Review Article, J Genet Disor Genet Rep Vol: 7 Issue: 3

Non-Viral Vectors for Cystic Fibrosis Therapy: Recent Advances

Faisal Qaisar, Anum Habib, Noor Muhammad and Zia ur Rehman^*

Department of Biotechnology and Genetic Engineering, Kohat University of Science and Technology, Kohat 26000, Khyber Pakhtunkhwa, Pakistan

*Corresponding Author : Zia ur Rehman
Department of Biotechnology and Genetic Engineering, Kohat University of Science and Technology, Kohat 26000, Khyber Pakhtunkhwa, Pakistan
Tel: +92 3337757504
E-mail: [email protected]

Received: June 28, 2018 Accepted: October 19, 2018 Published: October 26, 2018

Citation: Qaisar F, Habib A, Muhammad N, Rehman Z (2018) Non-Viral Vectors for Cystic Fibrosis Therapy: Recent Advances. J Genet Disor Genet Rep 7:3. doi: 10.4172/2327-5790.1000179

Abstract

Keywords: Gene therapy; Cystic fibrosis; Nanocarriers; Cationic lipids; Cationic polymers

Introduction

Gene therapy is the transfer of genetic material in to the host cells for the treatment and prevention of inherited diseases and cancers. Since its time of discovery [1], gene therapy is been used for the treatment of genetic disorders through correction of mutated genes. Recent studies on other diseases such as cancers [2], autosomal dominant disorder, autosomal or X-linked recessive single gene disorder [3] and many other disorders had shown that they can potentially be treated through gene therapy approaches [4]. Monogenic, autosomal recessive disorders, for example cystic fibrosis, are good candidates for gene therapy trials. This can be attributed to the fact that only single gene needs to be corrected in a mutated cell to regain its normal function [5]. Since time, when CFTR gene was first discovered [6], many efforts have been made to develop gene delivery systems for somatic gene therapy of cystic fibrosis. Cystic fibrosis (CF) is a common life shortening genetic disorder, characterized by chronic lung inflammation resulting in reduced life expectancy to 45 years at best [7]. It is one of the most common autosomal recessive disorders among Caucasian population affecting 1 in 2000 individuals [8] and approximately 70,000 individuals worldwide each year [9]. The disorder is resulted from a mutation in CFTR gene located on chromosome 7q31.2. The gene encodes a protein of 1480 amino acid residues known as cystic fibrosis transmembrane conductance regulator (CFTR) [10,11] mainly located in the apical membrane of epithelial cells. Mutations in CFTR gene disrupt the cAMP (cyclin adenosine monophosphate) regulated chloride channel formed by CFTR protein and also interfere with the regulation of other ion channels [12]. This results in an imbalanced movement of ions and water across the airway epithelium thus leading to the accumulation of sticky mucus, chronic bacterial infection and inflammation of epithelial cells. Subsequently, these cause clinical aberrations and altered phenotype [13-16]. Presently, over 1500 mutations have been identified in CFTR gene [17] and are categorized into 6 classes shown in Table 1 [18-19].

Table 1: Six classes of mutati

ons in CFTR protein that lead to CF phenotype [20-23].

The current review will first generally highlight various approaches that can be applied in the gene therapy of CF. The later part of the review will mainly focus on nonviral methods, particularly, cationic lipids and polymers that are currently used for CF therapy. Additionally, various extracellular and intracellular barriers that these nonviral vectors have to cross before they can show their therapeutic effects are highlighted. Finally summarizes the various recent biochemical approaches used to cross these barriers efficiently. Finding ways to increase the intracellular delivery and subsequently, gene transfer efficiency of cationic nanocarriers will allow us to use them for therapeutic purposes of various inherited disease as well as cancers.

Gene Therapy for Cystic Fibrosis

Cystic fibrosis is widely regarded as a model genetic disorder for the gene therapy studies mainly because of four reasons; (1) it is a monogenic disorder (2) it is a recessive disorder in which heterozygotes are phenotypically normal (3) lungs are mainly affected that are comparatively easily accessible for treatment (4) it is a progressive disease with a nearly normal phenotype at birth, offering a therapeutic window. It is suggested that 5-10% of normal functioning CFTR gene expression is required for the treatment of disease [24]. However, it is not clear that whether majority of the airway epithelial cells have to express 5-10% CFTR function or just a small amount of cells expressing higher levels of CFTR would be sufficient for the treatment.

Nucleic acid fragment encoding CFTR protein should be transferred into the affected cell in order to regain its normal function. For the purpose, naked DNA have been delivered directly into the cells [25]. However, naked DNA molecules have several problems associated with them, when used for the therapy of CF. For example; they are larger in size and hydrophilic in nature which severely hampers their intracellular delivery. In addition, they are easily degraded by nuclease enzymes [26,27]. Therefore, people in the gene therapy field have always been interested in the development of ideal nanocarrier systems that ensure both, efficient delivery of CFTR gene into the target cells as well as maximum protection against both intra and extracellular enzymes.

Various types of vectors have been used in clinical trials to deliver CFTR cDNA into cells and in vivo (CFTR gene is 6.5 kb which can be reduced to 4.45 kb by using its cDNA [28]). These can broadly be grouped into viral vectors and nonviral vectors. In both cases plasmid DNA is used having three important components including; complementary CFTR DNA (cDNA), an upstream 5` promoter region which regulates transcription of gene of interest and downstream 3` terminator region which influences RNA splicing, polyadenylation and post-translational processing.

Therapeutic applicability of a particular vector is determined by numerous factors including but not limited to: vector carrying capacity, its efficiency, associated immunogenicity and possible mutagenesis. Viral vectors have higher efficiency of carrying nucleic acid fragments; however, their applicability is largely hampered by their higher immunogenicity, possible mutagenesis and potential risks in immune compromised individuals. Nonviral vectors, on the other hand, are lesser immunogenic and can carry large DNA fragments, however, their transfection efficiency is lower compared to their viral counterparts.

