Non Viral Vectors in Gene Therapy- An Overview

Non-viral vectors are simple in theory but complex in practice. Apart from intra cellular and extracellular barriers, number of other challenges also needs to be overcome in order to increase the effectiveness of non-viral gene transfer. These barriers are categorized as production, formulation and storage. No one-size-fits-all solution to gene delivery, which is why in spite of various developments in liposome, polymer formulation and optimization, new compounds are constantly being proposed and investigated. In this review, we will see in detail about various types of non-viral vectors highlighting promising development and recent advances that had improved the non-viral gene transfer efficiency of translating from “Bench to bedside”.

Gene therapy is defined as the procedure used to treat or improve the health condition of the patient by modifying the patient’s cells genetically [1]. It provides an unique approach to treat both inherited and acquired diseases by delivering a therapeutic gene material and its associated regulatory elements into the nucleus; in order to correct the loss of function caused by mutation or to express the deficient gene product at physiologic levels [2]. It is well documented that almost all human diseases occur due to defect in either a single gene or set of genes due to mutation.

Gene therapy is considered as an alternative for enzyme /protein replacement therapy. The disadvantages like in vivo clearance and manufacturing cost faced by the replacement therapy makes gene therapy a potential alternative for various rare genetic disorders.

In spite of various methods or types of gene therapy, the therapy starts with the identification of mutant gene which is responsible for the cause of the disease. The next step is cloning the identical healthy gene. This is called therapeutic gene or transgene. The therapeutic gene is tailored to the need i.e. to augment or suppress or repair. Once the therapeutic gene is produced it is loaded in a vehicle called vector. The function of the vector is to deliver the therapeutic gene to the patient target cell. After the vector reaches the target cell, it delivers the genetic material to the nucleus. In the nucleus the genetic material gets integrated into DNA and corrects the defective or mutated gene. The most critical step in achieving gene therapy is choosing the vectors. Sequential key steps in gene therapy are shown in [Table/Fig-1].

[Table/Fig-1]:

Schematic illustration of key steps in gene therapy

Vectors are vehicles that ferry the genetic material into a wide variety of cells, tissues and whole organs. The optimal vector and delivery system depends on the target cells and its characteristics, duration of expression and the size of the genetic material to be incorporated in the vector [3,4].The present vectors used for gene therapy are broadly classified as Viral vectors, Non-viral vectors and engineered vectors. The non-viral vectors are Naked DNA, particle based and chemical based. They are administered by direct administration (plasmid DNA/Naked DNA)/ chemical /physical. Most of cardiovascular clinical trials use non-viral vectors as a mode of gene transfer.

The efficiency of transfecting host cells is relatively high with viral vectors compared to non-viral methods. The main drawbacks of using virus vectors are its immunogenicity and cytotoxicity. The first related fatality of gene therapy clinical trial was related to the inflammatory reaction to the viral vector (Adenovirus). Additional cause of concern over using viral gene transfer vehicle is the phenomenon known as insertional mutagenesis i.e. ectopic chromosomal integration of viral DNA disrupts the expression of tumour suppression gene or activates oncogene leading to the malignant transformation of cells. Due to its demonstrated reduced pathogenicity, low cost and ease of production, non-viral vectors have important safety advantage over viral approaches. The major advantage of using non-viral vectors is its bio-safety. However the application of non-viral gene transfer have been ignored for a long time in past because of their poor efficiency of delivery thereby low transient expression of their transgenes [2]. Non-viral vectors have drawn significant attention due to its less immunotoxicity. Use of non-viral vectors in clinical trials increased from 2004 to 2013 while that of viral vector saw significant decrease. Advances in efficiency, specificity, gene expression duration and safety led to an increased number of non-viral vector products entering clinical trials.

Unfortunately none of the currently available non-viral vectors fulfills ideal vector properties. This has led to research focus on suitable ideal vector delivery system.

