Journal: The CRISPR Journal

Article Title: Automated Good Manufacturing Practice-Compatible CRISPR-Cas9 Editing of Hematopoietic Stem and Progenitor Cells for Clinical Treatment of β-Hemoglobinopathies

doi: 10.1089/crispr.2022.0086

Figure Lengend Snippet: Study and optimization of electroporation conditions using CliniMACS Prodigy electroporator. (A) Small-scale pulse optimization using DsRed mRNA to determine the transfection efficiency (gray) and viability (white), n = 2. (B) Upscale BCL11A transfection using two of the most suitable electroporation settings in small-scale optimization led to moderate InDel efficiencies, n = 1. (C) Upscale comparison of different conditions to further optimize transfection efficiency. BCL11A editing is provided in InDel rate after Sanger sequencing of the PCR product (gray). Viability was determined by flow cytometry 2 days post-transfection (white), n = 2. Nonsignificant differences were observed for editing efficiencies in Mann–Whitney tests (p > 0.05). (D) Influence of different RNP concentrations (2250 pmol/mL, light gray; 4500 pmol/mL, dark gray; 6750 pmol/mL, black) with increasing cell concentrations (5 × 106 to 1.5 × 107 cells/mL) on the BCL11A editing rate, n = 1. (E) Same data as in (D), but displayed as editing rate in relation to RNP concentration per 106 cells, n = 1. (F) Effect of the electroporation volume in CliniMACS Prodigy EP-2 cuvette on the editing performance (gray) and viability (white), n = 2. Nonsignificant differences were observed for editing efficiencies and viabilities in Mann–Whitney tests (p > 0.05). (G) RNP stability controlling freshly prepared RNP (control) versus RNP recovered after a process run and storage time of 60 min, n = 1. (H) Effect of the RNP incubation time on the editing rate (light gray) and viability (white), n = 2. Nonsignificant differences were observed for editing efficiencies and viabilities in Mann–Whitney tests (p > 0.05). (I) Average editing rate for thawed HSPCs from different donors, n = 4. (J) Comparison of BCL11A transfection efficiency after cultivation in the CliniMACS Prodigy system versus a classical cell incubator, n = 1. HSPCs, hematopoietic stem and progenitor cells; PCR, polymerase chain reaction; RNP, ribonucleoprotein.

Article Snippet: Upscale HPSC electroporation The cuvette of the CliniMACS Prodigy EP-2 was manually filled with 600 μL of CD34+ cells in the CliniMACS Electroporation Buffer (Miltenyi Biotec) at a concentration of 5 × 106 to 1.5 × 107 cells/mL with either 30 μg/mL eGFP mRNA (Miltenyi Biotec) or 150–900 pmol ribonucleoprotein (RNP) complex per million cells.

Techniques: Electroporation, Transfection, Comparison, Sequencing, Flow Cytometry, MANN-WHITNEY, Concentration Assay, Control, Incubation, Polymerase Chain Reaction

Journal: The CRISPR Journal

Article Title: Automated Good Manufacturing Practice-Compatible CRISPR-Cas9 Editing of Hematopoietic Stem and Progenitor Cells for Clinical Treatment of β-Hemoglobinopathies

doi: 10.1089/crispr.2022.0086

Figure Lengend Snippet: Large-scale BCL11A editing of HSPCs using CliniMACS Prodigy system with electroporator compared with NTC, NTCe, and upscale controls using the CliniMACS Prodigy EP-2 cuvette. (A) BCL11A editing at the genomic level (n = 1 with technical replicates), cellular viability, and recovery at day 2 after electroporation (n = 1). (B) CFU assay of large-scale samples compared with upscale samples. Total colonies counted for 250 seeded HSPCs (left) and proportion of different colonies, n = 1 with technical replicates. (C) Erythroid differentiation staining on days 7, 14, and 21. Positive rate by flow cytometry for CD34, CD36, CD235a, CD71, CD233, and CD49d, mock electroporated cells (NTCe) (dark gray), upscale control electroporation (light gray), and cells electroporated by the CliniMACS Prodigy process (white), n = 1. (D) HbF levels of electroporated samples measured by flow cytometry on days 7, 14, and 21. (E) HbF/(HbF+HbA0) ratio as determined by HPLC analysis of normal control cells, HbF expressing control cells, and processed cells after additional erythroid differentiation: mock electroporated cells (NTCe), upscale control electroporated cells, and cells electroporated by the CliniMACS Prodigy process, n = 1. (F) HPLC chromatograms of HbF expressing control cells and electroporated samples after in vitro erythroid differentiation. BFU-E, burst-forming unit-erythroid; CFU, colony-forming unit; CFU-G, CFU-granulocyte; CFU-GEMM, CFU-granulocyte erythrocyte macrophage megakaryocyte; CFU-GM, CFU-granulocyte macrophage; CFU-M, CFU-macrophage; HbF, fetal hemoglobin; HPLC, high-performance liquid chromatography; NTC, nontransfected controls; NTCe, electroporated nontransfected controls.

