An Efficient Low Cost Method for Gene Transfer to T Lymphocytes

Component: Device; Description: Lonza Nucleofector II (square-wave pulse electroporation) with generic 0.2 cm cuvettes (Mirus Biotech)

Component: In-House Buffers; Description: 7 formulations (1M, 1SM, 1S, 2M, 2S, 3P, 3S) with different compositions and osmolarities (detailed in Table S1)

Component: Optimal Buffer (Human); Description: 1SM — highest electroporation score in primary human T cells; used for CAR transfection

Component: Optimal Buffer (Murine); Description: 2S — highest electroporation score in mouse T cells

Component: Transposon System; Description: Sleeping Beauty (SB) transposon for stable genomic integration

Component: Transposon Plasmids; Description: pT2-GFP (reporter), pT3-GFP, pT3-20z (CD20-specific CAR)

Component: Transposase; Description: SB100X (hyperactive transposase, 0.5–2 μg)

Component: Programs; Description: Human: U-14 (resting T cells, optimal); Murine: X-001; Jurkat: X-005

Component: CAR Construct; Description: 20z CAR (CD20 antigen binding domain, CD8α hinge, CD3ζ signaling domain) — codon-optimized, inserted into pT3 backbone

Component: Activation; Description: Human: anti-CD3 (OKT-3, 1 μg/mL) + anti-CD28 (0.5 μg/mL) post-electroporation, or irradiated L388 feeder cells; Murine: pre-activation 24 h with anti-CD3 (2C11) + anti-CD28 (37.51)

Parameter: Cell Types; Details: • Jurkat T cell line (clone E6-1)<br>• Primary human PBMCs from healthy donors (n=2 donors)<br>• Primary murine T lymphocytes from lymph nodes (C57BL/6, BALB/c, B10A strains)

Parameter: Culture Conditions; Details: RPMI 1640 + 10% FCS + L-Glu + 2-ME + Pen/Strep; human rIL-2 (50 U/mL) for primary lymphocytes

Parameter: Plasmid Mass Tested; Details: 0–20 μg transposon + 0–2 μg SB100X transposase

Parameter: Electroporation Conditions; Details: 10⁷ cells/100 μL buffer; 0.2 cm cuvettes; programs: U-14 (human), X-001 (murine), X-005 (Jurkat)

Parameter: Controls; Details: • Mock electroporated cells (no DNA)<br>• Lonza commercial kits (Human T Cell Nucleofector Kit, Mouse T Cell Nucleofector Kit)<br>• Untransfected cells for viability normalization

Parameter: Replicates; Details: Two separate experiments in triplicate; values expressed as mean ± SEM

Analysis Category: Transgene Expression; Methods: Flow cytometry (FACSCalibur®): GFP expression (direct), CAR expression (anti-Fab biotin + streptavidin-PECy5), CD20 staining (anti-CD20 APC)

Analysis Category: Cell Viability; Methods: • Trypan blue exclusion<br>• FSC vs SSC gate (viable cells) — confirmed by 7AAD staining (Figure S1)<br>• Viability normalized to mock-electroporated control (100%)

Analysis Category: Subset Analysis; Methods: CD4/CD8 staining (human); analysis of transgene expression in T cell subsets

Analysis Category: Cytotoxicity Assay; Methods: 4-hour co-culture with CD20+ NALM-6 GFP+ target cells (E/T ratios: 6:1 to 0.18:1); percentage of GFP+ cells measured by flow cytometry; specific lysis calculated

Analysis Category: Statistical Analysis; Methods: • Unpaired Student's t-test (comparisons between Lonza kit and in-house buffers)<br>• One-way ANOVA with Tukey post test (multiple buffer comparisons)<br>• p < 0.05 considered significant

Analysis Category: Electroporation Score; Methods: Formula: (Viability% × Expression%) / division factor (to fit graph scale) — combined measure of performance

Buffer: 3P; GFP Expression (Day 1): Highest; Viability (Day 20): >80%; Score: Best overall

