Precision Medicine, CRISPR, and Genome Engineering Moving from Association to Biology and Therapeutics

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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.