Immune cell engineering for in vitro cell therapy
A technology of immune cells and cell engineering, applied in blood/immune system cells, receptors/cell surface antigens/cell surface determinants, using vectors to introduce foreign genetic material, etc., can solve problems such as difficulties
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Embodiment 1
[0270] Example 1: Efficacy and multifaceted functionalization of primary human immune cells
[0271] Evaluate SOLUPORE TM Delivery Methods Ability to deliver the model cargo, GFP (green fluorescent protein) mRNA, to primary human T cells. Due to the variety of manufacturing processes for T cell therapy, including a variety of cell culture systems, PBMC (peripheral blood mononuclear cell)-initiated and CD3 + (Epitope 3) Purified T cell cultures, each isolated from three human donors. For PBMC-initiated and CD3 + In purified T cells, GFP expression at 24 hours was 65-75% and 40-50%, respectively, and cell viability was greater than 70% ( Figure 1A and Figure 1B ).
[0272] SOLUPORE was next evaluated using the Cas9 (CRISPR-associated endonuclease Cas9 (Cas9)) protein-ribonucleoprotein (RNP) complex TM Efficiency of delivery methods for functional cargo, the complexes were designed to target the TRAC (T cell receptor alpha) and PDCD1 (programmed cell death protein 1) ge...
Embodiment 2
[0273] Example 2: Dual Delivery and Sequential Delivery of Multiple Goods
[0274] The next generation of immune cell therapy products will require multiple modifications, which means the use of transfection techniques to deliver multiple cargoes. However, such engineering is only useful if the health and function of the cells are not adversely affected by the delivery method. Therefore, evaluating SOLUPORE TM Delivery methods deliver the efficacy of two goods simultaneously or sequentially. Maintenance of cell viability was also assessed.
[0275] double cargo delivery
[0276] To test the concept of dual cargo delivery, SOLUPORE was used TM The delivery method or electroporation method simultaneously delivered CD19 (cluster of differentiation 19) CAR (chimeric antigen receptor) mRNA and GFP mRNA to stimulated T cells from 3 donors. 24 hours after transfection, use SOLUPORE TM 68.7±4.1% of cells delivered by the delivery method were CD3 positive and CAR positive, an...
Embodiment 3
[0281] Example 3: Cytokine Release Shows Minimal Cell Disturbance
[0282] The cargo delivery studies in Example 2 above show that with SOLUPORE TM The delivery method for transfection is efficient and has minimal impact on cell viability. However, although delivery methods such as electroporation have also been reported to have minimal effects on T cell viability, such methods can also stress cells, leading to unexpected changes in gene and protein expression and ultimately cell function. Therefore, the effect of transfection on cytokine release and immune gene expression in T cells (Example 4) was evaluated.
[0283] SOLUPORE was first detected using multiplex analysis (Luminex) TM Whether the delivery method results in nonspecific release of cytokines from T cells. This experimental set contains 11 human analytes: IFN-γ (interferon γ), IL-2 (interleukin 2), TNFα (tumor necrosis factor α), IL-8 (interleukin 8), GM-CSF (granulocyte-macrophage colony stimulating factor),...
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