However, for optimal use, this technology must be carried using an efficient delivery system.
An efficient delivery system implies:
- A high penetration rate into target cells
- A high level of editing of the targeted gene
- The preservation of the viability and the original cell phenotype
- An all-in-one delivery system (gRNA guide(s) + Cas9)
- A transient Cas9 expression by mRNA delivery, since it has been demonstrated that compared to a long-term viral Cas9 expression, a transient Cas9 expression can substantially reduce off-target genome editing in cells(1,2).
With these needs in mind, Vectalys has created a new RNA delivery system named LentiFlash®. Through a fast and transitory RNA expression, this non-integrative particle is particularly suitable to carry CRISPR-Cas9 into any cell type while preserving their original cell phenotype and viability.
The data below, gathered by Vectalys and shown during the AACR and ASGCT annual meetings, demonstrate that the combination of LentiFlash® with the CRISPR-Cas9 technology can be applied to several therapeutic fields.
LentiFlash® carrying CRISPR-Cas9 for cancer immunotherapy:
PD-L1 is a ligand expressed at the surface of tumor cells. In the tumor microenvironment, PD-L1 recognize PD1, its receptor at the surface of T cells. This interaction, one of the most important immune checkpoint, leads to an inhibition of T cells. Tumor cells use this way to evade the immune response through inhibition of the T cells activation(3) (illustration adapted from Servier Medical Art).
Recently, the blockade of PD1/PD-L1 interaction has shown promising results in cancer immunotherapy overcoming the tumor cells escape to T cells immune response. Based on these data, Vectalys has used the LentiFlash® delivery system to carry CRISPR-Cas9 into human primary T cells and efficiently disrupt PD1(3) (illustration adapted from Servier Medical Art).
This bar graph shows the high efficiency of LentiFlash-CRISPR-Cas9 in editing PD1 in human primary T cells. Indeed, a PD1 disruption rate of 65,9% is reached with a dose of 5 pg p24 per cell.
Figure 3: highly efficient disruption of PD1 in human primary T-cells (data gathered by Vectalys)
Thanks to the knock-out of PD1, T cells will not be able to interact with PD-L1 and will be effective against the tumor. Therefore, this strategy is a promising approach for future immunotherapeutic programs.
LentiFlash® carrying CRISPR-Cas9 for antiviral strategies:
CXCR4 works as a co-receptor for HIV, allowing virus entry into CD4+ cells. This bar graph shows the high efficiency of LentiFlash-CRISPR-Cas9 in editing CXCR4 in human primary T cells. Indeed, a CXCR4 disruption rate of more than 98,7% is reached with a dose of 5 pg p24 per cell.
Figure 4: Highly efficient disruption of CXCR4 in human primary T cells (data gathered by Vectalys)
Thanks to the knock-out of CXCR4, T cells will not allow the entry of HIV 1. This approach paves the way to new antiviral strategies.
A safe cell engineering:
By using highly purified and concentrated LentiFlash particles, human primary T lymphocytes are efficiently transduced without affecting viability and the original cell phenotype is preserved (data gathered by Vectalys).
Figure 5: Preservation of the original cell phenotype.
Material & methods
1. Activated T cells are transduced by a range of LentiFlash particles (from 0 to 5 pg of p24/cell) carrying Cas9 and a sgRNA targeting either the human PD1 or the human CXCR4 gene.
2. Transduction is performed once (T1) or twice (T2).
3. LentiFlash delivering Cas9 alone is used as a control of PD1 or CXCR4 editing.
4. PD1 or CXCR4 expression is analyzed by FACS 4 days after the 2nd transduction, as well as the viability and expression of TCR and CD25 on the cells.
The CRISPR approach using LentiFlash RNA delivery leads to a highly efficient and dose-dependent gene knock-out into human T lymphocytes, without any genomic integration.
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- Hao Yin, Chun-Qing Song, Joseph R Dorkin, Lihua J Zhu, Yingxiang Li, Qiongqiong Wu, Angela Park, Junghoon Yang, Sneha Suresh, Aizhan Bizhanova, Ankit Gupta, Mehmet F Bolukbasi, Stephen Walsh, Roman L Bogorad, Guangping Gao, Zhiping Weng, Yizhou Dong, Victor Koteliansky, Scot A Wolfe, Robert Langer, Wen Xue, Daniel G Anderson. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat Biotechnol. Author manuscript; available in PMC 2017 May 9. Published in final edited form as: Nat Biotechnol. 2016 Mar; 34(3): 328–333. Published online 2016 Feb 1. doi: 10.1038/nbt.3471
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- Laurence Zitvogel, Guido Kroemer. Targeting PD-1/PD-L1 interactions for cancer immunotherapy. Oncoimmunology. 2012 Nov 1; 1(8): 1223–1225. doi: 10.4161/onci.21335.