Stable expression of large transgenes via the knock-in of an integrase-deficient lentivirus

CAR’TCR-T cells co-expressing CD33-CAR and dNPM1-TCR as superior dual-targeting approach for AML treatment

“…To achieve this, high-capacity viral vectors or implementation of novel gene-transfer technologies would be required to achieve sufficient transduction and expression levels. 35 , 36 Interestingly, ex vivo analysis at study endpoint showed that Triple-T mainly consisted of CAR-T cells, suggesting that CAR’TCR-T cells display reduced persistence. Continuous CD33-targeting in AML elevates the risk of myelosuppression or severe sinusoidal obstruction syndrome, attributed to on-target off-tumor toxicity against myeloid progenitors or Kupffer cells, respectively.…”

Non-viral Intron Knockins Enable Simplified and Flexible Targeting of Endogenous Genes

“…Classical selection methods such as positive selection using fluorescently conjugated antibodies and cell sorting (FACS), positive selection with antibodies conjugated to magnetic beads, or drug selection using resistance genes have recently been supplemented by selection strategies taking advantage of the ability to target transgenes to specific endogenous loci, such as by integration within essential genes [5][6][7][8] . However, each of these selection methods has significant drawbacks: (1) positive selections require direct cellular manipulation (such as antibody binding), which can leave bulky reagents bound to the cell surface after selection, inhibiting later cellular functions and presenting potential immunogenic antigens, (2) drug selections expose cells to toxic compounds and require expression of similarly potentially immunogenic foreign resistance genes, (3) both require additional genetic material beyond the desired therapeutic genetic sequence to be added to facilitate the selection, taking up large portions of the limited amounts of DNA deliverable into cells 9 , and (4) knockins to essential genes lose the benefits of choosing optimal endogenous gene regulation (e.g.…”

Applications of CRISPR technology in cellular immunotherapy

“…To tackle this, many delivery systems that involve either pseudoviral or nonviral components have been developed recently. A recent paper applied an integrase‐deficient lentivirus (pseudovirus) encoding a payload flanked by homology arms and “cut sites” to insert the payload upstream and in‐frame with an endogenous essential gene, followed by the delivery of a CRISPR‐associated ribonucleoprotein (RNP) complex via electroporation 75 . With this system, large payloads such as CRISPR activation components could be stably expressed in primary human T cells.…”