Buffer: 1M, 1SM, 1S, 2M, 2S; GFP Expression (Day 1): Comparable; Viability (Day 20): Recovered; Score: Similar scores

Buffer: Long-term expression (3P); GFP Expression (Day 1): ~35% GFP+ at day 20; Viability (Day 20): -; Score: Stable integration

Parameter: GFP expression (24h); In-House Buffer (1SM): Mean 45% (41-59%); Lonza Kit: Slightly higher; Significance: Slight difference

Parameter: Cell viability; In-House Buffer (1SM): >50%; Lonza Kit: Comparable; Significance: ns

Parameter: Electroporation score; In-House Buffer (1SM): 43.5; Lonza Kit: 64.4; Significance: p < 0.05

Parameter: Transgene retention (Day 7); In-House Buffer (1SM): ~30%; Lonza Kit: -; Significance: -

Parameter: Expression in CD4+/CD8+; In-House Buffer (1SM): Both subsets; Lonza Kit: -; Significance: NK cells showed no expression

Buffer: 2S; GFP Expression: ~38% (36-42%); Viability: Highest; Score: 52.8

Buffer: 1M; GFP Expression: >35%; Viability: Lower; Score: -

Buffer: Lonza kit; GFP Expression: Similar expression; Viability: Lower; Score: 47.4

Buffer: Pre-activation; GFP Expression: Required (4× higher than resting); Viability: >30% vs <10%; Score: -

Buffer: Strain variation; GFP Expression: C57BL/6, BALB/c, B10A; Viability: No substantial variation; Score: -

Time Point: Day +1; 20z CAR Expression: 34.7%; Absolute Cell Number: -

Time Point: Day +10; 20z CAR Expression: 63.3%; Absolute Cell Number: -

Time Point: Day +20; 20z CAR Expression: 66.4%; Absolute Cell Number: >5 × 10⁷ cells

Time Point: Day +30; 20z CAR Expression: 83.8%; Absolute Cell Number: -

Time Point: Cytotoxicity; 20z CAR Expression: Specific lysis against CD20+ NALM-6; Absolute Cell Number: CAR+ T cells showed target-specific killing; mock electroporated cells did not

1. Stable integration not formally demonstrated: "In this report we did not unequivocally demonstrate the integration of transgenes in host cell DNA"—long-term expression (GFP to day 20, CAR to day 30) supports stable integration but was not formally proven (e.g., by PCR or Southern blot).

2. Murine long-term expression not achieved: "We were unable to achieve long term expression of the transgene when SB transposase was used under the conditions tested (data not shown)"—significant limitation for stable gene transfer in mouse T cells.

3. NK cell transfection failed: "GFP expression was not detected in NK cells"—the main cell population expressing transgene are T lymphocytes; NK cells were refractory under tested conditions.

4. Activated human T cells ineffective: "Electroporation of activated human T cells was ineffective with all the buffers and programs tested resulting in large loss of cell viability"—resting T cells must be electroporated, then activated (delayed activation protocol).

5. Primary cell donor variability: Studies performed with only 2 human donors and 3 mouse strains; variability across different donors not systematically characterized.

6. No in vivo validation: All experiments performed in vitro; no demonstration of CAR-T cell function in animal models or clinical samples.

7. No head-to-head cost analysis: While the method is described as "low cost," the actual cost savings compared to commercial kits are not quantified in the paper.

8. Polymer addition attempted but unsuccessful: Addition of PEG or Poloxamer-188 did not improve electroporation efficiency in mouse T cells—the authors note "Testing of additional polymers may increase the efficiency of the protocol and is currently under evaluation."

9. Plasmid mass toxicity: Higher plasmid masses (20 μg) resulted in decreased initial viability (nearly 30% loss compared to 4 μg conditions) requiring careful optimization.

10. No comparison to viral transduction: The method is compared only to commercial electroporation kits, not to the "gold standard" retroviral/lentiviral transduction methods (though the authors note these require 2-3 weeks for vector production).

Report prepared based on the published PLOS ONE article. For full experimental details, buffer compositions, and supplementary figures, please refer to the original publication.