360 17 Synthetic Biology in Immunotherapy and Stem Cell Therapy Engineering
interference with the intricate balance between immunostimulatory and immu-
nosuppressive signaling creates inherent risks for autoimmune dysfunction
[96–98]. Safety concerns thus demand gene expression control systems that can
be regulated by physician-administered drugs or by molecules specific to the
tumor microenvironment. To address this challenge, several ligand-responsive
regulatory systems have been developed to control the production of potent
cytokines or suicide proteins by engineered T cells [99, 100]. In an early example
of mammalian synthetic biology, small molecule-responsive ribozyme switches
were inserted in the 3′ untranslated region of transgenes encoding IL-2 and
IL-15, resulting in posttranscriptional control of cytokine production in a rapid,
reversible manner in vitro as well as ligand-dependent modulation of T-cell pro-
liferation in vivo [99]. Notably, the ribozyme switch is modularly composed with
well-defined sensing, actuating, and information-transmission domains that can
be independently modified for the specific application of interest. For example,
RNA aptamers to a wide variety of ligands including nucleic acids, small mole-
cules, and proteins have been generated in vitro [101], and ribozyme switches
tailored for ligands specific to the disease of interest can be rationally designed
by incorporating the appropriate RNA aptamers. In the context of cytokine
regulation for T-cell therapy, ribozyme switches can be designed to respond to
physician-administered drugs or to molecules known to be overexpressed by
tumor cells, thus increasing the specificity and safety of this cell-based therapeu-
tic strategy.
As an alternative to the regulation of growth-promoting cytokines, the expres-
sion of suicide genes that can rapidly and precisely eliminate engineered T cells
provides a means to prevent runaway immune responses. The most commonly
used suicide gene is the herpes simplex virus I-derived thymidine kinase
(HSV-TK). Originally developed as a method to deplete donor T cells that cause
graft-versus-host disease after allogeneic bone marrow transplantation, HSV-TK
expression confers sensitivity toward the small molecule drug ganciclovir, thus
enabling selective depletion of T cells that have been engineered to transgeni-
cally express HSV-TK [102]. However, HSV-TK-mediated cell depletion is often
incomplete, and the strategy precludes the use of ganciclovir as an antiviral drug
for cytomegalovirus infections, a common and often fatal complication of bone
marrow transplants [103]. Taking an alternative approach, researchers have
developed chimeric suicide genes that fuse pro-apoptotic proteins with dimeri-
zation domains to induce apoptosis upon the administration of a chemical
ligand [104]. For example, an inducible caspase 9 suicide system has been con-
structed by fusing an inactive pro-caspase 9 monomer to FKBP [105]. Upon
administration of the chemical inducer of dimerization (CID) molecule AP1903,
the FKBP domains homodimerize, resulting in the cross-linking and activation
of the tethered caspase 9 domains, which in turn induce apoptosis in cells
expressing this suicide system (Figure 17.4). The inducible caspase 9 system has
been tested in an adoptive T-cell therapy trial and demonstrated the ability to
eradicate >90% of engineered T cells within 30 min of AP1903 administration,
effectively eliminating graft-versus-host disease symptoms without recurrence
[100]. Combining several of the technologies summarized previously, research-
ers have engineered second-generation CD19 CAR-T cells equipped with