A novel dual-targeting delivery system for specific delivery of CRISPR/Cas9 using hyaluronic acid, chitosan and AS1411

Abstract

A facile method was designed that can specifically deliver CRISPR/Cas9 into target cells nuclei and reduce the off-target effects. A multifunctional delivery vector for FOXM1 knockout was composed by integration of cell targeting polymer (hyaluronic acid) and cell and nuclear targeting group (AS1411 aptamer) on the surface of nanoparticles formed by genome editing plasmid and chitosan (CS) as the core (Apt-HA-CS-CRISPR/Cas9). The data of cytotoxicity experiment and western blot confirmed this issue. The results of flow cytometry analysis and fluorescence imaging demonstrated that Apt-HA-CS-CRISPR/Cas9 was significantly internalized into target cells (MCF-7, SK-MES-1, HeLa) but not into nontarget cells (HEK293). Furthermore, the in vivo studies displayed that the Apt-HA-CS-CRISPR/Cas9 was strongly rendered tumor inhibitory effect and delivered efficiently CRISPR/Cas9 into the tumor with no detectable distribution in other organs compared with naked plasmid. This approach provides an avenue for specific in vivo gene editing therapeutics with the lowest side effect.

Introduction

Cancer is one of the most conventional causes of mortality in the world. Multimodal therapy is the mainstay of cancer treatment that includes surgery, radiotherapy and chemotherapy. A critical hurdle in cancer treatment is the resistance of tumor cells to diverse therapeutic forms caused by gene mutations and genetic abnormalities (Francies et al., 2020; Matano et al., 2015).

Gene therapy is one of the most promising ways and effective strategies to resolve this problem (Naldini, 2015). As it is well known, the novel gene-editing method relying on the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR associated protein 9 (Cas9) system has great potential for the treatment of human genetic disorders and cancers, and many studies have attempted to harness its mechanism for clinical applications (Long et al., 2016; Mali et al., 2013; Suzuki et al., 2016). The CRISPR/Cas9 enzyme machine consists of two components: the single-guide RNA (sgRNA) that is utilized for the target site recognition and Cas9 nuclease that targets predefined genomic positions through guide RNA (sgRNA) to introduce double-strand breaks (DSBs) in the genomic specific site (Cong et al., 2013; Hsu et al., 2014; Mali et al., 2013). Cas9 is navigated to a 5′-NGG-3′ protospacer adjacent motif (PAM) sequence, and cleaves DNA strands through the HNH and RuvC nuclease domains, then the break is healed by error-prone nonhomologous end-joining (NHEJ) or precise homology-directed repair (HDR) (Chen et al., 2017; Sternberg et al., 2015). The repair of DSB by NHEJ can generate the small insertions and deletions at the genomic sequence of interest, which permanently knockout the disease-causing mutations (Doudna & Charpentier, 2014; Maggio & Goncalves, 2015). The repair of DSB based on homology-directed repair (HDR) can cause the creation of precise changes in the target sequences, which correct the underlying disease-causing genes (Matsumoto et al., 2020; Porteus, 2015; Song & Stieger, 2017). Owing to its simplicity, high efficacy and specificity, the CRISPR/Cas9 system holds immense promise for effectively gene editing to cure genetic and cancer diseases fundamentally (Nihongaki et al., 2015; Rouet et al., 2018; Sánchez-Rivera & Jacks, 2015; Wu et al., 2015). However, off-target effects and poor stability can happen.

A desirable delivery of CRISPR/Cas9 system into the target cell nucleus is of special importance, which has superior biosecurity, high specificity, and capable to overcome some barriers such as endosomal entrapment barrier, enzymatic degradation hurdle, immune response barrier, and intracellular targeting (Chen et al., 2017; Klann et al., 2017). For this purpose, non-viral vectors have attracted the attention as delivery vehicles, because of being cost-effective, inert to immune responses, no or low toxicity, no limitation for DNA cargo size, excellent membrane permeability and avoiding transgene integration (Li et al., 2020; Nyamay'Antu, Dumont, Kedinger, & Erbacher, 2019; Qi et al., 2020). The non-viral delivery vectors cover different materials, including cell exosomes (Kim et al., 2017; Lin et al., 2018), synthetic polymers (Li et al., 2019; Luo et al., 2018), lipid based materials (Luo et al., 2018; Sun, Schur, et al., 2020), metal containing nanoparticles (Alsaiari et al., 2018; Wang et al., 2017) and nucleic acid based nanoparticles (Sun, Wang, et al., 2020). More recently, cationic polymers have been considered for gene delivery, including poly-l-lysine (PLL) (Wu & Wu, 1987), polyethylenimine (PEI) (Boussif et al., 1995) and chitosan (CS) (He, Liu, Peng, Zhuo, & Cheng, 2018; Zhang et al., 2020).