Barriers for Cystic Fibrosis Gene Therapy

In order to deliver their payload and consequently show their effects, the vectors have to cross various anatomical and cellular barriers in efficient manner. Extracellular matrix which surrounds the epithelial and endothelial cells lining are regarded as anatomical barriers while plasma membrane, endosomal membrane, cytoplasmic constituents and nuclear membrane are cellular barriers. Collectively, these two significantly affect the overall efficiency of gene transferring systems. For example, in blood circulation, DNA loaded nanocarriers are cleared by phagocytes such as Kupffer cells in liver and macrophages in the spleen. Moreover, nucleases present in blood and extracellular matrix can also degrade the unprotected nucleic acid fragments. Furthermore, the situation is worsened in case of CF, where a thin mucus layer covering the airway epithelium in lungs, provides an additional barrier. Although, the main role of mucus layer is to trap invading foreign particles, it significantly lowers the efficiency of gene delivery systems. Situations are further aggravated in the later stages of cystic fibrosis patients when lung airways are filled with sputum (mixture of saliva and mucus) thus further lowering the transfection efficiency of nanocarriers [29].

Among cellular barriers, cell membrane is a first barrier for various nonviral nanocarriers. Most of the nonviral nanocarriers exploit various cellular endocytic mechanisms to get entry into the cells, although, alternate mechanisms have also been suggested. For example, direct fusion of nanocarrier with plasma membrane, injecting its genetic payload into the cell, have been suggested [30]. Once inside the cells, through either of the endocytic mechanisms, the nanocarriers are enclosed by endosomal membrane and thus provides an additional barrier to the successful transfection process. Several escape mechanisms have been suggested for the release of macromolecules from endosomes. Nanocarriers that are unable to escape from endosomes are eventually digested in lysosomes thus further hampering their transfection efficiency. Although, endosomal escape often leads to the release of free nucleic acid fragments, alternatively, escape of entire complex consisting of nucleic acids and nanocarriers into the cytoplasm has also been suggested [31,32]. Depends on the type of application and nucleic acid fragments, they should either stay free in cytoplasm in order to bind with target RNA (in case of siRNA etc.) or they should travel to the nucleus for efficient gene expression [33]. Cytoplasm by itself is greatly studded by huge number of proteins, nucleic acid digesting enzymes and network of cytoskeleton. Therefore, transport through cytoplasm is another huge task that a nanocarrier has to achieve. Nuclear envelope is another fundamental barrier that nanocarriers have to cross. The double membrane of nucleus greatly affects the transfection efficiency of nanoparticles. Overall this implies that gene therapy is a multistep process and nanocarriers have to efficiently cross various barriers in order to show their therapeutic effect (Figure 1). In following sections we will first briefly summarize various approaches adopted to efficiently cross these barriers. In later sections, we will discuss, exclusively, various chemical modifications made in nanocarriers to cross various cellular barriers in efficient manner.

Gene Therapy Systems

There are two essential components in current gene therapy protocol: 1) a therapeutic gene and 2) a vector system that delivers it to the target tissues or cells. Although an effective therapeutic gene construct with proper regulatory element is equally important, much efforts have been made to develop an efficient and safer vector system. Various vector systems used for gene delivery can be broadly grouped into viral and non-viral vectors [24,34].

Viral vectors used in gene therapy

In most of currently available gene therapy tools, various viral vectors are predominant. Genetically altered viral vectors, in which amplification potential is removed, are used. Due to their intrinsic infection ability, modified viral vectors have enhanced efficiency and in case of insertion vectors, long term expression of desired gene. Recombinant viruses such as adenovirus, adeno-associated virus, lentivirus and helper-dependent adenovirus have been applied for gene delivery in cystic fibrosis (Table 2) [35,36].

Table 2: Various viral vectors that can be used for gene therapy of cystic fibrosis.

Adenovirus vector: Adenovirus, used as a vector, has been isolated from a vast variety of species and has more than hundred serotypes. Among adenoviruses, serotype 2 and 5 are mostly used for gene delivery in cystic fibrosis patients. This can be attributed to the facts that humans are mostly exposed to these serotypes implying that they have higher gene-transfer efficiency [37]. Additionally, it also has the capacity to deliver large DNA fragments which is prerequisite in case of CFTR gene. Furthermore, since adenovirus serotype 2 and 5 have lower tissue specificity, until and unless targeted, they can be used to deliver genes to wide range of tissues [37]. However, due to severe host immunological responses and its ability to cause serious side effects in patients, the use of adenovirus vector in CF gene therapy is largely limited [38-40].

Adeno-associated viral vector: Adeno-associated viral vector are safer compared to adenovirus vectors since they are lesser toxic and are also replication deficient [41]. Moreover, AAV also has the ability to specifically integrate its genome into host DNA, therefore, it can be used to deliver gene to the targeted site. However, their main drawback in cystic fibrosis gene therapy is that they can carry only a small piece of nucleic acid (up to 4.8 kb) into the host genome which is not desired in case of CF [42,43].

Helper-dependent adenoviral vector: Helper-dependent adenoviral vector (HdAd) is consisted of two vectors, the helper vector and another vector. The helper vector contains viral genes required for replication and another vector consists of several fragments including: gene of interest ends of viral genome and packaging recognition signal. HdAd is superior version of adenovirus in which many limitations have been removed. Higher packaging capacity, lower immunogenicity and reduced toxicity are advantages of using HdAd vectors in gene delivery [44-46].

Lenti viral vector: Lentivirus is a subclass of retroviruses having higher efficiency of gene delivery in both dividing and non-dividing cells. Moreover, they have the greater capacity to deliver larger (8 kb) nucleic acid fragments. Furthermore, their ability to stably express gene of interest in cells and lower immunogenicity make them suitable candidates for gene therapy of cystic fibrosis [47-50].

