## Toward Engineering \(\gamma\delta\) T Cells: Transduction Strategies  
The emergence of synthetic biology has offered a broader set of tools for cellular engineering and reprogramming immune cells. Many engineering strategies have been developed for \(\alpha\beta\) T cells which might be also used for modifying \(\gamma\delta\) T cells. To date, retrovirus has been widely used to transduce \(\gamma\delta\) T cells mainly due to availability of packaging cell lines which can be stably transduced to produce high virus titer [129]. High transduction efficiencies are achieved using a moloney murine leukemia virus-based vector, SFG [97], along with the RD114 [130] or GALV envelope vectors. To enter the cell nucleus, most retroviruses require the dissolution of the nuclear envelope which only occurs during mitosis. Therefore, retroviruses application becomes mainly restricted to highly proliferating cells [131]. Nevertheless, this has not limited their application for V\(\delta\)2 \(\gamma\delta\) T cells, since they can be activated prior to transduction. Other subsets of \(\gamma\delta\) T cells such as V\(\delta\)1, can be potentially transduced following Con-A driven expansion, but the results has been variable and less predictable [42]. The use of viruses always runs the risk of insertional mutagenesis [132]. Lentiviruses hence have received more attention because of their safer insertional profile [131]. [PERSON] et al. compared two different lentiviral vectors for transducing \(\gamma\delta\) T cells and showed a higher transduction efficiency (65% vs. 42%) with simian immunodeficiency virus (SIV) vector compared to human immunodeficiency virus (HIV)-based vector using the same envelope (VSV G) [62]. [PERSON] et al. established a lentiviral-based protocol for transducing \(\gamma\delta\) T cells after expansion with IPP and IL-2 [58]. To assure that viral gene integration does not occur at the oncogenic loci, while exploiting the potential advantage of targeted integration at a specific loci (TCR\(\alpha\)), other editing technologies such as CRISPR/Cas9 have been designed and tested to improve engineered chimeric antigen receptor (CAR) \(\alpha\beta\) T cells function [133; 134]. This strategy has not yet been tested in \(\gamma\delta\) T cells. The use of non-viral vectors avoids many of the defenses that cells normally employ against viral vectors. Non-viral transduction methods such as the Sleeping Beauty (SB) Transposon system uses transposase enzymes to insert a new transgene randomly into the target cell [135; 136]. Although, transduction efficacy using SB technique has been lower than viruses [137], it provides selective advantages for particular hosts. SB uses electroporation for gene transfer, hence does not require a proliferative status. [PERSON] at al. used SB to transfer a CD19 CAR gene into a polyclonal population of \(\gamma\delta\) T which were subsequently expanded using CD19\({}^{+}\) artificial APCs (aAPCs). These CAR \(\gamma\delta\) T cells expressing different \(\gamma\delta\) genes, displayed enhanced killing of CD19\({}^{+}\) leukemia xenografts in mice compared with untransduced \(\gamma\delta\) T cells. SB, unlike proliferation-based transduction methods, did not skew the \(\gamma\delta\) T cell population toward a particular subset [138]. This may be advantageous when aiming to maintain a particular V\(\gamma\)/V\(\delta\) subset such as non-V82 \(\gamma\delta\) T cells in order to harness their tropism toward epithelial cells for treating epithelial tumors. While viral or transposon-based techniques provide stable transduction and therefore long-term persistence of engineered T cells, transient CAR expression strategies have been suggested to reduce CAR T cell toxicity. mRNA transduced CAR \(\alpha\beta\) T cells were used to target mesothelin in patients [139], although repeated infusions of CAR T cells were required to replenish the reservoir of circulating CAR T cells as expression was lost in few days [43].