Among cationic polymers, CS, the natural marine polymer derived from chitin, is a biocompatible, biodegradable, and relatively nontoxic polymer (Khademi et al., 2020; Onishi & Machida, 1999). It is found that CS is a favorable candidate for gene delivery (He et al., 2019; Zhang et al., 2020). It is worth noting that cationic polymers, such as CS due to the capability of capture protons by their amino group have displayed the ability of endosomal escape which is known the proton sponge effect. This principle is mediated by the acidic pH of endosomes that triggers the influx of chloride ion and H₂O, and subsequently leads to osmotic swelling and rupture of the endosome (Khademi et al., 2020; Lai & Wong, 2018). Nevertheless, despite non-viral vectors have made great achievements in overcoming many obstacles, but still suffer from their fairly low targeted delivery efficiency which is a bottleneck for achieving clinical application (Shi et al., 2018). In this regard, aptamers have commonly considered as an amazing means of targeted delivery and rapid targeted entry into the tumor cells endosome and nucleus. AS1411 aptamer, a G-rich oligonucleotide, as a targeting agent can specifically recognize and bind to nucleolin in cell-surface and nucleus of many tumor cell lines, including lung, breast, prostate, and cervical cancer cells (He et al., 2020; Sun et al., 2015).

FOXM1 aptamer (FOXM1 Apt) has attracted interest as a promising anticancer to inhibit FOXM1 protein. FOXM1 protein, a member of the fork head/winged-helix family, is an oncogenic transcription factor upregulated in numerous cancers. Pan-cancer analyses have illustrated that the aberrant overexpression of FOXM1 protein is significantly correlated with cancer cell proliferation, tumorigenesis and metastasis. It was revealed that cell invasion and migration through knockdown of FOXM1 by siRNA could be suppressed (Wang et al., 2013). It has been demonstrated that FOXM1 can augment the expression of stem cells markers like multi-drug resistance proteins including ATP-binding cassette superfamily G2 (ABCG2) and lead to drug resistance (Bergamaschi et al., 2014). Also, the downregulation of FOXM1 protein leads to the inhibition of cell proliferation (Barger et al., 2019; Xiang et al., 2017). As such, we recently showed the synergic cytotoxic effects of FOXM1 Apt and doxorubicin (DOX) in cancer therapy (Abnous et al., 2018).

Hyaluronic acid (HA), a hydrophilic and anionic natural polymer, with a linear structure comprises of repeated units of glucuronic di-saccharide and N-acetyl glucosamine. It has the advantages of targeting capability of Cluster of Differentiation 44 (CD44) receptors, bioavailability, and good biocompatibility. So, HA is a good choice for targeted delivery systems (Parashar et al., 2019). CD44 proteins as cell surface adhesion molecules are overexpressed by nearly all tumors of epithelial origin, including lung and breast (Mattheolabakis et al., 2015).

Inspired by these achievements, we designed two highly efficient platforms for therapeutic FOXM1 aptamer and CRISPR/Cas9 genome editing based on natural polymers by self-assembly in an aqueous medium to achieve efficient cancer treatment both in vitro and in vivo. Our delivery system carries no risk of transgene integration and easily avoided inadvertent immune activation. AS1411 Apt and HA were self-assembled onto the surface of nanovector as targeting agents to facilitate the transporting of CRISPR/Cas9 into cancer cells via overexpressed nucleolin and CD44 on the cancer cell surface. Also, AS1411 can shuttle the nanoparticle to the nucleus of cancer cells through binding to its ligand, nucleolin. This system comprises of the genome editing plasmid and CS as the core and HA as the shell which is decorated with AS1411 Apt (Scheme 1). It should be pointed out that the introduction of CS as a positively charged polysaccharide in the core of the system eased the conjugation of the negatively charged HA on the nanoparticle surface, besides plasmid condensation caused by CS. Also, the presence of HA as a mucopolysaccharide and targeting agent, could remarkably promote the efficient genome editing in target cancer cells by the introduction of a dual-targeting delivery system. The CRISPR/Cas9 system utilized in this investigation is for FOXM1 knockout. The overexpression of FOXM1 is generally observed in different kinds of tumor cells because of a series of genetic and epigenetic incidents (Liu et al., 2019; Wang et al., 2010). Our study developed a new method to effectively transfer the CRISPR/Cas9 plasmid into tumor cells and restore their functions.