Article Snippet: Upscale HPSC electroporation The cuvette of the CliniMACS Prodigy EP-2 was manually filled with 600 μL of CD34+ cells in the CliniMACS Electroporation Buffer (Miltenyi Biotec) at a concentration of 5 × 106 to 1.5 × 107 cells/mL with either 30 μg/mL eGFP mRNA (Miltenyi Biotec) or 150–900 pmol ribonucleoprotein (RNP) complex per million cells.

Techniques: Electroporation, Colony-forming Unit Assay, Staining, Flow Cytometry, Control, Expressing, In Vitro, High Performance Liquid Chromatography

Journal: Nature

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

doi: 10.1038/s41586-018-0326-5

Figure Lengend Snippet: a, Except where explicitly noted otherwise, we use viability to refer to the number of live cells in an experimental condition (expressed as a %) relative to an equivalent population that went through all protocol steps except for the actual electroporation (No electroporation control). We use the term efficiency to refer to the percentage of live cells in a culture expressing the “knocked in” exogenous sequence (such as GFP). Finally, the total number of cells positive for the desired modification was calculated by multiplying the efficiency by the absolute cell count. Methodological changes that maximized efficiency were not always optimal for the total number of positive cells, and vice-versa. b, Double-stranded (ds)DNA, both circular (plasmid) and linear, when electroporated into primary human T cells, caused marked loss in viability with increasing amounts of template. Co-delivery of an RNP caused less reduction in viability post electroporation. Of note, in these experiments no loss in viability was seen with short single-stranded (ss)DNA oligo donor nucleotides (ssODNs). c, RNPs must be delivered concurrently with DNA to see increased viability. T cells from two donors were each electroporated twice with an eight-hour rest in between electroporations. While two electroporations so closely interspersed caused a high degree of cell death, delivery of the RNP and linear dsDNA template could be delivered separately. Initial RNP electroporation did not protect from the loss of viability if dsDNA was delivered alone in the second round of electroporation. d, We determined whether the order of adding reagents influenced targeting efficiency and viability. In wells where the RNP and the DNA HDR template were mixed together prior to adding the cells (1. RNP + HDRT; 2. + Cells), there was a marked increase in targeting efficiency. e, Note, with the high concentration of dsDNA used in these experiments, viability was higher if the RNP and cells were mixed first and the DNA template was added immediately prior to electroporation (1. RNP + Cells; 2. + HDRT). Taken together, these data likely suggest that pre-incubation of the RNP and HDR template, even for a short period, increased the amount of DNA HDR template delivered into the cell, which increased efficiency but decreased viability. However, viability after RNP and dsDNA HDR template pre-incubation was still higher than was observed with dsDNA HDR template electroporation by itself ( b ). 5 μg of dsDNA HDR temple was used in ( c-e ). f, Primary human T cells were cultured for 2 days using varying combinations of anti-CD3/CD28 T cell receptor (TCR) stimulation and cytokines prior to electroporation of RAB11A targeting RNP and HDR template, followed by varying culture conditions post-electroporation. g, Among the RNP and HDR template concentrations tested here, optimal GFP insertion into RAB11A was achieved at intermediate concentrations of the RNP and dsDNA HDRT. h, Arrayed testing of electroporation pulse conditions showed that, in general, conditions yielding higher HDR efficiency decreased viability. EH115 was selected to optimize efficiency, while still maintaining sufficient viability. i, Diagrammatic timeline of non-viral genome targeting. Approximately one week is required to design, order from commercial suppliers, and assemble any novel combination of genomic editing reagents (gRNA and the HDR template). Two days prior to electroporation, primary human T cells isolated from blood or other sources are stimulated. dsDNA HDR templates can be made easily by PCR followed by a SPRI purification to achieve a highly concentrated and pure product suitable for electroporation. On the day of electroporation, the gRNA (complexed with Cas9 to form an RNP), the HDR template, and harvested stimulated T cells are mixed and electroporated, a process taking approximately 1.5 hours. After electroporation, engineered T cells can be readily expanded for an additional 1-2 weeks. Viability was measured 2 days following electroporation and GFP expression was measured at day 4. Graphs display mean ( b, c, g, h ) and/or individual donor values ( b-h ) in n=2 independent healthy donors ( b-h ). For d, e, and h one representative donor is shown.