The major technical limitations or critical steps in attaining a successful gene therapy are categorized into [5–8]: efficiency of vector transport and unloading into target cells, perseverance, activity, immune response, regulatory issues and ethical concerns and commercialization. These different stages pose a big challenge to gene therapy to be efficiently treating the disease. The cost of gene therapy creates an image that it is meant for the affluent. This was clearly evident with the first commercialized gene therapy Aliopogene tiparvovec for Lipoprotein Lipase deficiency in November 2013. The estimated treatment cost for LPLD gene therapy is about 1.6million/patient [9]. This tends to be the major hurdle in commercializing the gene therapy if proven successful.

Non-viral vectors are generally used to transfer following types of nucleic acids [10,11].

I. Physical Methods: Gene therapy researchers are more attracted towards physical means of transferring gene material as it is simpler. These methods employ physical force to counteract the membrane barrier of the cells thus facilitating intracellular delivery of the genetic material.

1. Needle: The genetic material of interest is administered through a needle carrying syringe into tissue or systemic injection from a vessel. Without any carrier it is the simplest and safest method of gene transfer. Attractive candidate tissues are muscle, skin, liver, cardiac muscle and solid tumours. However, the efficiency is low due to rapid degradation by nucleases in serum and cleared by mononuclear phagocyte system [6,12,13].

2. Ballistic DNA: Particle bombardment, micro projectile gene transfer or gene gun are the other terms used for ballistic DNA. This method was first used as gene transfer technique to plants. The method is based on the principle of delivering DNA coated heavy metal particles by crossing target tissue at a certain speed. The sufficient speed is achieved by high voltage electronic discharge, spark discharge or helium pressure discharge. Gas pressure, particle size, dose frequency are the critical parameters in determining the efficiency of gene transfer. Gold, tungsten and silver are used as metal particles and they are typically 1 μm diameter. The major advantage of gene gun is precise delivery of DNA doses. It is most commonly used in gene therapy research in ovarian cancer [6,12,14].

3. Electroporation: The other terms used for electroporation are gene electro injection, gene electro transfer, electrically mediated gene therapy, electro gene transfer. Applying an electric field that is greater than the membrane capacitance will cause charges of opposite polarity to line up on either side of cell membrane thus forming a potential difference at a specific point on the cell surface. As a result membrane breakdown form a pore and allows the molecule to pass. Pore formation occurs in approximately 10 nanoseconds. The pore of the membrane can be reversible based on the field strength and pulse duration. If it is reversible cells remain viable, otherwise cell death results. Irreversible electroporation is used in cancer treatment to destroy cancer cells. The permeability of the membrane to the gene transfer is controlled by the amplitude and duration of pulse. Currently used field strength are either high field strength [>700V/cm] or low field strength {<700V/cm} with short pulses (microseconds) or long pulses (milliseconds). Target tissue determines this combination of variables. Generally cancer cells require low field strength with long pulse, whereas muscle cells need short pulse with high field strength. Electroporation has emerged as a reliable physical method for delivering plasmid DNA. The therapy can be delivered by intradermally, intramuscularly or as intratumoural [6,12,14-16].

4. Sonoporation: Sonoporation is a noninvasive site specific technique which utilizes ultrasound wave to temporarily permeablize the cell membrane to allow cellular uptake of DNA. Genetic material of interest is incorporated within micro bubble and administered into systemic circulation. This is followed by external application of ultrasound. The ultrasound wave’s cavitate the micro bubble within the microcirculation of target tissue, produces bio effects that result in deposition of targeted transfection of therapeutic gene. Micro bubbles are composed of gas filled core [air/nitrogen/inert gas] with high molecular weight such as per fluorocarbon or sulfur hexafluoride. The outer shell consists of biocompatible compounds like lipids, proteins or synthetic biopolymers. Micro bubbles resemble red blood cells in circulation (mean diameter of 2-4 μm). Sonoporation technique is generally used in brain, cornea, kidney, peritoneal cavity, and muscle and heart tissues. The gene products are paired with micro bubbles in any of following three ways [17].

5. Photoporation: This physical method utilizes single laser pulse to generate transient pores on a cell membrane to allow DNA to enter into the cell. Focal point and pulse frequency of the laser controls the efficiency. It is claimed that the level of transgene expression is identical to that of electroporation. This technique lacks documented evidence [18].

