Elsevier

Biomaterials

Volume 218, October 2019, 119329
Biomaterials

Review
Innovative nanotechnologies for enhancing nucleic acids/gene therapy: Controlling intracellular trafficking to targeted biodistribution

https://doi.org/10.1016/j.biomaterials.2019.119329Get rights and content

Abstract

Nanomedicine promises to play an important role in next generation therapy, including Nucleic acid/Gene therapy. To accomplish this, innovative nanotechnologies will be needed to support nanomedicine by controlling not only the biodistribution but also the intracellular trafficking of macromolecules such as RNA/DNA. A multifunctional envelope-type nano device (MEND) was developed to meet this requirement. We herein provide an update regarding the functions of the MEND system focusing on the introduction of different functional biomaterials that enhance efficiency. The octaarginine (R8) peptide enhances cellular uptake and controls intracellular trafficking to induce synergism in transgene expression. The R8 was also used for developing a MITO-Porter system for mitochondrial targeting. The function of the MITO-Porter system was extended by developing a mitochondrial reporter gene for mitochondrial gene therapy. For efficient in vivo gene delivery, new pH-sensitive lipids have been introduced to achieve controlled biodistribution and to enhance endosomal escape. For example, the CL4H6 lipid exerts a more efficient in vivo gene silencing than that of ONPATTROTM, a preparation that has been approved by the US Food and Drug Administration. We further summarize new technologies that have been successfully applied to cancer immunotherapy leading to the introduction of a new strategy based on the concept of the Cancer-Immunity Cycle.

Introduction

In August 2018, the US Food and Drug Administration (FDA) approved Patisiran (ONPATTROTM by Alnylam) as the first small interfering RNA (siRNA) drug. This event moved the concept of nucleic acids/gene therapy from research to clinical reality, thus raising the hopes that it will be the next generation therapy for the treatment of currently non-curable diseases. Patisiran is a lipid-based nanomedicine in which siRNA is encapsulated in lipid nanoparticles composed of DLin-MC3-DMA, a very efficient pH-sensitive cationic lipid developed by Alnylam [1]. It is administered in the form of an intravenous infusion which efficiently delivers siRNA to hepatocytes. The emergence of Patisiran represents a breakthrough in the area of nucleic acids therapy based on a combination between RNA interference (RNAi) and nanotechnology [2]. It opens a new era of rapid development of future nanomedicines based on nucleic acids. This nanomedicine requires a sound nanotechnology to control, not only the biodistribution, but also the intracellular trafficking of macromolecules such as RNA and DNA, which cannot reach their sites of action by themselves [3]. To meet this requirement, we have been developing a multifunctional envelope-type nano device (MEND) for efficiently releasing nucleic acids-based nanomedicine [4, 5]. The MEND is a lipid-based system encapsulating different nucleic acids, and is designed to provide protection and control of biodistribution of its encapsulated macromolecules. The MEND can be modified with various functional devices for targeting specific tissues as well as for controlling intracellular trafficking. Compared to polymeric systems, the presence of a lipid layer provides more protection for the encapsulated nucleic acids. In addition, the lipid layer makes it easier to modify the surface with various ligands or peptides. Furthermore, the lipid layer plays an important role in enhancing the endosomal escape of nucleic acids through fusion with the endosomal membranes.

Octaarginine (R8), a cationic cell penetrating peptide (CPP), was introduced on the surface of the MEND to enhance cellular uptake in the early phase of our studies. Since then, it was found that R8 not only non-specifically enhances cellular uptake, but it also has other functions in enhancing the activity of other functional devices that make up the MEND system. A remarkable enhancement/synergism can be achieved when R8 is properly combined with targeting ligands, fusogenic peptides and pH-sensitive cationic lipids [6]. The value of R8 extend to additionally play a critical role in developing a MITO-Porter system for targeting mitochondria. Although mitochondria are important organelles as a target of therapy, there are only a few delivery systems that can be used for mitochondrial delivery. We developed a MITO-Porter system to deliver different cargos into mitochondria based on a membrane fusion mechanism to induce gene expression as well as silencing in mitochondria [7]. Developing a new mitochondrial DNA (mtDNA) was essential for achieving gene expression in mitochondria, since there was no reporter gene for mitochondria and the codon is different between mitochondria and the nucleus.

Although R8-based MENDs have achieved a high gene expression and silencing in vitro, the efficiency of these MENDs in vivo needed to be further improved. The most critical point was the low ability of the R8-MEND to target specific tissues in vivo. It was essential to expand the function of the MEND system to control its biodistribution after in vivo administration for achieving efficient targeted therapy. To achieve this, new types of pH-sensitive cationic lipids such as YSK05/12-C4/13-C3 were developed to control biodistribution and increase the efficiency of endosomal escape of lipid nano particles (LNPs) [8]. These lipids have an important advantage over R8-based systems because MENDs prepared with these lipids carry a neutral charge under physiological conditions. These nearly neutral MENDs were successfully used to efficiently deliver plasmid DNA and siRNA to hepatocytes after intravenous injection. A YSK library was generated by altering the hydrophilic head groups and hydrophobic scaffolds. The CL4H6 lipid was the most efficient pH-sensitive cationic lipid for liver siRNA silencing. The CL4H6 containing LNP (CL4H6-LNP) induced gene silencing in the mouse liver more efficiently than DLin-MC3-DMA, which is used in ONPATTROTM.