Overall, recombinant viral vectors are effective tools in gene therapy for cystic fibrosis due to their higher transfection efficiencies. However, there are still many problems associated with viral vectors including (1) difficulty in production (2) increased immune response (3) limited packaging capacity (4) mutagenesis due to random insertion [51]. These all, lowers the therapeutic applicability of viral vectors for the cystic fibrosis.

Nonviral Gene Delivery Methods

Nonviral gene delivery systems exploit all other methods except viruses for the delivery of genetic materials into the cells. These include both physical and chemical methods. Electroporation, gene gun, needle injection etc. are regarded as physical methods, while cationic lipids and polymers in addition to some others are grouped into the chemical methods.

Table 3,4 provides an overview of various physical and chemical methods that can potentially be applied in CF gene therapy.

Table 3: Various physical approaches used for gene therapy.

Table 4: Various chemical approaches used for gene therapy.

Physical methods

Several serious issues have been shown to be associated with viral vectors when used in gene delivery trials including their: potential immunogenicity and cytotoxicity, insertional mutagenesis, toxin production and limited nucleic acid packaging ability [32,82]. Alternatively, physical methods including naked DNA delivery through needle injection, gene gun and electroporation have been suggested as substitutes to the viral methods. Easy manufacturing, storing and potential safety benefits potentiate their application in cystic fibrosis therapy.

Needle injection: Genes of interest are delivered to the target site through needle injection, which are manipulated using atomic force microscope. The method has higher transfection efficiency and apparently there are no irreparable damages. In addition, since the technique does not utilize any vector for the nucleic acid transfer, it is widely regarded as one of the simplest and safest methods of gene transferring. However, there are still some issues associated with them, for example free nucleic acids are degraded intramuscularly, thus largely reducing its transfection efficiency [27]. Moreover, transfection achieved through this method is also limited to the needle surroundings.

Gene gun: In this approach, the gene of interest is attached to heavy metal and then transferred to a specific site with speed achieved through helium pressure discharge [67,83]. Delivery of precise DNA doses to the targeted area is one of the main advantages of this system. However, greater cell damage during the process is the huge disadvantage of the method, hampering its applicability in CF therapy.

Electroporation: Electric pulses are applied in electroporation technique to generate transient pores in the plasma membrane so that the gene of interest can be transferred into the target cells. The method is efficient and reproductive if the associated parameters are optimized [84]. However, like other methods, electroporation also has limitations including but not limited to: huge cell damage due to high voltage, lower transfection rate and limited accessibility of electrodes to internal organs [64,84].

The above mentioned physical methods used in gene delivery of cystic fibrosis minimize various side effects associated with viral vectors such as potential immunogenicity and carcinogenesis etc. In addition, these methods deliver gene of interest to single or multiple targeted cells at specific site effectively thus minimizing the risks of dispersion of transfected genes to other areas. However, in addition to these advantages, there are some problems linked with physical methods. For instance, in case of a gene that needs to be transferred to the nucleus, the transfection efficiency is significantly low as the nuclear membrane and potential nuclease degradation are the main limiting factors. In addition, higher cell damage, difficulty in large scale delivery and necessity of costly instruments critically limits the use of physical methods in gene therapy of cystic fibrosis.

Chemical-based nonviral vectors

Chemical-based nonviral gene delivery systems use synthetic or natural compounds for transferring of genes to the targeted area. Advantages of these chemical based vectors is that they are lesser toxic and lesser immunogenic compared to their viral counterparts. Additionally, they can be easily targeted to some specific cells or tissues by decorating their outer covering with cell or tissue specific ligands. Furthermore, they can be easily synthesized and importantly, due to their lower toxicity they can be repeatedly administered with minimum off-target effects.

Cationic lipids: Cationic lipids-based gene transfer was first achieved by Felgner et al. [85] in 1987. Afterwards, many lipid-based gene delivery systems have been developed all sharing the same basic structure consisting of hydrophilic head, hydrophobic tail and a linker between them. The positively charged head groups are necessary for binding of lipids to the negatively charged phosphate groups in nucleic acids. Primary, secondary, tertiary amines and quaternary ammonium salts are predominantly used as head groups. Alternatively, several other head groups: for example guanidine and imidazole have also been suggested. Eighty nine pyridinium based compounds, with primary compound named 1-(2.3-dioleoyloxypropyl)-2, 4, 6-trimethyl pyridinium-lipid having a pyridinium group as its head group instead of amine or quaternary ammonium groups have been developed [86,87]. On the other hand, most of the hydrophobic tails are made up of aliphatic chains, cholesterol or other types of steroids rings. On the contrary, ether, carbamate and amide bonds are frequently used as linkers connecting hydrophilic heads with hydrophobic tails. Some commonly used cationic lipids in gene therapy with modifications are summarized in Table 5. Moreover, recent modifications made in these cationic lipids for enhanced gene delivery are diagrammatically shown in Figure 2.

Table 5: Some commonly used cationic lipids for gene delivery with some recent modifications made in them to enhance their transfer ability.

Liposomes and lipoplexes: Lipid platforms are suitable for carrying nucleic acid fragments by encapsulating them in liposomes core. Liposomes are easily prepared using reverse-phase evaporation. The method involves amphiphilic lipid hydration which leads to the multilamellar vesicles formation. Subsequently, the multilamellar vesicles are converted into unilamellar vesicles upon sonication. Liposomes when mixed with negatively charged nucleic acid fragments, form lipoplexes. Positively charged lipoplexes surrounds negatively charged DNA, due to electrostatic interactions, thus providing protection against extracellular and intracellular digestions. Transfection efficiency depends, among other factors, on the structure of liposomes (which includes geometric shape, number of charged groups per molecule, lipid anchor nature and linker), charged ratio used for DNA-lipid complexes formation and properties of colipids [88].