6. Magnetofection: It is based on the hypothesis of magnetically targeted drug delivery. The technique is based on coupling therapeutic gene to magnetic nanoparticle. This complex is introduced in the cell culture. The field gradient produced by rare, earth electromagnets which are placed below cell culture increases sedimentation of complex and increases transfection speed. In case of in vivo, the therapeutic gene-magnetic particle complex is administered intravenously. Using strong high gradient external magnets, the complex is captured and held at the target. The genetic material is released by enzymatic cleavage of cross linking molecule, charge interaction or degradation of the matrix. This technique is mostly used in invitro research for transfecting primary cells and cells that are difficult to transfect by other means [6,19,20].

7. Hydroporation: It is also called as hydrodynamic gene transfer. The technique uses hydrodynamic pressure to penetrate the cell membrane. Hydrodynamic pressure is created by injecting large volume of DNA solution in a fraction of time. This creates increased permeability of capillary endothelium and forms pores in plasma membrane encircling parenchyma cells. The therapeutic gene of interest can reach the cell through these pores and these membrane pores are closed later thus keeping the genetic material inside the cell. This technique is most commonly used for gene therapy research in hepatic cells [12,21].

8. Mechanical Massage: It is based on the hypothesis that mechanical massage of liver generates transient membrane defects for a few minutes, which facilitates plasmid DNA to enter into hepatic cells by diffusion. But it was highly debatable and studies are scarce in using mechanical massage as a mode of gene transfer [15].

II. Chemical Carriers: Chemical vectors are broadly classified into inorganic particles, lipid based, polymer based and peptide based. They are generally categorized as [15,22].

Chemical non-viral nucleic acid delivery systems are generally DNA/Cationic lipid (Lipoplexes), DNA/cationic polymer (Polyplexes) and DNA/cationic Polymer/cationic Lipid (Lipopolyplexes)[10].

A. Inorganic particles [6]: They are generally nanoparticles that can be engineered by varying in size, shape and porosity in order to escape from reticulo endothelial system or to protect an entrapped molecule from degradation. Calcium sulphate, silica, gold, magnetic compounds, quantum dots. Carbon nanotubes, fullerenes, supra molecular system are most studied in this category.

1.Calcium phosphate: Calcium phosphate particles were the first ones to be used in this system. It is biocompatible and biodegradable. Calcium plays a vital role in endocytosis and has the advantage of being readily absorbed and it poses high binding affinity. However calcium phosphate nano crystals grow with time reducing its capacity to store. This is later overcome by adding magnesium.

2.Silica: It is the major component of widely used materials like sand and glass by humans. Its relative ease of functionalizing makes it attractive to use as gene delivery vehicle. The most commonly used silica as gene delivery agent is obtained by functionalizing nanoparticles with amino silicanes due to its low toxicity. Its decreased delivery efficiency in the presence of serum containing media due to the interaction between serum proteins is a major limiting factor.

3.Gold: The properties like ease of preparation, unlimited surface characterization and inert nature attracted researchers towards gold nanoparticles. Gold nanoparticles have strong absorption of light near infra-red region. The near infra-red light can penetrate deeply into tissues. Modifying the surface of gold with DNA can be used to transfect the cell by using photo thermal effect. Thermal denaturation induced by photo thermal effect helps to control the release of gene. Studies had proved that transfection efficiency with gold is comparable to lipoplexes comparatively with lower toxicity in vitro. However, major concern is its high chemical stability, so it is not easily dissolved in cell resulting in accumulation in cell which may harm the cell growth.

Magnetic nanoparticles (supermagnetic iron oxide mostly magnetite), fullerenes (soluble carbon molecules), carbon nanotubes (cylindrical fullerenes), quantum dots (semi conduction nanomaterial) and supramolecular systems all claimed some promising result in invitro and animal models. Surface of these inorganic nanoparticles can be coated to facilitate DNA binding. The hypothesis is that small particle size can efficiently by pass most of the physiological and cellular barriers and produce higher transfection efficiency. Still studies require on long-term safety, surface functionalization effect of type, size, and shape on transfection efficiency to accelerate their clinical application.

