A foundation for universal t-cell based adoptive immunotherapy

Elahe Kamali,1,* Fatemeh rahbarizadeh,2 Zohreh hojati,3 Mehdi shamsara,4

Abstract


Introduction

The patient-specific autologous therapy could be a significant limiting factor in the large-scale exploitation of car technology. these limitations would be circumvented by the use of premanufactured “off-the-shelf” engineered t cells. to create car-t therapy more attainable, it is extremely favorable to develop an allogeneic adoptive transfer strategy, in which universal car-t cells derived from healthy donors can be exploited to treat many patients. we are using the crispr/cas9-mediated editing approach to develop a process for manufacturing of t cells deficient in expression of some alloreactive antigens.

Methods

Grnas design using crispr design tools, preparation of grna expression constructs, preparation and cloning of the grnas oligo inserts into the pspcas9(bb)-2a-gfp and pspcas9(bb)-2a-puro plasmids for co-expression with cas9. sequence validation of crispr plasmids.

Results

Grnas were designed and cloned to recognize and cleave the coding sequences of human trac and cd52 genes. two or three colonies were picked to check for the correct insertion of grnas. the sequence of each colony was verified by sequencing to check the insertion of 20-nucleotide guide sequence between the u6 promoter and the remainder of the grna scaffold.

Conclusion

The sequence verified clones were selected to transfect the cell lines of interest.

Keywords

Universal t cell, gene editing, crispr/cas9, adoptive immunotherapy