AAVS1 site‐specific integration of the CAR gene into human primary T cells using a linear closed‐ended AAV‐based DNA vector

Abstract

Background Use of chimeric antigen receptor (CAR) T cells has become a promising strategy in cancer immunotherapy. However, safety in clinical application is also one of the most controversial issues. Methods In the present study, we investigated the application of a non‐viral site‐directed vector (CELiD [closed‐ended linear duplex DNA]) dependent on adeno‐associated virus (AAV) genomes for the purpose of safe CAR‐T engineering. We co‐electroporated CD19‐CAR encoding “CELiD” vectors with plasmid pCMV‐Rep into human T cells and ensured stably transfected CAR‐T cells by G418 selection. The efficiency of AAVS1 site‐specific integration was analyzed by a real‐time polymerase chain reaction. Results CAR‐T cells engineered by CELiD vectors could be established within 20 days with up to 22.8% AAVS1 site‐specific integration efficiency. CAR expression and cytokine secretion of CAR modified T cells were evaluated in vitro. Abundant effector cytokines were produced by the CAR‐T cells engineered by CELiD vectors compared to control T cells and the killing efficiency of target cells was estimated to as high as 75% in vitro. Conclusions With the help of the AAV‐derived CELiD vector, CAR genes were preferentially integrated into the AAVS1 site. This technology could be utilized in human T cell modification and remove the safety constraints of CAR‐T therapy.

An In Vitro Study on Prestin Analog Gene in the Bullfrog Hearing Organs

“…Our alignment results were consistent with former comparative peptide sequence analyses of mammalian prestins that were much more conserved with only minor changes, while prestins were quite variable among vertebrate species like the bony fish, amphibians, and birds [26]. We examined the electrophysiological properties from HEK cells transfected with the fPres-EGFP protein fusions by a site-specific gene transfer at the human AAV site 1 (AAVS1) [27][28][29][30]. Transgene expression is influenced by the integration site and some random insertions or transient transfections which can interfere with genes or disturb their transcription, while site-specific integration can minimize variations between different cells and constructs [31,32].…”

“…We examined the electrophysiological properties from HEK cells transfected with the fPres-EGFP protein fusions by a site-specific gene transfer at the human AAV site 1 (AAVS1) [ 27 – 30 ]. Transgene expression is influenced by the integration site and some random insertions or transient transfections which can interfere with genes or disturb their transcription, while site-specific integration can minimize variations between different cells and constructs [ 31 , 32 ].…”

Discovery and validation of human genomic safe harbor sites for gene and cell therapies

“…The development of technologies for predictable, durable, and safe expression of desired genetic constructs (i.e., transgenes) in human cells will contribute significantly to the improvement of gene and cell therapies ( Bestor, 2000 ; Ellis, 2005 ), as well as to the advancement of protein manufacturing ( Lee et al., 2019 ). One prominent beneficiary of such technologies is genetically engineered T cell therapies, which require the genomic integration of transgenes encoding novel immune receptors ( Chen et al., 2020 ; Richardson et al., 2019 ); another example is gene therapies for highly proliferating tissues, such as inherited skin disorders, in which entire wild-type (WT) gene copies must be integrated into epidermal stem cells ( Droz-Georget Lathion et al., 2015 ; Hirsch et al., 2017 ). Advances in genome editing using targeted integration tools ( Maeder and Gersbach, 2016 ) already allow precise genomic delivery and sustained expression of transgenes in certain cellular contexts, such as chimeric antigen receptors (CARs) integrated into the T cell receptor alpha chain locus in T cells ( Eyquem et al., 2017 ), and coagulation factors delivered to hepatocytes using recombinant adeno-associated viral (rAAV) vectors ( Barzel et al., 2015 ).…”

Site-specific transgene integration in chimeric antigen receptor (CAR) T cell therapies

“…On the other hand, this indicates that the transgene integrated into the AAVS1 region shows strong expression which remains stable in CAR NK cells derived from iPSC [ 149 ]. Moreover, CAR insertion into the AAVS1 site disrupts the phosphatase 1 regulatory subunit 12C (PPP1R12C) gene and the consequences of its haploinsufficiency or deactivation in some cells have been investigated [ 150 ]. So far, it has been elucidated that AAVS1 is a special site where the integrated CAR transgene can be stably expressed without pathogenicity in engineered human T/NK cells [ 151 ].…”