18
The crossover of the CRISPR multiplex editing techniques to CAR-T therapy is
a new and exciting area of active investigation. It has been shown that up to five
genes can be simultaneously disrupted in mouse embryonic stem cells with high
efficiency CRISPR-Cas9; specifically, CAR-T cells with either two or three gene
disruptions (TRAC, B2M +/− PD-1) and analysis of in vivo and in vitro antitumor
function. Using CAR-T cells targeting the B-cell antigen CD19, chosen for its
expression by nearly all B-cell malignancies and restriction in normal tissues to
expression in mature and precursor B cells, plasma cells, and follicular dendritic
cells [ 11 ]. It was shown that anti-CD19 CARs were capable of activating T cells in
a CD19-specific mechanism that could kill CD19+ primary leukemia cells in vitro
[ 174 , 175 ].
1.6.2 iPSCs
Reprogramming of somatic cells has allowed the creation of patient-specific induced
pluripotent stem cells (iPSCs). They have the unique properties of self-renewal,
large scale expansion, and ability to differentiate into endoderm, mesoderm, ecto-
derm, or even to hematopoietic stem cells (HSCs) in the presence of stromal cell
co-culture or hematopoietic cytokines [ 176 – 178 ]. In as early as the 1960s, it was
shown that a pluripotent state could be generated through the reprogramming of
fully differentiated cells; essentially, it was demonstrated early on that totipotency
could be achieved through alterations in the epigenetic profile [ 178 ]. Subsequent
somatic nuclear transfer (SCNT), including the “Dolly” experiment, and cell fusion
experiments revealed the presence of somatic cell-inducing cytoplasmic diffusible
transacting factors in the oocyte/ESC in addition to the proof of reprogrammable
terminally differentiated cells.
These results paved the way for one of the landmarks papers by Takahashi and
Yamanaka in 2006, which showed the possibility of ectopic expression of a distinct
and small set of transcription factors via retrovirus integration into differentiated
cells. By identifying and serially reducing this set of genes into the minimal set of
factors (Klf4, Sox2, Oct4, Myc) and demonstrating the retention of embryonic stem
cell properties in these now ‘induced pluripotent stem cells’ (iPSCs), they set the
stage for subsequent research on refining and implementing various methodologies
to edit and induce functional pluripotency in a range of differentiated human cell
types. The Yamanaka experiments additionally resolved and avoided the ethical
debate around the use of stem cells sans human embryos [ 178 ]. Figure 1.3 demon-
strates the process for ex vivo modification of somatic cells to iPSCs and ultimate
correction of disease mutations by genome editing.
Studies using CRISPR/Cas9 editing in the transformation of iPSCs generated
from somatic cells have demonstrated homologous recombination-based gene cor-
rection that could provide new avenues for treating certain genetic disorders, includ-
ing β-thalassemia and Duchenne muscular dystrophy, as mentioned before [ 179 ].
J.E. DiCarlo et al.