Colipids: Most commonly used colipids are dioleoylphosphatidylethanolamine (DOPE) and cholesterol. These colipids often act as “helper” lipids facilitating cationic lipids in transferring nucleic acid fragments on larger extent. Although some lipids require DOPE for transfection there are also others, which have double fatty acid chain capable of forming bilayer, do not require helper lipids for transfection. The presence of DOPE reduces cationic lipid to DNA charge ratio which is required for maximal transfection. Moreover, its addition also reduces cytotoxicity of cationic lipids. Furthermore, DOPE has also been shown to have fusogenic property, facilitating lipoplexes in endosomal escape. Cholesterol, another colipids, on the other hand, has been suggested to stabilize cationic lipid membrane against destructive activity of serum components thus improving transfection (Figure 3) [88].

Disadvantages of cationic lipids: Cationic lipids carry large surface charge contributing to their very short blood circulation time [89]. Moreover, they can potentially form large aggregates which lead to their systemic elimination. This can be attributed to the facts that large positively charged aggregates interact strongly with negatively charged serum molecules and cellular components [90]. Lipoplexes, after injection, reaches to lungs cells in about 60 min. This also includes crossing of pulmonary vasculature, which offers a first passage effect. Consequently, pulmonary vascular endothelial cells and some airway epithelial cells are predominant cells that are transfected with lipoplexes [91-93].

Barriers based recent advances in lipoplexes: Lipoplexes have already been successfully used for the gene delivery to various cell lines both in in vitro and in vivo conditions. However, various problems have been shown to be associated with lipid based gene delivery systems. Among these, acute toxicity and triggered host-immune response are the major issues [93,94].

Recent approaches, for example, surface coating of lipoplexes with hydrophilic and charge neutral polymers such as chitosan have significantly reduced their nonspecific interactions thus leading to their reduced toxicity [95]. Moreover, multiple issues associated with lipoplexes have recently been resolved using modified form of lipidbased carrier system called Vaxfectin. Vaxfectin is synthesized by combining cationic lipid (GAP-DMORIE) with neutral phospholipid (DPyPE). The liposomes thus formed, have shown improved gene delivery as well as DNA-based vaccination to the targeted sites [96]. Additionally, surfaces of liposomes and PEI have also been decorated with cell penetrating peptides also referred to as Protein Transduction Domain (PTDs) to improve their gene delivery efficiency [97,98]. Currently, over 25 clinical trials, utilizing PTDs-mediated lipid based gene delivery systems are approved as safer nanocarriers for gene delivery [99].

Lipoplexes, have been shown to form aggregate and non-specifically adsorb to non-targeted tissues thus severely hampering their applicability in cystic fibrosis therapy. These can be attributed to overall massive positive charge present on their surface [100]. For the purpose, PEGylation of cationic lipoplexes have been shown to reduce their interaction with serum protein in blood stream and also helps them in avoiding their recognition by immune system[101,102]. Subsequently, these resulted in their increased circulation time and facilitated their targeted delivery of genes [103]. Alternatively, surface of cationic liposomes is also covered with polyanions which ultimately reduced their nonspecific interactions thus leading to their increased in vivo circulation [104].

Among various prerequisites for gene therapy, targeted gene delivery is fundamentally important. For the purpose, various modifications are applied. Surfaces of liposomes, for instance, are decorated with iron-saturated transferrin [77], folic acid [105,106] and RGD [78] for specific gene delivery. In addition, protamine sulphate, has recently been applied to condense genetic material encoding for desired protein. Condensed DNA thus obtained is subsequently encapsulated in PEGylated liposomes. In this case, Calf thymus DNA is used which provide extra negative charge thus facilitates the formation of compact nanoparticle in the core, while PEGylated liposomes provided surface protection against digestion and aggregation, improving overall transfection efficiency [107,108]. In agreement, decorating the surface of liposomes with MPG resulted in enhanced expression of HPV16 E7 gene in vitro [109]. In addition, a cardiac glycoside such as Strophanthidins, are also attached to the surface of liposomes. Attachment of Strophanthidins is helpful in binding nanoparticles to the cell surface by targeting Na+/ K+ ATPase receptor. Subsequently, this improves delivery of nucleic acids to a number of cells originating from different organs such as lungs, prostate, ovary, breast, and pancreas [79].

Once inside the cells, the lipoplexes has to escape from endosomes. This implies that endosomal degradation and entrapment is among the major obstacles for the successful application of lipoplexes. Various modifications have been made in lipoplexes in order to promote their endosomal escape and the transfection efficiency. For example, Arukuush et al. [110] has recently modified lipoplexes with endosomal membrane penetrating peptide TP10, named as NickFects. The resulting complex can efficiently transfect a large variety of cell lines thus offering a potential applicability in CF therapy [110]. Notably, this modified particle appears not only efficient in crossing endosomal membrane but is also suggested to be useful for mammalian protein production system to express and produce recombinant proteins [110]. More recently tocopherol-based cationic lipids are prepared having pH sensitive dipeptide head groups. Introduction of cationic dipeptide head groups appreciably affects the physicochemical and biological properties of cationic lipids. Moreover, cationic head group of lipids are modified using imidazole group with pH buffering ability while using histidine as a building block in the preparation. The resulted nanocarrier has the ability to deliver nucleic acid with high efficiency and also reduces cytotoxicity [111].

Finally, the delivery of nucleic acids should reach to the nucleus, in order to replace mutated gene with normal functioning one. Lipoplexes are mostly decorated with NLS (nuclear localization signals), derived from short peptide sequences of viral proteins, in order to transfer gene to the nucleus [67]. Additionally, cell-specific modifications have also been made in lipoplexes in order to enhance their efficiency in particular types of cells. For instance, liposomes are modified with hyaluronic acid (HA), instead of PEG, for their applications in ocular gene therapy [112]. HA is a glycosaminoglycan, ubiquitously expressed in mammalian cells and have several sites of chemical modifications making it more attractive chemical in gene delivery applications. Electrostatic HA-coating increases intravitreal mobility of cationic nanocarriers without affecting its cellular uptake and transfection efficiency [113]. In addition, arginine rich lipids are synthesized which were later on modified with guanidine or imidazolerich margin. The resulting lipoplexes have been shown to have increased DNA binding abilities, decreased cytotoxicity and nineteen folds higher transfection efficiency compared to unmodified lipoplexes [114].

