Nontoxic nanopore electroporation for effective intracellular delivery of biological macromolecules

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Abstract

We present a simple nanopore-electroporation (NanoEP) platform for delivery of nucleic acids, functional protein, and Cas9 single-guide RNA ribonucleoproteins into both adherent and suspension cells with up to 80% delivery efficiency and >95% cell viability. Low-voltage electric pulses permeabilize a small area of cell membrane as a cell comes into close contact with the nanopores. The biomolecule cargo is then electrophoretically drawn into the cells through the nanopores. In addition to high-performance delivery with low cell toxicity, the NanoEP system does not require specialized buffers, expensive materials, complicated fabrication processes, or cell manipulation; it simply consists of a generic nanopore-embedded water-filter membrane and a low-voltage square-wave generator. Ultimately, the NanoEP platform offers an effective and flexible method for universal intracellular delivery.

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Most cited references 22

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Progress and problems with the use of viral vectors for gene therapy.

Clare Thomas, Anja Ehrhardt, Mark Kay (2003)

Gene therapy has a history of controversy. Encouraging results are starting to emerge from the clinic, but questions are still being asked about the safety of this new molecular medicine. With the development of a leukaemia-like syndrome in two of the small number of patients that have been cured of a disease by gene therapy, it is timely to contemplate how far this technology has come, and how far it still has to go.

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Reprogramming human T cell function and specificity with non-viral genome targeting

Theodore L Roth, Cristina Puig-Saus, Ruby Yu … (2018)

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes 1 using recombinant viral vectors, which do not target transgenes to specific genomic sites 2,3 . In addition, the need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells through homology-directed repair (HDR) 4,5 . Here, we developed a CRISPR-Cas9 genome targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (> 1kb) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we apply this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, demonstrating improved signaling function. Second, we replace the endogenous T cell receptor (TCR) locus with a new TCR redirecting T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognize tumour antigen and mount productive anti-tumour cell responses in vitro and in vivo. Taken together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

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Delivery technologies for genome editing

Hao Yin, Kevin J Kauffman, Daniel Anderson (2017)

Genome editing has emerged as an attractive approach to therapeutically manipulate gene expression. Here, Anderson and colleagues provide an overview of genome-editing platforms, focusing on the methods and challenges of intracellular biomacromolecule delivery. Preclinical and clinical trials involving genome-editing technologies are also discussed.