Efficient transgenesis and homology-directed gene targeting in monolayers of primary human small intestinal and colonic epithelial stem cells

Background & Aims2D monolayers of primary intestinal and colonic epithelial cells represent next-generation in vitro models of the gut. Efficient transgenesis and gene-editing in human intestinal stem cells (hISCs) would significantly improve utility of these models by enabling generation of reporter and loss/gain-of-function hISCs, but no published methods exist for transfecting 2D hISC monolayers. Electroporation has proven effective in other difficult-to- transfect cells; thus we applied this method to hISCs.MethodsTwenty-four electroporation parameters were tested, and the optimal condition for efficiency and viability was validated on hISCs from six anatomical regions along the small intestine and colon. PiggyBac™ transposase and Cas9 ribonucleoprotein (RNP) complexes were used for stable genomic integration of reporter genes. High-throughput methods for clone isolation, expansion, and screening were developed. An hISC OLFM4-emGFP reporter was generated and validated by qPCR, organoid assays, and hISC compartmentalization on a planar crypt-microarray (PCM) device.ResultsMaximum electroporation efficiency was 79.9% with a mean survival of 65%. Transfection of 105 hISCs produced ∼142 (0.14%) stable transposase-mediated clones. Transfection of OLFM4-targetting RNPs yielded ∼35% editing and 99/220 (45%) of antibiotic-resistant colonies analyzed expressed emGFP. OLFM4-emGFP hISCs applied to PCMs remained emGFP+ and proliferative in high-Wnt3a/R-spondin3/Noggin zones yet differentiated to emGFP-/KRT20+ cells outside engineered crypt zones. OLFM4-emGFP levels correlated with endogenous OLFM4. Olfm4-emGFPhigh cells were LGR5high/KRT20low, and demonstrated high organoid-forming potential.ConclusionsElectroporation of hISCs is highly efficient for stable transgenesis and transgenic lines can be generated in 3-4 weeks. Workflows mirror conventional culture methods, facilitating rapid integration into established tissue-culture operations. OLFM4high is a robust hISC marker with functional properties in culture.

535 Electroporation of B cells is correlated with cell size change during B cell expansion

BackgroundPrimary B cells are an important target for investigation and transfection of B cells is considered difficult. Electroporation is a very useful technology for transfection but its application on B cells has been unsatisfactory with low efficiency and low viability. The first reason is the small size of B cells compared to cell lines and the second reason is the low abundance of B cells in human PBMC. Since we had previous exprience with T cell electroporation, we sought to extend our knowledge on electroporation to B cells.MethodsHere we studied the B cell electroporation in PBMC samples and found that it is preferrable to electroporate the B cells in the PBMC mixture and B cells can be purified after electroporation if necessary. In this fashion, the total cell number in electroporation is boosted by other cell types in the PBMC and it helps B cell electroporation. Furthermore, we studied expanded B cells and found that they have a larger size than unstimulated B cells and the size increase is correlated to a decrease in electroporation voltage, consistent with the electroporation principle that larger cells need a lower voltage.ResultsWhen B cells are expanded, the electroporation efficiency is similar to common cell lines and it becomes easy to do gene expression or genomic modification.ConclusionsOur studies elucidated the mechanism of difference between unstimulated B cells and expanded B cells and could be useful in helping the research on B cells.

A novel gene expression system for Ralstonia eutropha based on the T7 promoter

Background: Ralstonia eutropha (syn. Cupriavidus necator) is a model microorganism for studying metabolism of polyhydroxyalkanoates (PHAs) and a potential chassis for protein expression due to various advantages. Although current plasmid systems of R. eutropha provide a basic platform for gene expression, the performance of the expression-inducing systems is still limited. In addition, the sizes of the cloned genes are limited due to the large sizes of the plasmid backbones.Results: In this study, an R. eutropha T7 expression system was established by integrating a T7 RNA polymerase gene driven by the PBAD promoter into the genome of R. eutropha, as well as adding a T7 promoter into a pBBR1-derived plasmid for gene expression. In addition, the essential DNA sequence necessary for pBBR1 plasmid replication was identified, and the redundant parts were deleted reducing the expression plasmid size to 3392 bp, which improved the electroporation efficiency about 4 times. As a result, the highest expression level of RFP was enhanced, and the L-arabinose concentration for expression induction was decreased 20 times. Conclusions: The R. eutropha T7 expression system provides an efficient platform for protein production and synthetic biology applications.

