Medicine by Alexandros G. Sfakianakis: Advancing CAR T Therapies with CRISPR/Cas9

Τρίτη 18 Σεπτεμβρίου 2018

Advancing CAR T Therapies with CRISPR/Cas9

By Victoria Osinski

Scientists have turned to gene editing techniques to modify patients' T cells to combat cancer, but are often limited by factors including cost, low cell yields, or availability of expertise for therapy development. Interest lies in developing "off-the-shelf", universal, donor cells, which require thorough T cell modifications to make this approach safe and feasible. Currently, scientists are leveraging the use of the gene editing tool clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to advance CAR T therapies.

"Off-the-shelf" donor T cells requires a combinatorial approach

Patients undergoing immunotherapies risk developing many complications including graft-vs-host disease, cellular aplasia, and cytokine release syndrome¹. In order to reduce these risks, the following modifications must be made to autologous or allogeneic cells:

To prevent rejection, histocompatibility genes must be removed from the donor cells
To target cells toward a cancer-specific antigen and increase efficacy, endogenous T cell receptors (TCRs) must be modified.
To further enhance the functional performance of the donor cells, other signaling pathways in T cells should be modified to enhance activation or reduce inhibitory signals in the cells²

CRISPR/Cas9 can be harnessed to accomplish all of these modifications.

Expression of Flag-tagged SpCas9 in Hela cells, doxycycline treated or untreated ICC Immunocytochemistry/Immunofluorescence: CRISPR-Cas9 Antibody (6G12) - C-Terminus [NBP2-52398] - CRISPR-Cas9 Antibody (6G12) [NBP2-52398] - Hela cells or Hela cells expressing Flag-tagged SpCas9 under the control of the PTight (Tet-ON) promoter were treated for 24h with 1ug/ul Doxycycline, fixed and permeabilized with Methanol/Acetone and blocked in 2% BSA in PBS for 2 hours at RT. Cells were stained with 6G12 hybridoma supernatant (diluted 1:10) at 4C o/n, followed by incubation with anti-mouse-AF488 coupled secondary antibody for 1 hour at RT. Nuclei were counter-stained with Hoechst 33342.

CRISPR/Cas9 is being used to develop new CAR T therapies

CRISPR/Cas9 relies on an RNA-guided endonuclease, Cas9, activity to induce doublestrand breaks (DSBs) followed by insertions or deletions created by the non-homologous end-joining pathway to disrupt a specific DNA sequence. The guiding RNA sequence(s) must (1) complement a specific DNA sequence to which Cas9 is targeted, (2) bind to Cas9, and (3) contain a protospacer-associated motif (PAM) at the 3' end of the complement sequence in order to activate Cas9. The type II CRISPR/Cas9 system utilizes a single guide RNA sequence that incorporates all three of these components. This technology has been successfully applied to primary T cells^2,3,4,5. Additionally, the first ever FDA-approved clinical study using CRISPR/Cas9 to develop CAR T cells is ongoing^3,6, which will assess safety of these genetically modified cells in humans⁶.

Advantages of CRISPR/Cas9

CRISPR/Cas9 is cost effective, integration-free, and has the capacity for mutating multiple genes in a single system². Other gene editing methods such as Zinc-finger Nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been used for targeted mutagenesis in CAR T cell development, but these protein-based methods require engineering and optimization approaches more extensive than those required by the RNA-guided mechanisms of CRISPR/Cas9^2,3.

Remaining challenges

One of the major challenges in successfully using CRISPR/Cas9 is efficient delivery of its components to primary T cells. T cells require stimulation to take up nucleotides and proteins, but there is a limited dose- and time-window during which T cells can handle such stimulation before they become exhausted. Lentivirus, adeno-associated virus, and electroporation have all been used, but none have 100% efficiency². An additional challenge is assessing the extent of off-target mutagenesis that occurs with CRISPR/Cas9. Various sequencing methods and computational analysis approaches are available for use, but each has its limitations². No need to worry yet, however, as there are additional systems and approaches yet to be tested.

Learn more about CAR T Cell immunotherapy

Victoria Osinski, Doctoral Candidate
University of Virginia
Victoria studies cellular mechanisms regulating vascular growth during peripheral artery disease and obesity.

References

Tat T, Li H, Constantinescu C, Onaciu A, Chira S, Osan C, Pasca S, Petrushev B, Moisoiu V, Micu W, Berce C, Tranca S, Dima D, Berindan-Naegoe I, Shen J, Tomuleasa C, Qian L. Genetically enhance T lymphocytes and the intensive care unit. Oncotarget. 2018;9(23):16557-16572.
Ren J, Zhao Y. Advancing chimeric antigen receptor t cell therapy with CRISPR/Cas9. Protein Cell. 2017; 8(9):634-643.
Singh N, Shi J, June CH, Ruella M. Genome-editing technologies in adoptive T cell immunotherapy for cancer. Curr Hematol Malig Rep. 2017;12:522-529.
Liu S, Zhao Y. CRISPR/Cas9 genome editing: Fueling the revolution in cancer immunotherapy. Current Research in Translational Medicine. 2018;66:39–42.
Ren J, Liu X, Fang C, Jiang S, June CH, Zhao Y. Multiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition. Clin Cancer Res. 2017;23(9):2255-2266.
Reardon S. First CRISPR clinical trial gets green light from US panel. Nature. 2016. doi:10.1038/nature.2016.20137.

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