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

(Dana P.) #1

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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.
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