A novel gene expression system for Ralstonia eutropha based on the T7 promoter

Background: Ralstonia eutropha (syn. Cupriavidus necator) is a model microorganism for studying metabolism of polyhydroxyalkanoates (PHAs) and a potential chassis for protein expression due to various advantages. Although current plasmid systems of R. eutropha provide a basic platform for gene expression, the performance of the expression-inducing systems is still limited. In addition, the sizes of the cloned genes are limited due to the large sizes of the plasmid backbones.Results: In this study, an R. eutropha T7 expression system was established by integrating a T7 RNA polymerase gene driven by the PBAD promoter into the genome of R. eutropha, as well as adding a T7 promoter into a pBBR1-derived plasmid for gene expression. In addition, the essential DNA sequence necessary for pBBR1 plasmid replication was identified, and the redundant parts were deleted reducing the expression plasmid size to 3392 bp, which improved the electroporation efficiency about 4 times. As a result, the highest expression level of RFP was enhanced, and the L-arabinose concentration for expression induction was decreased 20 times. Conclusions: The R. eutropha T7 expression system provides an efficient platform for protein production and synthetic biology applications.

Compact Square-Wave Pulse Electroporator with Controlled Electroporation Efficiency and Cell Viability

The design and development of a compact square-wave pulse generator for the electroporation of biological cells is presented. This electroporator can generate square-wave pulses with durations from 3 μs up to 10 ms, voltage amplitudes up to 3500 V, and currents up to 250 A. The quantity of the accumulated energy is optimized by means of a variable capacitor bank. The pulse forming unit design uses a crowbar circuit, which gives better control of the pulse form and its duration, independent of the load impedance. In such cases, the square-wave pulse form ensures better control of electroporation efficiency by choosing parameters determined in advance. The device has an integrated graphic LCD screen and measurement modules for the visualization of the current pulse, allowing for express control of the electroporation quality and does not require an external oscilloscope for current pulse recording. This electroporator was tested on suspensions of Saccharomyces cerevisiae yeast cells, during which, it was demonstrated that the application of such square-wave pulses ensured better control of the electroporation efficiency and cell viability after treatment using the pulsed electric field (PEF).

A novel gene expression system for Ralstonia eutropha based on the T7 promoter

Background: Ralstonia eutropha (syn. Cupriavidus necator) is a model microorganism for the metabolism of polyhydroxyalkanoates (PHAs) and a potential chassis for protein expression. Although current plasmid systems for R. eutropha provide a basic platform for gene expression, the performance of the induction systems is still limited. In addition, the sizes of the cloned genes is limited due to the large sizes of the plasmid backbones.Results: In this study, an R. eutropha T7 expression system was established by integrating a T7 RNA polymerase gene driven by the PBAD promoter into genome of R. eutropha, and cloning the T7 promoter into a pBBR1-derived plasmid for gene expression. In addition, the essential sequence necessary for pBBR1 plasmid replication was identified, and the redundant parts were deleted, reducing the expression plasmid size to 3392 bp, and the electroporation efficiency was improved 4 times. As a result, the highest expression level of Rfp was enhanced slightly, and the L-arabinose concentration necessary for induction was decreased 20 times. Conclusions: R. eutropha with the T7 expression system provides an efficient platform for protein expression and synthetic biology applications.

Clinical-Scale Production of CAR-T Cells for the Treatment of Melanoma Patients by mRNA Transfection of a CSPG4-Specific CAR under Full GMP Compliance

Chimeric antigen receptor (CAR)-T cells already showed impressive clinical regressions in leukemia and lymphoma. However, the development of CAR-T cells against solid tumors lags behind. Here we present the clinical-scale production of CAR-T cells for the treatment of melanoma under full GMP compliance. In this approach a CAR, specific for chondroitin sulfate proteoglycan 4 (CSPG4) is intentionally transiently expressed by mRNA electroporation for safety reasons. The clinical-scale protocol was optimized for: (i) expansion of T cells, (ii) electroporation efficiency, (iii) viability, (iv) cryopreservation, and (v) potency. Four consistency runs resulted in CAR-T cells in clinically sufficient numbers, i.e., 2.4 × 109 CAR-expressing T cells, starting from 1.77x108 PBMCs, with an average expansion of 13.6x, an electroporation efficiency of 88.0% CAR-positive cells, a survival of 74.1% after electroporation, and a viability of 84% after cryopreservation. Purity was 98.7% CD3+ cells, with 78.1% CD3+/CD8+ T cells and with minor contaminations of 1.2% NK cells and 0.6% B cells. The resulting CAR-T cells were tested for cytolytic activity after cryopreservation and showed antigen-specific and very efficient lysis of tumor cells. Although our work is descriptive rather than investigative in nature, we expect that providing this clinically applicable protocol to generate sufficient numbers of mRNA-transfected CAR-T cells will help in moving the field of adoptive cell therapy of cancer forward.