The hydrophobic tail of a pH-sensitive cationic lipid influences siRNA transfection activity and toxicity in human NK cell lines
Graphical abstract
Introduction
Adaptive cell therapy (ACT) is a powerful therapeutic against cancer. ACT typically shows a strong antitumor effect due to the direct administration of this to CD8+ killer T cells and natural killer (NK) cells. Chimeric antigen receptor (CAR) T cell therapy against blood cancers is now in clinical use. NK cell therapy promises to be the next generation ACT (Xin Yu et al., 2019). NK cell therapy involves the use of autologous/allogenic NK cells and human NK cell lines, and, compared with T cell therapy, has the advantage of low graft-versus-host (GVH) reactions (Cheng et al., 2012). Additionally, using cell lines permits an off-the-shelf operation to be conducted. NK-92 cells are the only human NK cell line tested in a clinical trial stage (Arai et al., 2008, Tonn et al., 2013, Boyiadzis et al., 2017). An important feature of NK-92 cells is that they lack nearly all inhibitory receptors, thus allowing their continuous activation (Klingemann et al., 2016). This makes NK-92 cells a potential platform for NK cell therapy.
For enhancing the capabilities of ACT, modifying gene expression is an essential technology. The success of CAR-T cell therapy has accelerated CAR-NK cell research (Reindl et al., 2020). Regarding the control of gene expression, gene silencing by small interfering RNA (siRNA) represents an attractive approach, because some types of siRNA medicines have already been approved. Viral vectors are often employed in genetic modification and gene silencing and this approach has been confirmed to be successful (Reindl et al., 2020, Matosevic, 2018). However, producing viral vectors for clinical use is costly and a high level of safety is needed. In contrast, only a few non-viral vectors have been used in drug development, because such non-viral vectors frequently have a low transfection efficiency (Reindl et al., 2020). Viral vectors and electroporation systems generally deliver siRNA to NK-92 cells (Braunschweig et al., 2011, Jiang et al., 2011, Wang et al., 2018). In contrast, there no reports of the use of non-viral vectors for delivering siRNA to human NK cells including NK-92 cells. A low efficiency of cellular internalization and endosomal escape appeared to be the cause of it. Because non-viral vectors have superior features such as high safety, low cost, and ease of handling, the development of efficient non-viral vectors such as lipid nanoparticles (LNPs) represents an urgent issue.
We previously reported on the development of a LNP for siRNA transfection to immune cells (Warashina et al., 2016, Nakamura et al., 2016, Nakamura et al., 2018, Endo et al., 2019, Nakamura et al., 2020). The siRNA-loaded LNP contains a YSK12-C4 (YSK12-LNP), a pH-sensitive cationic lipid, and shows high efficiency of endosomal escape by disrupting the endosomes (Warashina et al., 2016). Additionally, the YSK12-LNP has a high affinity in human immune cell lines, resulting in significant levels of gene silencing (Nakamura et al., 2016). However, the YSK12-LNP shows significant cytotoxicity in NK-92 cells at the dosage needed to induce a gene silencing effect (Nakamura et al., 2016). Although decreasing the level of YSK12-C4 also reduced the degree of cytotoxicity, the magnitude of the reduction was insufficient (Nakamura et al., 2018). It will be necessary to change the intracellular trafficking of LNP to achieve the dissociation between gene silencing activity and toxicity.
