17.4 Challenges and uture utlook 363technologies are poised to enable the generation of universal, off-the-shelf
cellular therapeutics that lack antigenic markers to induce immune rejection, a
development that would significantly reduce the time and financial costs associ-
ated with producing personalized supplies of therapeutic cells for each patient
[118]. If efforts in whole-genome synthesis and the construction of artificial cells
come to fruition, cellular therapeutics may eventually consist of fully synthetic
cells with precisely controlled functions.
Despite the myriad possibilities that synthetic biology inspires, real obstacles
need to be overcome in moving from model systems to real-world applications
in health and medicine. First, most synthetic biological systems demonstrated
to date have been designed to function in microorganisms such as yeasts and
bacteria rather than mammalian cells. Although some studies have shown
transportability across organisms [99, 119], significantly more experience will
be required in mammalian cell engineering to achieve the level of efficiency in
system assembly, integration, and characterization that is now possible in
microorganisms.
Second, despite the variety of synthetic circuits that have been reported, a rela-
tively small number of parts (e.g., the tet-inducible promoter, the theophylline
aptamer, fluorescent protein outputs, or acyl-homoserine lactone (AHL)-based
quorum sensing components) have been reused in a large number of designs,
reflecting a need to expand the inventory of biological parts. In particular,
cellular therapeutics development will require new outputs that execute thera-
peutic functions at precisely defined activity levels [120], a significantly more
complex task than ON/OFF control of fluorescent protein outputs. Similarly,
new sensors need to be developed to respond to therapeutically relevant inputs
such as disease-associated metabolites or FDA-approved drugs rather than oft-
used but clinically unacceptable inputs such as theophylline or isopropyl β-d-1-
thiogalactopyranoside (IPTG).
Third, given the paramount importance of safety in medical applications, any
synthetic system applied to cellular therapeutics must perform with consistency
and precision in the face of heterogeneities that are inevitable in the human body
and particularly in diseased cells. Unlike model systems in which parameters
such as input ligand concentration and cell density can be precisely controlled,
clinical applications in which heterogeneous cell populations harvested from
patients need to be quickly genetically modified, expanded, and reinfused in bulk
into the patients require a high level of robustness such that the system would
generate predictable and consistent outputs without the benefit of extensive cell-
population refinement or well-defined ranges of input signal strength. In this
regard, researchers are actively investigating genetic engineering strategies that
can enable synthetic components to interface more robustly with host cell physi-
ology. In contrast to random insertion of CAR transgenes via viral transduction,
site-specific integration of a CD19 CAR into the T-cell receptor α chain (TRAC)
locus of primary human T cells resulted in antigen-stimulated regulation and
greater uniformity of CAR expression [121, 122]. These site-specifically modi-
fied T cells exhibited reduced tonic signaling, delayed T-cell exhaustion, and
enhanced antitumor potency [121], underscoring the importance of being able
to tune the expression level and signaling strength of synthetic systems.