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Progress in Neurobiology. The Yin and Yang of nucleic acid-based therapy in the brain. Checking for direct PDF access through Ovid. We discuss the challenges for the molecular therapy in the brain and we highlight the renaissance of nucleic acid-based drugs to treat neurodegenerative diseases. We describe the potential therapeutic effects that can be obtained by inhibiting Yin and increasing Yang gene expression.

The Yin side of nucleic acid-based therapy: we summarize the current technologies and we provide examples of their applications to treat to neurodegenerative diseases in mouse models and in clinical trials. We provide examples of activatory RNA therapy in neurodegenerative disorders in mouse models and in clinical trials. We conclude with our vision on the future perspectives of nucleic acid-based therapy in the brain. In , first human gene therapy was conducted, targeting adenosine deaminase deficiency via retrovirus-mediated delivery system [ 1 ].

Since then, the number of clinical trials has gradually increased, and approximately trials have been globally undertaken or approved until November [ 2 ]. Viral vector-based delivery resulted in a high level of gene expression for a long period; however, carcinogenesis and lethal immune reaction were reported [ 3 , 4 , 5 ].

Numerous researchers have been attempting to overcome these serious obstacles to enable safe and efficient therapy. For this purpose, the improvement of viral vector has been extensively studied in the last decade, and in addition, nonviral vector-based gene delivery method has developed with great promise. As expected, it resulted in less antigenicity and less chance of integration into the human genome than viral vector; therefore, it can be regarded as a biologically safer method than viral vector-based gene delivery method.

However, the period of transgene expression tends to be limited. This chapter focuses on nonviral vector-based delivery method, which could be used for the nucleic acid-based therapy. In these methods, a transgene is not integrated into the host genome; hence, gene expression is transient. The last section of this chapter outlines the recent progress in the HGD, which enables the highest level of delivery efficiency among nonviral vector-based approaches and the clinical application utilizing the well-established method of catheter insertion into the vessels in the multiple organs.


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This section focuses on gene delivery methods using nonviral vector-based approach. Nucleic acids loaded in artificial or natural cargos or in naked condition are transferred to target cells. The characteristics of various gene deliveries are briefly described in Table 1. Lipofection, a cationic lipid-mediated approach, is widely used in numerous in vitro and in vivo studies. The first study reporting lipofection was published in [ 6 ].

Molecules comprising hydrophilic head, linker, and hydrophobic anchor form a spherical structure. The positively charged hydrophilic head plays a role in condensing the negatively charged DNAs. It also helps in establishing an electrostatic interaction with the negatively charged cell membrane.

As a result, it promotes the cellular uptake of DNA-loaded liposome lipoplex , endosomal escape, and subsequent release of the condensed DNAs into the cytoplasm. On the contrary, the hydrophobic anchor protects DNAs from degradation by nucleases. Liposome is a popular carrier to deliver even large-sized transgene; it is easy to prepare and modify and is utilized in numerous laboratories worldwide. Nevertheless, there are several drawbacks for its use in gene therapy.

It has difficulty in achieving therapeutic level of transgene expression, shows no tropism to desired cells, and exhibits a short life span. Furthermore, the positively charged head has cell toxicity. An inflammatory response occurs when unmethylated CpG DNA is transported, which is one of the obstacles that need to be addressed. Various strategies to achieve high level of safety and efficiency, such as introduction and improvement of polyethylene glycol [ 7 ] and cell-specific targeting ligand on the surface of the liposome, have been extensively studied.

Development of a promising linker also improves stability, biodegradability, and transfection efficiency and reduces cytotoxicity [ 8 ]. Lipofection has been utilized in 4. Because secretory fluid becomes viscous, the patient may experience repeated respiratory infection and, finally, respiratory failure. In , other clinical trials for genitourinary cancers and solid tumors reportedly used the truncated forms of the RB gene and p53 gene with docetaxel, respectively [ 11 , 12 ].

Cationic polymer is an artificially synthesized vehicle, and various types of polymer have been studied. DNA condensed in cationic polymer polyplex acquires tolerance to enzymatic degradation, which results in stability in the blood.

Cellular uptake is via receptor-mediated endocytosis, which leads to a high level of transfection activity. Clinical trials using this approach for cystic fibrosis and ocular degenerative disease have been reported [ 13 , 14 ]. Nevertheless, the stability of polyplex and persistent positive charge leads to high cytotoxicity. Because cationic polymer is easy to prepare and improve, various constructs, such as polyethylenimine, polyamidoamine, polyallylamine, chitosan, dendrimers, cationic proteins, and peptides, have been studied to overcome the obstacles. Lipopolyplex comprises polycation cationic polymer or peptide and condensed DNA with lipid shell and is divided into diverse categories according to the combination and ternary structure.

Its advantages are of both lipoplex and polyplex, that is, more efficient transfection and less cytotoxicity. Previous study [ 15 ] and reviews [ 16 , 17 ] have described the strategy, variety, and preparation of lipopolyplex. Exosome is a kind of extracellular vesicle secreted by various cells. It comprises a lipid bilayer with several surface antigens derived from the parent cell. Moreover, exosome is known to have organ and cell tropism; however, the mechanism is not completely clarified.

This indicates that exosome plays a role in intercellular communication. Cancer cells as well as healthy cells secrete exosome.