Most of these lipoplexes have shown their increased applicability in specific types of cells, implying that CF affected cells can potentially be targeted using specific nanocarriers. Therefore, it is urgently required to synthesize lipoplexes keeping in mind the physicochemical properties of CF cells.

Cationic polymers: Cationic polymers have been extensively used for gene delivery mainly because of their versatility and easy manipulation. Although, mostly cationic polymers are used for gene therapy, other types such as hydrophobic biodegradable polymers and negatively charged alginate have also been applied [115,116]. Polymers differ from cationic lipids both in structure and chemical composition, lacking hydrophobic tails and are completely soluble in water. Moreover, they interact totally differently with DNA and their intracellular behaviors also vary from their cationic lipids counterparts [117,118]. Synthetic polymers are attractive tools for gene therapy because of their easy and low cost production as well as their availability in wide range of structures [118]. Importantly, when required, they can easily be modified. Furthermore, modified polyplexes reduces various side effects including cytotoxicity and nonspecific binding, indicting their utility in gene delivery systems with higher transfection efficiency [119].

Polyplexes: Polyplexes are formed due to electrostatic interactions between cationic polymers and anionic DNA. They are more stable and relatively smaller in size as compared to their lipoplexes counterparts [120]. Among various synthetic polymers, Polyethylenimine (PEI) is fundamentally important for various gene therapy applications including CF. This can partially be attributed to the fact that PEI is rich in amine groups which are unprotonated at neutral pH. Upon acidification in endosomal compartments, excessive protonation of amine groups takes place leading to their swelling and subsequent endosomal escape [121,122]. Cytotoxicity and gene transfer ability of PEI depends, among many factors on, molecular weight of PEI, polymer structure (liner or branched), and ratio of amine group of PEI to phosphate of DNA (N/P ratio). For example, PEI with low molecular weight and optimum N/P ratio has been shown to be more efficient and lesser toxic compared to high molecular weight PEI [123]. Moreover, branched PEI have been shown to have high toxicity and low transfection efficiency compared to linear PEI [123,124]. In addition to PEI, poly(L-lysine) (PLL) is another cationic polymer can potentially be used for CF gene therapy. Along with physical properties similar to PEI, PLL also show has high transfection efficiency (Table 6) [125,126].

Table 6: Some commonly used polymers for gene delivery.

Disadvantages of Polyplexes: Polyplexes used in CF gene therapy systems have various issues associated with them thus limiting their therapeutic applicability. For example, branched polyplexes possess higher toxicity and lower transfection efficiency as compared to linear polyplexes[80,127]. Moreover, polyplexes have been shown to form large aggregates upon in vivo administration, further questioning their applicability in CF gene therapy [80]. Furthermore, non-targeted polyplexes bind to various non-specific areas thus showing potential off-target effects [128]. This can be attributed to their net positive charge, which can efficiently bind to the negatively charged cell surfaces of various cells [81]. Keeping in mind these multiple issues, various modifications have been made in these polyplexes to ensure their future applicability in CF therapy. Some of these recent modifications are summarized in the following section.

Recent advances in polymers: The fundamental properties of polymers which make them potential alternatives to viral vectors are their easier functionalization, protectability of cargo and biodegradability upon administration. In earlier trials, polymer like PLL (poly-L-lysine) was used as vector. However, its poor transfection efficiency seriously limits its in vivo applicability. This can be attributed to the fact that the characteristic amine-groups of PLL are positively charged at physiological pH. Consequently, this lowers the buffering capacity of PLL thus severely reducing its endosome escape ability [33]. Among various modifications of PLL, PEGylation is fundamentally important. It ensures both enhanced endocytosis and reduced cytotoxicity of PLL. PEGylated PLL used in trials of cystic fibrosis has already shown more promising results compared to its unmodified form [129]. Earlier, it has been shown that PLL tends to form aggregate upon administration to the cells resulting in huge cell toxicity and subsequent cell death [80]. More recently this problem was solved by combining PLL with palmic acid (PA). Mixing of PA with PLL not only prevent PLL from forming aggregates but also improve its transfection efficiency. In fact an increase in transfection efficiency due to addition of PA can be attributed to the fact that PA increases the interaction of nanocarriers with plasma membrane [130].

PEI (poly-ethyleneimine) is another widely used cationic polymer used for gene delivery. PEI is routinely used as transfection agent in in vitro experiments. However, their huge positive charge makes them cytotoxic and non-biodegradable [118] making their in vivo applications in question. Particularly, low molecular weight PEI (LMW PEI), which is routinely used, is less efficient as compared to high molecular weight forms of PEI(HMW PEI) [131]. Recently, a strategy has been developed in converting LMW PEI into HMW PEI. The idea is to allow the cells to convert the modified HMW PEI, after transfection, into segments that would be easily eliminated from the cells [132,133]. For the purpose, phenyl boronic acid (PBA) conjugated with sugar molecule is linked to LMW PEI, through cross linking, thus converting it into HMW PEI [134]. The resulting particles are then transferred into target cells where the bond between PBA and sugar is disrupted by acidic pH in endosomes or intracellular ATP thus leading to efficient release of genetic payload [135]. Additionally, in order to protect nucleic acid from nuclease degradation, PEI has been conjugated with polyamidoamines leading to enhanced efficiency [136].

Cell membrane is among the major barriers to the gene therapy of CF. For the purpose, surface of the PEI is decorated with cell penetrating peptides (CPPs). The basic amino acid present in the structure of CPPs hugely increases the intracellular delivery of Polyplexes [137]. Alternatively, a domain from herpes simplex virus, WT peptide has been attached to the surface of polyplexes leading to their increased cellular uptake [138].

