Elsevier

Journal of Controlled Release

Volume 197, 10 January 2015, Pages 1-9
Journal of Controlled Release

Intrathecal injection of a therapeutic gene-containing polyplex to treat spinal cord injury

https://doi.org/10.1016/j.jconrel.2014.10.027Get rights and content

Abstract

Spinal cord injury (SCI) is a serious clinical problem that suddenly deprives patients of neurologic function and drastically diminishes their quality of life. Gene introduction has the potential to be effective for various pathological states of SCI because various proteins can be produced just by modifying nucleic acid sequences. In addition, the sustainable protein expression allows to maintain its concentration at an effective level at the target site in the spinal cord. Here we propose an approach using a polyplex system composed of plasmid DNA (pDNA) and a cationic polymer, poly{N′-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} [PAsp(DET)], that has high capacity to promote endosome escape and the long-term safety by self-catalytically degrading within a few days. We applied brain-derived neurotrophic factor (BDNF)-expressing pDNA for SCI treatment by intrathecal injection of PAsp(DET)/pDNA polyplex. A single administration of polyplex for experimental SCI provided sufficient therapeutic effects including prevention of neural cell death and enhancement of motor function recovery. This lasted for a few weeks after SCI, demonstrating the capability of this system to express BDNF in a safe and responsible manner for treatment of various pathological states in SCI.

Introduction

Spinal cord injury (SCI) is a serious clinical problem that suddenly deprives patients of neurologic function and drastically diminishes their quality of life [1], [2]. Initial mechanical trauma to the spinal cord is followed by secondary injury, which is the progression of tissue damage for several days due to delayed neural cell death around the original site of injury [3], [4], [5]. Because initial injury has already occurred before hospital arrival, the therapeutic target has been focused on attenuation of secondary injury. However, there are few pharmacological strategies showing solid evidence of therapeutic effectiveness in clinical settings. One reason for this is the complex pathophysiology of secondary injury such as inflammatory responses, a variety of molecules inhibiting regeneration, and a lack of trophic support [6], [7], [8], [9], [10]. In addition, the pathophysiology is constant or increased for several days. Thus, therapeutic molecules need to be effective and sustainable for various pathological states in secondary injury. However, it is not only difficult to choose adequate therapeutic molecules, but it is also difficult to maintain the concentration of the molecules at an effective level at the target site in the spinal cord [11].

Gene introduction has the potential to be effective for the treatment of SCI because various proteins can be produced just by modifying nucleic acid sequences. In addition, sustainable protein expression resulting from an introduced gene is apparently beneficial for regulating the complex pathophysiology of SCI. For this purpose, viral vectors such as herpes simplex virus vectors had been evaluated [12]. However, viral vectors still have problems surrounding their genotoxicity and immunogenicity, hampering their administration for acute and serious states of SCI [13], [14], [15].

A non-viral system that can introduce genes to express therapeutic molecules has high potential for the treatment of SCI. Since neural tissue is mostly composed of highly differentiated, non-dividing cells, the system needs to have a high level of safety so as not to impede long-term neural function after gene introduction.

In this context, we propose an approach using a polyplex system composed of plasmid DNA (pDNA) and cationic polymers [16], [17]. The polyplex system can protect pDNA from nuclease attack and increase cellular uptake through electrostatic interactions between a cationic polyplex and the anionic cell membrane. One of the critical barriers in achieving good gene expression is the inefficient translocation from endosomes to the cytoplasm. The capacity of polymers to buffer acidic environments in the endosomes, typically represented by polyethylenimine (PEI) [18], has been intensely investigated to increase the efficiency of translocation, although the toxicity of the polymers has hampered their application for therapeutic purposes [19]. Our original cationic polymer, poly{N′-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} [PAsp(DET)] [20], [21], effectively solves the safety issues by self-catalytically degrading within a few days, minimizing the cumulative toxicity caused by the polymers remaining in the cells [22]. Besides its high capacity to promote endosome escape, it was revealed by a pharmacogenomic analysis that PAsp(DET) did not alter the expression profiles of endogenous genes after introduction into cells, confirming the long-term safety of PAsp(DET) in not affecting innate cell function [23], [24].

The purpose of this study is to investigate the availability of the polyplex system for the treatment of SCI. As a proof-of-concept study, we used Brain-derived neurotrophic factor (BDNF) as a therapeutic agent. BDNF is a member of neurotrophins, which have gained much attention for exerting diverse effects as a trophic support in treating SCI [25], [26]. In animal studies, recombinant BDNF protein showed therapeutic effects by enhancing neural cell survival and axonal regeneration in SCI by intrathecal infusion [27], [28]. However, multiple or continuous administrations using a catheter are usually required to maintain effective concentrations of BDNF protein at the injured site, and this would cause complications such as scar formation at the catheter tip, which can lead to infusion failure and damage to the spinal cord caused by the catheter itself [29], [30].

In this study, we applied BDNF-expressing pDNA for SCI treatment by intrathecal injection of PAsp(DET)/pDNA polyplex. As shown later, a single administration of polyplex provided sufficient therapeutic effects including prevention of neural cell death and enhancement of motor function recovery. This lasted for a few weeks after SCI, demonstrating the capability of this system to express BDNF in a safe and responsible manner for treatment of various pathological states in SCI.

Section snippets

Materials

Plasmid DNA (pDNA) encoding luciferase (pGL4.13: Promega, Madison, WI, USA) and brain-derived neurotrophic factor (BDNF) (pUNO1-hBDNFa: InvivoGen, San Diego, CA, USA) were amplified in competent DH5α Escherichia coli and purified using NucleoBond Xtra EF (Nippon Genetics, Tokyo, Japan). The pDNA concentration was determined by reading the absorbance at 260 nm. Linear polyethyleneimine (LPEI) (Exgen 500, in vivo; MW = 22,000) was obtained from MBI Fermentas (Burlington, ON, Canada). Lipofectamine

Evaluation of transgene expression after intrathecal injection of pDNA/PAsp(DET) polyplex and other carriers

The gene introduction capacity and safety was evaluated by administering luciferase-expressing pDNA using cationic polymers (PAsp(DET) or LPEI), lipid (Lipofectamine), or naked pDNA. For each condition, an identical amount of pDNA was administered into normal mice by intrathecal injection from the interlaminar space between L4 and L5, followed by evaluation of luciferase expression by IVIS™ Imaging System.

One day after administration, the luciferase expression was well detected for polyplexes

Discussion

The aim of this study was to demonstrate the feasibility of the PAsp(DET)/pDNA polyplex system which can introduce genes with high efficacy and safety for the treatment of SCI. While a number of therapeutic agents including BDNF have been identified for SCI, there remains a severe problem of how to deliver these agents to the target region of the injured spinal cord. In the case of peptides and proteins, the effects tend to be too short due to limited duration in the tissues. Therefore,

Acknowledgments

This work was financially supported in part by JSPS KAKENHI Grant-in-Aid for Scientific Research (B) (grant number 24300170 (K.I.)), the Center of Innovation (COI) Program and the S-innovation program from Japan Science and Technology Agency (JST), and the JSPS Core-to-Core Program, A. Advanced Research Networks. We thank Ms. Katsue Morii, Satomi Ogura, Asuka Miyoshi, and Sae Suzuki (The University of Tokyo) for technical assistance.

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