The success of Onpattro (patisiran), a first siRNA therapeutic using LNP for targeting the liver required substantial effort. These efforts were largely focused on the structure of the pH-sensitive cationic lipid that was used in the system (Heyes et al., 2005, Zimmermann et al., 2006, Akinc et al., 2008, Semple et al., 2010, Jayaraman et al., 2012, Sato et al., 2016, Sato et al., 2019). In addition, the impact of head and tail groups in a pH-sensitive cationic lipid were demonstrated using cancer cells in vitro (Wang et al., 2007, Malamas et al., 2013). Such pH-sensitive cationic lipid based LNPs generally have a neutral charge at physiological pH such as in the blood circulation and culture medium, whereas, in endosomes, the LNPs adopt a cationic charge, which enhances the delivery of siRNA to the cytosol. However, the impact of the structure of the pH-sensitive cationic lipid for delivering siRNA to NK cells has not been extensively studied. We recently constructed a lipid library consisting of YSK12-C4 that had been systematically derivatized as a benchmark (Sato et al., 2019). In this study, we investigated the effect of lipid structure on gene silencing activity and toxicity in human NK cell lines. The hydrophilic head group of lipids largely affects the apparent pKa of LNP formulations, and lipids having an amino moiety substituted with a methyl group usually show elevated gene silencing activity. In addition, the hydrophobic tail of the lipids can also influence gene silencing activity as well as cell viability. We found that an LNP prepared using a pH-sensitive cationic lipid, CL1H6 (CL1H6-LNP), had an increased gene silencing and cell viability, compared with the YSK12-LNP. Interestingly, the intracellular trafficking of CL1H6-LNP was quite different from that of YSK12-LNP. The YSK12-LNP appeared to escape from the endosomes via a high degree of membrane fusion, leading to membrane disruption, whereas the CL1H6-LNP appeared to escape from endosomes via only mild membrane fusion. This suggests that this difference resulted in a decreased level of cytotoxicity. Collectively, the CL1H6-LNP has promising capability as a non-viral vector for delivering siRNA to human NK cell lines.
Section snippets
Cell lines and animals
The culture methods for NK-92 (American Type Culture Collection, Manassas, VA) and KHYG1 cells (Japanese Cancer Research Resources (JCRB) cell bank, Osaka, Japan) are described in the Supplementary data. Female C57BL/6 mice (7–9 weeks old) were maintained under SPF conditions. The experiments were approved by the Animal Committee of Hokkaido University (#16-0014).
LNP preparation
The method used to prepare the LNPs was described in a previous report (Warashina et al., 2016, Nakamura et al., 2016, Nakamura et
Impact of the hydrophilic head group on gene silencing activity and cytotoxicity
The structure of the hydrophilic head group of lipids largely influences the apparent pKa of the LNP (Jayaraman et al., 2012, Sato et al., 2019). In addition to YSK12-C4, CL1H6, CL15H6 and CL4H6 were also evaluated in this study. The CL1H6 has the same hydrophilic head group as YSK12-C4 and an oleate structure as a hydrophobic tail (Fig. 1) (Sato et al., 2019). The apparent pKa values of YSK12-LNP and CL1H6-LNP were 8.00 and 8.20, respectively (Fig. S1). The CL15H6 and CL4H6 have different
Discussion
In the case of delivering siRNA to the liver, the structure of the pH-sensitive cationic lipid has been the major focus of investigations (Heyes et al., 2005, Zimmermann et al., 2006, Akinc et al., 2008, Jayaraman et al., 2012, Sato et al., 2016, Sato et al., 2019). However, the impact of the structure of the pH-sensitive cationic lipid for delivering siRNA to NK cells has not been extensively studied. We demonstrate herein that a pH-sensitive cationic lipid (CL1H6) in which an amino moiety is
Conclusions
We herein report on the importance of the structure of a pH-sensitive cationic lipid on gene silencing activity and cytotoxicity in human NK cell lines. The CL1H6-LNP (25/75) was found to function as a potent LNP for delivering siRNA to NK-92 cells. Cationic properties at neutral pH and a hydrophobic tail with an oleate structure are key factors for the improvement of gene silencing activity and cell viability. The use of CL1H6 avoids endosomal disruption, resulting in a decreased level of
CRediT authorship contribution statement
Takashi Nakamura: Conceptualization, Methodology, Formal analysis, Writing – original draft, Writing – review & editing, Visualization, Supervision, Project administration, Funding acquisition. Taisei Nakade: Validation, Investigation, Writing – review & editing. Koharu Yamada: Validation, Investigation, Writing – review & editing. Yusuke Sato: Investigation, Resources, Writing – review & editing. Hideyoshi Harashima: Conceptualization, Writing – review & editing, Supervision, Project
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported, in part, by JSPS KAKENHI (Grant Number 20H03373) (T.N.) and the Special Education and Research Expenses from the Ministry of Education, Culture, Sports, Science and Technology (H.H.). We also appreciate the helpful advice of Dr. Milton S. Feather for examining the English in this manuscript.
Takashi Nakamura, Hideyoshi Harashima, Yusuke Sato, Koharu Yamada and Taisei Nakade have patent pending to Hokkaido University.
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These authors contributed equally to this work.