PEI is proved to be an efficient gene delivery vehicle that triggers endosomal escape via pH buffering-effect. The so called proton-sponge effect results both in; endosomal escape and dissociation of cargo from the polymers [139]. However, cationic property of PEI can potentially induce cytotoxicity which hampers its therapeutic applicability. For the purpose, a promising approach is to adjoin anionic polymer with cationic PEI [140]. In agreement, incorporation of γ-PGA group to PEI resulted in enhanced gene delivery with minimal cytotoxicity [141].

After delivery to the target site, the efficiency of nanocarriers is also dependent upon the dissociation of polymer from the nucleic acid fragments. For example, early release may result in the degradation of nucleic acid by cytoplasmic nucleases while the late release will results in non-efficient gene expression [142]. Therefore, optimized conditions should be maintained in order to ensure enhanced nucleic acids release from polymers and subsequent higher gene expression [142]. Nuclear membrane is one of the fundamental barriers in gene therapy strategies which should be efficiently crossed by nanocarriers for the optimum gene expression [143]. In recent studies, a modification has been made in PEI by coupling it to large T-antigen peptide of SV40 which acts as NLS (nuclear localizing signals). Resulted PEI has shown improved nuclear transport and hence higher gene expression [144].

PPA (polyallylamine) is another polymer used for gene delivery to target cells. Although, currently used for various delivery applications, PPA still carries various issues that need to be addressed. For example, it is less efficient in both crossing the plasma membrane and escaping from endosomes. Moreover, it also shows increased toxicity once supplied to the cells [145]. These issues are recently solved by modifying PAA with bromoalkane derivatives. Inclusion of bromoalkane derivatives into PAA increases its hydrophobicity thus leading to its improved interaction with both cell and endosomal membrane. Subsequently, these greatly enhances the transfection efficiency of PAA [146].

Dendrimers are type of polymers having highly branched structures. PAMAM (polyamidoamine) is a type of dendrimer which is widely used for gene delivery because of its high transfection efficiency. However, toxicity is a major problem associated with PAMAM hampering its therapeutic applicability in CF therapy [147,148]. Recently, researchers have formulated various nitrogen-core poly(propyl etherimine) (PETIM) dendrimer-DNA formulations. Complexes thus obtained were investigated for their applications in clinical trials. Overwhelmingly, they showed lower toxicity and efficient gene delivery compared to their unmodified counterpart [149]. In addition to PETIM dendrimers, normal dendrimers have also been modified using tri-ethanolamine, which greatly increased its transfection efficiency [150].

PLGA (poly-lactic-co-glycolic acid) is biodegradable polymers used for the gene delivery mainly because of its stability and ability to protect DNA from degradation during in vivo application [132]. Nucleic acid fragments are encapsulated in PLGA using doubleemulsion solvent evaporation method. Lower cellular interaction and less efficient endosomal escape are the main problems faced by PLGA polymer during gene delivery process [151]. To solve these problems, recently a modification has been made in PLGA. The polymer is modified with biocompatible chitosan and other cationic components which increases its overall positive charge. This lead to the enhanced PLGA cellular interaction subsequently increasing its transfection efficiency [151-154].

Increasing the hydrophobicity of nonviral vectors enhances their interactions with cells thus enhancing their cellular uptake and subsequent transfection efficiency [155]. For example, synthesis of chitosan with a number of hydrophobic modifications improved its cellular uptake [156]. More recently, researchers have utilized trimethylated-chitosan, a chitosan based nanoparticle, for transferring nucleic acid into the cells. The modified complexes have shown high transfection efficiency even compared to PEI/DNA Polyplexes [157,158].

Besides to different modification made in the various already available polymers such as PLL, PEI, PLGA, chitosan, dendrimers, and PPA, other formulations have been made to develop novel polyplexes with improved transfection ability. For crossing cell membrane barrier effectively, membrane disruptive peptides, such as melittin, are attached on the surface of polymers. Moreover decoration of polymers with virally derived peptides such as TAT, Antennapedia and HGP have also showed promising results in crossing cell membrane. Overall these all resulted in polymers with improved gene delivery [159,160].

Release from endosome is a key step for the successive gene delivery because the nanoparticles that is unable to escape from endosomes and subsequently degraded there. In order to help nonviral vectors to escape efficiently from endosome, recently researches have ligated the KALA sequences with nonviral vectors. The KALA has been shown to facilitate endosomal escape by disrupting its membrane [161]. However, how these disruptions affects the cell physiology is a fundamental question that needs to be answered.

Nuclear membrane along with nuclear pore complex (NPC) is considered as selective barrier that severely limit the overall transfection efficiency of polyplexes for those nucleic acid fragments that need to be delivered into the nucleus. Recently Liashkovich et al. [162] have functionalized the surface of polyplexes with trans-1,2- cyclohexanediol (CHD) and importin β. Importin β has been shown to help nanocarriers in guiding towards NPC while CHD helps in leaking of NPC. Combined effects of both have greatly improved the overall efficiency of the modified Polyplexes [162].

Conclusion and Future Perspectives

Various strategies have been developed to deliver CFTR gene efficiently into target cells through carrier vectors. In recent years nonviral vectors have attracted much attention because of having the ability of gene delivery without the complication of immunogenicity and insertion mutation seen in viral vectors. In past few years the work continued in developing new nonviral vectors for gene delivery in cystic fibrosis. Further improvements are made to increase the efficiency and reduce toxicity of nonviral vectors are needed before clinical implication can be met. Many improvements are made in changing the composition of existent nonviral vectors to overcome the problems such as large surface charge, formations of aggregates, early clearances while other strategies are apply to improve gene delivery in cystic fibrosis by combining of nonviral vectors with different chemicals. It is imaginative that strategies that combine nonviral and viral vectors might be helpful to achieve more, efficient, long-lasting, and nontoxic gene delivery systems used for the treatment of cystic fibrosis.

Acknowledgment

Current study is a part of research projects funded by Higher Education Commission (HEC) Pakistan (No.20-3421/ NRPU/R&D/HEC/374 and NO: 21-185 SRGP/R&D/HEC/2015).