Article Snippet: Immediately prior to electroporation, de-beaded cells were centrifuged for 10 minutes at 90g, aspirated, and resuspended in the Lonza electroporation buffer P3 using 20 μL buffer per one million cells.

Techniques: Electroporation, Expressing, Sequencing, Modification, Cell Counting, Plasmid Preparation, Concentration Assay, Incubation, Cell Culture, Isolation, Purification

Journal: Nature

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

doi: 10.1038/s41586-018-0326-5

Figure Lengend Snippet: a, HDR mediated integration of a GFP fusion tag to the housekeeping gene Rab11A. b, Development and optimization of non-viral genome targeting for both cell viability and HDR efficiency. c, Insertion of a GFP fusion into the endogenous RAB11A gene using non-viral targeting in primary human CD4+ and CD8+ T cells. d, Average efficiency with the RAB11A-GFP HDR template was 33.7% and 40.3% in CD4+ and CD8+ cells respectively. e, Viability (number of live cells relative to non-electroporated control) after non-viral genome targeting averaged 68.6%. Efficiency and viability were measured 4 days following electroporation. Mean of n=12 independent healthy donors displayed ( d-e ). See also .

Article Snippet: Immediately prior to electroporation, de-beaded cells were centrifuged for 10 minutes at 90g, aspirated, and resuspended in the Lonza electroporation buffer P3 using 20 μL buffer per one million cells.

Techniques: Electroporation

Journal: Nature

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

doi: 10.1038/s41586-018-0326-5

Figure Lengend Snippet: a, Diagram of in vivo human antigen specific tumour xenograft model. 8 to 12 week old NSG mice were seeded with 1×10 6 A375 cells (human melanoma cell line; NY-ESO-1 antigen+ and HLA-A*0201+) subcutaneously in a shaved flank. Primary human T cells edited to express an NY-ESO-1 antigen specific TCR were generated (either through lentiviral transduction or non-viral TCR replacement), expanded for 10 days following transduction or electroporation, and frozen. Either a bulk edited population was used ( b,c ) or a NY-ESO-1 TCR+ sorted population ( d-f ) was used. At seven days post tumour seeding, T cells were thawed and adoptively transferred via retro-orbital injection. b, Two days following transfer of 5×10 6 bulk non-virally targeted T cells (~10% TCR+ NYESO-1+ (Red), ~10% TCR+ NYESO-1- (Orange), and ~80% TCR- NYESO-1- (Green), see ), NY-ESO-1+ non-virally edited T cells preferentially accumulated in the tumour vs. the spleen. n=5 mice for each of four human T cell donors. c, Ten days following transfer of 5×10 6 bulk non-virally targeted CFSE labeled T cells, NYESO-1 TCR+ cells showed greater proliferation than TCR- or TCR+NYESO-1- T cells, and showed greater proliferation (CFSE Low) in the tumour than in the spleen. At ten days post transfer TCR- and TCR+NYESO- T cells were difficult to find in the tumour . d, Individual longitudinal tumour volume tracks for data summarized in . 3×10 6 sorted NY-ESO-1 TCR+ T cells generated either by lentiviral transduction (Black) or non-viral TCR replacement (Red) were transferred on day 7 post tumour seeding and compared to vehicle only injections until 24 days post tumour seeding. Note that the same data for vehicle control data are shown for each donor in comparison to lentiviral delivery (above) and non-viral TCR replacement (below). e,f, In these experiments, seventeen days following T cell transfer ( d ), non-virally TCR replaced cells appeared to show greater NY-ESO-1 TCR expression and lower expression of exhaustion markers. Transfer of both lentivirally transduced and non-viral TCR replaced cells showed significant reductions in tumour burden on day 24. In this experimental model, non-viral TCR replacement showed further reductions compared to the lentiviral transduction , potentially due to knockout of the endogenous TCR, endogenous regulation of the new TCR’s expression, some difference in the cell populations amenable to non-viral vs lentiviral editing, or confounding variables in cell handling between lentiviral transduction and non-viral genome targeting. n=4 ( b ), n=2 ( d-f ), or n=1 ( c ) independent healthy donors in 5 ( b, c ) or 7 mice ( d-f ) per donor with mean ( b, e, f ) and standard deviation ( b ).

Article Snippet: Immediately prior to electroporation, de-beaded cells were centrifuged for 10 minutes at 90g, aspirated, and resuspended in the Lonza electroporation buffer P3 using 20 μL buffer per one million cells.

Techniques: In Vivo, Generated, Transduction, Electroporation, Injection, Labeling, Expressing, Knock-Out, Standard Deviation