In addition to hepatocyte targeting, various types of YSK lipids were successfully applied for cancer immunotherapy, which is an important strategy for killing cancer cells using the immune system [9]. Cancer immunotherapy has been recently validated by the successful development of OPDIVOĀ®, which is a well-known immune checkpoint inhibitor (ICI). The concept of the Cancer-Immunity Cycle was recently introduced to understand the dynamic mechanisms related to how the immune cells function in vivo to kill cancers [10]. We are currently developing a nano drug delivery system (DDS) for the targeted delivery of adjuvants to different immune cells based on the concept of the Cancer-Immunity Cycle.

In this review, we describe innovative nanotechnologies for enhancing nucleic acids/gene therapy through controlling intracellular trafficking as well as targeted biodistribution. The multiple roles of the R8 peptide in enhancing the activity of different MEND systems and in developing a MITO-Porter system for mitochondria targeting are discussed. Furthermore, current progress on mitochondrial delivery and the need to develop a reporter gene for mitochondria is introduced. The use of a series of original pH-sensitive cationic lipids developed in this laboratory in developing highly efficient MENDs for targeting hepatocytes is also discussed. We particularly focused on the structure-activity relationships of these lipids based on recent studies. Finally, we discuss the applicability of the MEND platform for targeting different immune cells for achieving successful cancer immunotherapy. The current state of knowledge and perspectives regarding Cancer-Immunity Cycle are comprehensively summarized.

Section snippets

The octaarginine peptide: multiple roles in improving MEND efficiency

Oligoarginines are short cationic peptides that represent simplified models of CPPs. The conjugation of oligoarginines to different high molecular weight drugs and drug carriers showed an improved drug delivery due to the ability of these peptides to bind to cellular membranes and to mediate efficient cellular uptake [11]. Among these oligoarginines, R8 proved to be the most efficient peptide [12]. The R8 peptide was employed as a functional device in the MEND platform to improve its

Significance of targeting mitochondria to develop an innovative therapy and current status.

In the previous section, it was stated that R8 peptide plays multiple roles in gene delivery in cultured cells and liver gene targeting in animal experiments. Here, another role of R8 for organelle targeting to regulate intracellular trafficking is discussed. Electrostatic interactions between cationic membrane-permeant peptides such as R8 and mitochondria was well known. Mitochondria, which contain their own genome, mtDNA, play various essential functions, which include ATP production, the

Development of potent cationic lipids for in vivo hepatic delivery of siRNA

Recent remarkable progress in the efficiency of siRNA delivery in vivo can be attributed to the rapid development of potent pH-sensitive cationic lipids as an alternative technology to the permanently cationic systems, including the R8 peptide, which was described in the previous sections. In this section, recent progress in the development of pH-sensitive cationic lipids and the related key parameters that affect physicochemical properties and activity of the LNPs are discussed.

siRNAs can

Understanding the current cancer immunotherapy

ICIs such as the cytotoxic T-lymphocyte associated protein 4 (CTLA-4) antibody, the programmed cell death-1 (PD-1) antibody and the programmed cell death ligand 1 (PD-L1) antibody have revolutionized the field of cancer therapy. Science regarding the approval of the CTLA-4 antibody for use in cancer immunotherapy has been developing at amazing pace. Dr. James P Alison and Dr. Tasuku Honjo who discovered CTLA-4 and PD-1, respectively, were awarded the 2018 Novel Prize in Physiology or Medicine.

Conclusion

In 2018, ONPATTROTM was approved by the FDA and EMA, which opened the year of Nucleic Acids Nanomedecine and new sophisticated systems will likely follow this success and some will be successfully transferred to clinical practice in accordance with the progress of science. We are interested in contributing to this strong wave with the cooperation of industrial power. It is essential to have a new technology to bridge between an innovative technology born in a laboratory in a University and a

Acknowledgment

This work was supported, in part, by a Grant-in-Aid for Young Scientists (A) (Grant No. 17H05052 to Y.S.), a Grant-in-Aid for Scientific Research (B) (Grant No. 17H02094 to Y.Y. and 17H03974 to T.N.), a Grant in Aid for Challenging Research (Exploratory) (Grant No. 17K20076 to Y.Y., 18K19888 to T.N. and 18K19889 to Y.S.) and the Special Education and Research Expenses (to H.H.) from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government (MEXT), Supporting

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