References

1. Friedmann T, Roblin R (1972) Gene therapy for human genetic disease? Science 175: 949-955.
2. Saadatpour Z, Bjorklund G, Chirumbolo S, Alimohammadi M, Ehsani H, et al. (2016) Molecular imaging and cancer gene therapy. Cancer Gene Ther 23: 1-5.
3. Hacein-Bey-Abina S, Hauer J, Lim A, Picard C, Wang GP, et al. (2010) Efficacy of gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 363: 355-364.
4. Banwait JK, Bastola DR (2015) Contribution of bioinformatics prediction in microRNA-based cancer therapeutics. Adv Drug Deliv Rev 81: 94-103.
5. Naldini L (2011) Ex vivo gene transfer and correction for cell-based therapies. Nat Rev Genet 12: 301-315.
6. Riordan JR, Rommens JM, Kerem BS, Alon N, Rozmahel R, et al. (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245: 1066-1073.
7. MacKenzie T, Gifford AH, Sabadosa KA, Quinton HB, Knapp EA, et al. (2014) Longevity of patients with cystic fibrosis in 2000 to 2010 and beyond: survival analysis of the Cystic Fibrosis Foundation patient registry. Ann Intern Med 161: 233-241.
8. Cuthbert AW (2011) New horizons in the treatment of cystic fibrosis. Br J Pharmacol 163: 173-183.
9. Cutting GR (2015) Cystic fibrosis genetics: from molecular understanding to clinical application. Nat Rev Genet 16: 45-56.
10. Pier GB (2012) The challenges and promises of new therapies for cystic fibrosis. J Exp Med 209: 1235-1239.
11. Bosch B, De Boeck K (2016) Searching for a cure for cystic fibrosis A 25-year quest in a nutshell. Eur J Pediatr 175: 1-8.
12. Lazrak A, Fu L, Bali V, Bartoszewski R, Rab A, et al. (2013) The silent codon change I507-ATC→ ATT contributes to the severity of the ΔF508 CFTR channel dysfunction. FASEB J 27: 4630-4645.
13. Patient Registry (2013) Cystic Fibrosis Foundation Annual Data Report 2013. Cystic Fibrosis Foundation.
14. Brennan ML, Schrijver I (2016) Cystic fibrosis: a review of associated phenotypes, use of molecular diagnostic approaches, genetic characteristics, progress, and dilemmas. J Mol Diagn 18: 3-14.
15. Cai ZW, Liu J, Li HY, Sheppard DN (2011) Targeting F508del-CFTR to develop rational new therapies for cystic fibrosis. Acta Pharmacol Sin 32: 693-701.
16. Collie JT, Massie RJ, Jones OA, LeGrys VA, Greaves RF (2014) Sixty‐five years since the New York heat wave: Advances in sweat testing for cystic fibrosis. Pediatr Pulmonol 49: 106-117.
17. Cystic Fibrosis Mutation Database (2015) Cystic Fibrosis Mutation Database, Statistics 2015. Sickkids.
18. Kreindler JL (2010) Cystic fibrosis: exploiting its genetic basis in the hunt for new therapies. Pharmacol Ther 125: 219-229.
19. Goetzinger KR, Cahill AG (2010) An update on cystic fibrosis screening. Clin Lab Med 30: 533-543.
20. Wang Y, Wrennall JA, Cai Z, Li H, Sheppard DN (2014) Understanding how cystic fibrosis mutations disrupt CFTR function: From single molecules to animal models. Int J Biochem Cell Biol 52: 47-57.
21. Accurso FJ, Rowe SM, Clancy JP, Boyle MP, Dunitz JM, et al. (2010) Effect of VX-770 in persons with cystic fibrosis and the G551D-CFTR mutation. N Engl J Med 363: 1991-2003.
22. Bompadre SG, Sohma Y, Li M, Hwang TC (2007) G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects. J Gen Physiol 129: 285-298.
23. Accurso FJ, Rowe SM, Clancy JP, Boyle MP, Dunitz JM, et al. (2010) Effect of VX-770 in persons with cystic fibrosis and the G551D-CFTR mutation. N Engl J Med 363: 1991-2003.
24. Sinn PL, Anthony RM, McCray Jr PB (2011) Genetic therapies for cystic fibrosis lung disease. Hum Mole Genet 20: R79-R86.
25. Ramamoorth M, Narvekar A (2015) Non viral vectors in gene therapy-an overview. J Clin Diagn Res 9: GE01-GE06.
26. Konstan MW, Ratjen F (2012) Effect of dornase alfa on inflammation and lung function: Potential role in the early treatment of cystic fibrosis. J Cyst Fibros 11: 78-83.
27. Al-Dosari MS, Gao X (2009) Nonviral gene delivery: principle, limitations, and recent progress. AAPS J 11: 671-681.
28. Tsui LC, Dorfman R (2013) The cystic fibrosis gene: a molecular genetic perspective. Cold Spring Harb Perspect Med 3: a009472.
29. Hida K, Lai SK, Suk JS, Won SY, Boyle MP, et al. (2011) Common gene therapy viral vectors do not efficiently penetrate sputum from cystic fibrosis patients. PloS One 6: e 19919.
30. Barichello JM, Kizuki S, Tagami T, Asai T, Ishida T, et al. (2011) Agitation during lipoplex formation improves the gene knockdown effect of siRNA. Int J Pharm 410: 153-160.
31. Erazo-Oliveras A, Muthukrishnan N, Baker R, Wang TY, Pellois JP (2012) Improving the endosomal escape of cell-penetrating peptides and their cargos: Strategies and challenges. Pharmaceuticals 5: 1177-1209.
32. Zhang Y, Satterlee A, Huang L (2012) In vivo gene delivery by nonviral vectors: Overcoming hurdles? Mol Ther 20: 1298-1304.
33. Mintzer MA, Simanek EE (2008) Nonviral vectors for gene delivery. Chem Rev 109: 259-302.