Series Introduction: Emerging clinical applications of nucleic acids

Integrin included in exosome reportedly determines organ tropism for metastasis. Exosome from metastatic lung tumor of breast cancer induced lung metastasis of breast cancer, which originally had metastatic ability only to the bone [ 18 ]. Recently, many researchers have been studying exosome as delivery system for cancer therapy. Surface antigens of exosomes are known to be modified directly and genetically. The exosomes from leukemia cells, marrow stromal cells, adipose-derived mesenchymal stem cells, breast cancer cells, and kidney cells including siRNA and miRNA were reported to be used for colorectal tumor, glioma, hepatocellular carcinoma, breast cancer, and chronic myelogenous leukemia [ 20 , 21 , 22 , 23 , 24 ].

Direct injection to the tissue is the simplest approach for the physical delivery of nucleic acid. The first report for delivery to muscle was published in [ 25 ]. Needle injection was expanded to the skin [ 26 ], heart muscle [ 27 ], liver [ 28 ], and tumor [ 29 ]. Currently, microneedle is studied as a minimally invasive delivery for skin disease and vaccination [ 30 , 31 ]. In a mouse study, siRNA delivery is reported to be effective for skin conditions with aberrant gene expression, such as alopecia, allergic skin diseases, hyperpigmentation, psoriasis, skin cancer, and congenital pachyonychia [ 33 ].

Gene gun is known as microprojectile bombardment, and the first study reporting its use was published in [ 34 ]. At first, this method was developed for gene delivery into plant cells. A bullet with the microparticles containing DNA is shot to a target cell, and gene delivery is achieved.


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On the basis of the principle of obtaining a driving force, a gene gun is divided into three major groups: powder gene gun [ 34 ], high-voltage electric gene gun [ 35 ], and gas gene gun [ 36 ]. The driving force moves the microparticles containing DNA toward a target tissue and penetrates the cell membrane. Because delivery efficiency and cell damage are two sides of the same coin, appropriate operating pressure is required.

A phase I clinical study was performed to treat melanoma using IL gene [ 37 ]. Although an attempt of combining delivery with microneedles reportedly enhanced the penetration depths of microparticles [ 38 ], gene gun may be more appropriate for delivery to the skin, such as for vaccination.

Nucleic Acid Hybridization and Probes

Sonoporation, using ultrasound [ 39 , 40 ], and electroporation, using electric pulse [ 41 ], increase the permeability of cell membrane for cellular uptake of nucleic acid. Magnetofection utilizes magnetic field to enable microparticles with nucleic acid to pass through the cell membrane [ 42 ]. These methods are used in combination with other methods, such as lipofection, to protect nucleic acid against degradation by nucleases.

REFERENCES | Pharmaceutical Perspectives of Nucleic Acid-Based Therapy | Taylor & Francis Group

To increase gene delivery efficiency of sonoporation, microbubbles were shown to be effective [ 43 ] and applied for delivery to cancer cells [ 44 , 45 ] and the central nervous system [ 46 , 47 ]. Clinical trials in phases I and II have been reported for the treatment of melanoma [ 48 , 49 , 50 ] and solid tumors [ 51 ]. HGD is one of the simplest methods for gene transfer. The efficiency of HGD is the highest among nonviral vector-based delivery methods, and its physical force to deliver the gene into the cells relies on a high level of flow rate and volume of the injected solution.

Since the first published reports in [ 52 , 53 ], many researchers have utilized this methodology for gene transfer in animal experiments, particularly in rodent studies. For its application in human, safety and efficacy of this approach have been extensively studied and improved. To date, various types of nucleic acid have been delivered by this approach in rodents as well as pigs [ 54 , 55 , 56 , 57 ], dogs [ 58 , 59 ], and rhesus monkeys [ 60 , 61 ]. The next section describes its principle and progress in human gene therapy.

HGD is achieved by the quick injection of a large amount of naked nucleic acid solution into the vein. In case of a rodent, the solution is injected from the tail vein. The most important step of successful gene delivery is a precise insertion of an injection needle into the tail vein.

The details of technical tips are described in Figure 1. The quick injection can transiently increase an intravenous pressure. Mechanical force by rapid increase in venous pressure allows nucleic acid to pass through the cell membrane into the cytoplasm and nucleus. Technical details of the tail vein injection in a mouse.

Review Series

The puncture can be performed from the top of the tail curve. Once the backflow is confirmed, a needle tip can be further inserted to the proximal side of the tail vein. Among various organs, the liver can achieve the highest level of gene expression because of the presence of the specific structure fenestra.

Fenestra is a small window in the sinusoidal vessel, and hepatocytes are partly exposed to the blood stream. In other words, hepatocytes can be directly affected by intravascular pressure. A rapid stream of hydrodynamic injection can wash out the blood in the sinusoid vessel transiently and thoroughly, and nucleic acid can reach the hepatocytes without degeneration by nucleases. A high intravascular pressure creates dimples on the surface of the hepatocyte and finally generates transient small pores. The nucleic acid is pushed into the hepatocyte through the transient pores Figure 2.

Although serum transaminase shows transient increase after a hydrodynamic injection, these values return to the background level within a short period. Considering the short life time of transaminase, an increase in serum transaminase is speculated to be caused by leakage from the transient pores. If the intravascular pressure is kept within an adequate range, this change in hepatocyte is reversible and does not result in apoptosis and necrosis; therefore, acute liver failure is not a concern.

Scheme of hydrodynamic gene delivery. The hepatocyte partly faces to the blood stream via the fenestra in the sinusoidal structure. A rapid stream of hydrodynamic injection has the blood in the sinusoid washed out transiently, and the nucleic acid can be delivered into hepatocytes without being degraded by nucleases. A high intravascular pressure makes dimples on the surface of hepatocyte, and finally generates transient pores. Nucleic acid is pushed into the hepatocyte through the transient pores.