNA in Cell Therapy

   https://doi.org/10.1038/s41598-020-63172-8  

   Chang, An-Yi ; Liu, Xuan ; Tian, Hong ; Hua, Liping ; Yang, Zhaogang ; Wang, Shengnian ( April 2020 , Scientific Reports)

   Standard electroporation with pulses in milliseconds has been used as an effective tool to deliver drugs or genetic probes into cells, while irreversible electroporation with nanosecond pulses is explored to alter intracellular activities for pulse-induced apoptosis. A combination treatment, long nanosecond pulses followed by standard millisecond pulses, is adopted in this work to help facilitate DNA plasmids to cross both cell plasma membrane and nuclear membrane quickly to promote the transgene expression level and kinetics in both adherent and suspension cells. Nanosecond pulses with 400–800 ns duration are found effective on disrupting nuclear membrane to advance nuclear delivery of plasmid DNA. The additional microfluidic operation further helps suppress the negative impacts such as Joule heating and gas bubble evolution from common nanosecond pulse treatment that lead to high toxicity and/or ineffective transfection. Having appropriate order and little delay between the two types of treatment with different pulse duration is critical to guarantee the effectiveness: 2 folds or higher transfection efficiency enhancement and rapid transgene expression kinetics of GFP plasmids at no compromise of cell viability. The implementation of this new electroporation approach may benefit many biology studies and clinical practice that needs efficient delivery of exogenous probes.

    

   more » « less
3. Optimized nonviral gene delivery for primary urinary renal progenitor cells to enhance cell migration

   https://doi.org/10.1002/jbm.a.36775  

   Liu, Gary W. ; Johnson, Soren L. ; Jain, Ritika ; Peeler, David J. ; Shankland, Stuart J. ; Pun, Suzie H. ( August 2019 , Journal of Biomedical Materials Research Part A)

   Progressive loss of glomerular podocytes during kidney disease leads to irreversible kidney failure, and is exacerbated by the fact that podocytes are terminally differentiated epithelial cells and unable to proliferate. Regeneration of lost podocytes must therefore derive from nonpodocyte sources. Human urine‐derived renal progenitor cells (uRPCs) are attractive podocyte progenitors for cell therapy applications due to their availability from patient urine and ability to migrate to injured glomeruli and differentiate intode novopodocytes after intravenous administration. Because gene delivery has emerged as an important strategy to augment the functionality and survival of cell therapies prior to injection, in this work we optimized nonviral gene delivery conditions (cell density, DNA dose, % FBS, and transfection material composition) to primary uRPCs. Using the cationic polymer‐peptide conjugate VIPER for gene delivery and theSleeping Beautytransposon/transposase constructs for gene integration, we optimized transfection parameters to achieve efficient transgene expression (up to 55% transfected cells) and stable transgene expression (>65% integration efficiency) lasting up to 10 days. With these methods, we transfected uRPCs to overexpress CXCR4, an important chemokine receptor that mediates uRPC migration to the kidneys after intravenous injection, and demonstrate that CXCR4‐uRPCs exhibit enhanced migration compared to mock‐transfected cells.

    

   more » « less
4. Nonviral gene delivery to T cells with Lipofectamine LTX

   https://doi.org/10.1002/bit.27686  

   Harris, Emily ; Zimmerman, Devon ; Warga, Eric ; Bamezai, Anil ; Elmer, Jacob ( February 2021 , Biotechnology and Bioengineering)

   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
5. Quinine copolymer reporters promote efficient intracellular DNA delivery and illuminate a protein-induced unpackaging mechanism

   https://doi.org/10.1073/PNAS.2016860117  

   Van Bruggen, Craig ; Punihaole, David ; Keith, Allison R. ; Schmitz, Andrew J. ; Tolar, Jakub ; Frontiera, Renee R. ; Reineke, Theresa M. ( December 2020 , Proceedings of the National Academy of Sciences)

   null (Ed.)

   Polymeric vehicles that efficiently package and controllably release nucleic acids enable the development of safer and more efficacious strategies in genetic and polynucleotide therapies. Developing delivery platforms that endogenously monitor the molecular interactions, which facilitate binding and release of nucleic acids in cells, would aid in the rational design of more effective vectors for clinical applications. Here, we report the facile synthesis of a copolymer containing quinine and 2-hydroxyethyl acrylate that effectively compacts plasmid DNA (pDNA) through electrostatic binding and intercalation. This polymer system poly(quinine- co -HEA) packages pDNA and shows exceptional cellular internalization, transgene expression, and low cytotoxicity compared to commercial controls for several human cell lines, including HeLa, HEK 293T, K562, and keratinocytes (N/TERTs). Using quinine as an endogenous reporter for pDNA intercalation, Raman imaging revealed that proteins inside cells facilitate the unpackaging of polymer–DNA complexes (polyplexes) and the release of their cargo. Our work showcases the ability of this quinine copolymer reporter to not only facilitate effective gene delivery but also enable diagnostic monitoring of polymer–pDNA binding interactions on the molecular scale via Raman imaging. The use of Raman chemical imaging in the field of gene delivery yields unprecedented insight into the unpackaging behavior of polyplexes in cells and provides a methodology to assess and design more efficient delivery vehicles for gene-based therapies. 

   more » « less