i. Cationic lipids: Hundreds of lipids have been developed for gene transfer. All of them share the common structures of positively charged hydrophilic head and hydrophobic tail with linker structure that connects both. The positively charged head group binds with negatively charged phosphate group in nucleic acids and form uniquely compacted structure called lipoplexes. Transfection efficiency depends on overall geometric shape, number of charged group per molecules, nature of lipid anchor and linker bondage. Lipoplexes due to their positive charge electrostatically interact with negatively charged glycoproteins and proteoglycans of cell membrane which may facilitate cellular uptake of nucleic acids. The positively charged lipids surrounding the genetic material help it to protect against intracellular and extracellular nucleases. However the problem lies with surface charge, this reduces the half-life of lipoplexes circulation in blood limiting its utility not beyond vascular endothelial cells. Neutral polymer like polyethylene glycol (PEG) is used as surface shielding to overcome the excessive charge and to prolong the half-life. Though considered to be of low toxicity, lipoplexes become cytotoxic beyond 3:1 ratio of lipid: DNA [6,12,15,20,23–25].

ii. Lipid Nano Emulsions: Lipid emulsion is a dispersion of one immiscible liquid in another stabilized by emulsifying agent. They are particles of around 200nm comprises of oil, water and surfactant. Lipid nano emulsion is considered to be superior to liposomes in scaling up, and stability, thus making it more serum resistant. The data shows they are less toxic than liposomes [6].

iii. Solid lipid nanoparticles: Solid lipid particles are made from lipid are solid at both room temperature and body temperature. It has advantages of both cationic lipids and lipid nano emulsions. It is shown that cationic solid lipid nanoparticle can effectively protect nucleic acid from nuclease degradation. It is currently the choice of delivery system for SiRNA [6].

iv. Peptide based: Peptide based vectors are considered advantageous over other non-viral vectors in tight compact and protecting DNA, target specific cell receptor, disrupting endosomal membrane and delivering genetic cargo into nucleus. Cationic peptides are rich in basic residues like lysine and/or arginine. Attaching peptide ligands to polyplex or lipoplexes enables vector to achieve specific target. Short peptide sequence taken from viral protein enables the vector to provide nuclear localization signal that assist transport of genetic material into nucleus. Due to these advantages peptides are frequently used to functionalize cationic lipoplexes or polyplexes [6].

v. Polymer based vectors [26]:

Cationic polymers mix with DNA to form nanosized complex called polyplexes. Polyplexes are more stable than lipoplexes. Polymers are categorized into natural and synthetic polymers.

Natural- proteins, peptides, polysaccharides.

Synthetic- Polyethylene mine (PEI), Dendrimers, and Polyphosphoesters.

a. Polyethylenimine (PEI): PEI is considered as a gold standard for in vivo and invitro gene transfer. Cationic polymers have high density amine groups which exert protein sponge effect that ultimately stops the acidification of endosomal pH. This leads to the influx of chloride within the compartment and increases the osmotic pressure, leading to the swelling and rupture of endosomal membrane.

b.Chitosan: It is a natural polymer based on cationic polysaccharide. One of the most studied non-viral vectors. It is nontoxic even at high concentrations. It is a linear cationic polysaccharide composed of glucosamine. The positive charge of chitosan electrostatically bind with negative charged DNA. On account of its mucoadhesive properties chitosan/DNA polyplexes are widely used in oral and nasal gene therapy. To effectively negotiate intracellular barriers, chitosan is conjugated to folic acid.

c. Poly (DL- Lactide) (PLA) and Poly ( DL-Lactide- co- glycoside) (PLGA): They are biodegradable polyesters undergo bulk hydrolysis thus providing sustained delivery. The degradation products are removed by citric acid cycle. PLGA is approved by FDA as vehicle for protein delivery. Less than 10 μm in size, they are easily phagocytosed by antigen presenting cell and inducing immune reaction

d. Dendrimers: Dendrimer molecules are symmetrical in size and shape with terminal group functionality. It binds to genetic material when positively charged peripheral groups interact with nucleic acids in physiological pH. due to nanometric size it can interact effectively with cell membranes, organelles, and proteins. The terminal amino group and positive charge density determine the toxicity profile.

e. Polymethacrylate Polymethacrylate are vinyl based polymer able to condense polynucleotides into nanometer size particle. But the transfection is limited due to their low ability to interact with membranes.

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