34. Griesenbach U, Alton EW (2013) Moving forward: cystic fibrosis gene therapy. Hum Mol Genet 22: R52-R58.
35. Biffi A, Bartolomae CC, Cesana D, Cartier N, Aubourg P, et al. (2011) Lentiviral-vector common integration sites in preclinical models and a clinical trial reflect a benign integration bias and not oncogenic selection. Blood 1: Blood-2010.
36. Arnberg N (2012) Adenovirus receptors: implications for targeting of viral vectors. Trends Pharmacol Sci 33: 442-448.
37. Wirth T, Ylä-herttuala S (2014) Gene therapy used in cancer treatment. Biomedicines 2: 149-162.
38. Brown NJ, Hirsch ML (2015) Adeno-associated virus (AAV) gene delivery in stem cell therapy. Discov Med 20: 333-342.
39. Freytag SO, Stricker H, Lu M, Elshaikh M, Aref I, et al. (2014) Prospective randomized phase 2 trial of intensity modulated radiation therapy with or without oncolytic adenovirus-mediated cytotoxic gene therapy in intermediate-risk prostate cancer. Int J Radiat Oncol Biol Phys 89: 268-276.
40. Morishita Y, Kobayashi K, Klyachko E, Jujo K, Maeda K, et al. (2016) Wnt11 gene therapy with adeno- associated virus 9 improves recovery from myocardial infarction by modulating the inflammatory response. Sci Rep 6: 21705.
41. Grieger JC, Samulski RJ (2012) Adeno-associated virus vectorology, manufacturing, and clinical applications. Methods Enzymol 507: 229-254.
42. Hirsch ML, Green L, Porteus MH, Samulski RJ (2010) Self-complementary AAV mediates gene targeting and enhances endonuclease delivery for double-strand break repair. Gene Ther 17: 1175-1180.
43. Vidovic D, Gijsbers R, Quiles-Jimenez A, Dooley J, Van den Haute C, et al. (2015) Noninvasive imaging reveals stable transgene expression in mouse airways after delivery of a nonintegrating recombinant adeno-associated viral vector. Hum Gene Ther 27: 60-71.
44. Van der Loo JC, Wright JF (2015) Progress and challenges in viral vector manufacturing. Hum Mol Gen 25: R42-R52.
45. Brunetti-Pierri N, Ng P (2016) Gene therapy with helper-dependent adenoviral vectors: lessons from studies in large animal models. Virus Genes 53: 684-691.
46. Ashtari M, Zhang H, Cook PA, Cyckowski LL, Shindler KS, et al. (2015) Plasticity of the human visual system after retinal gene therapy in patients with Leber’s congenital amaurosis. Sci Transl Med 7: 296ra110.
47. Zhou Q, Uhlig KM, Muth A, Kimpel J, Lévy C, et al. (2015) Exclusive transduction of human CD4+ T cells upon systemic delivery of CD4-targeted lentiviral vectors. J Immunol 195: 2493-2501.
48. Rees DC, Williams TN, Gladwin MT (2010) Sickle-cell disease. Lancet 376: 2018-2031.
49. Pala F, Morbach H, Castiello MC, Schickel JN, Scaramuzza S, et al. (2015) Lentiviral-mediated gene therapy restores B cell tolerance in Wiskott-Aldrich syndrome patients. J Clin Investig 125: 3941-3951.
50. Houghton BC, Booth C, Thrasher AJ (2015) Lentivirus technologies for modulation of the immune system. Curr Opin Pharmacol 24: 119-127.
51. Conese M, Ascenzioni F, Boyd AC, Coutelle C, De Fino I, et al. (2011) Gene and cell therapy for cystic fibrosis: From bench to bedside. J Cyst Fibros 10: S114-S128.
52. Campos SK, Barry MA (2007) Current advances and future challenges in Adenoviral vector biology and targeting. Curr GeneTher 7: 189-204.
53. Chen GX, Zhang S, He XH, Liu SY, Ma C, et al. (2014) Clinical utility of recombinant adenoviral human p53 gene therapy: Current perspectives. OncoTargets Ther 7: 1901-1909.
54. Santiago-Ortiz JL, Schaffer DV (2016) Adeno-associated virus (AAV) vectors in cancer gene therapy. J Control Release 240: 287-301.
55. Coura RD, Nardi NB (2008) A role for adeno-associated viral vectors in gene therapy. Genet Mol Biol 31: 1-11.
56. Yi Y, Jong Noh M, Hee Lee K (2011) Current advances in retroviral gene therapy. Curr Gene Ther 11: 218-228.
57. Real G, Monteiro F, Burger C, Alves PM (2011) Improvement of lentiviral transfer vectors using cis-acting regulatory elements for increased gene expression. Appl Microbiol Biotechnol 91: 1581-1591.
58. Piccolo P, Brunetti-Pierri N (2014) Challenges and prospects for helper-dependent adenoviral vector-mediated gene therapy. Biomedicines 2: 132-148.
59. Brunetti-Pierri N, Ng P (2011) Helper-dependent adenoviral vectors for liver-directed gene therapy. Hum Mol Genet 20(R1): R7-R13.
60. Tesson L, Usal C, Ménoret S, Leung E, Niles BJ (2011) Knockout rats generated by embryo microinjection of TALENs. Nat Biotechnol 29: 695-696.
61. Liu X, Fernandes R, Gertsenstein M, Perumalsamy A, Lai I, et al. (2011) Automated microinjection of recombinant BCL-X into mouse zygotes enhances embryo development. PLoS One 6: e21687.
62. Shirley SA, Heller R, Heller LC (2014) Gene therapy of cancer. Elsevier.
63. van Drunen Littel-van den Hurk S, Hannaman D (2010) Electroporation for DNA immunization: clinical application. Expert Rev Vaccines 9: 503–517.
64. Potter H, Heller R (2017) Transfection by electroporation. Curr Protoc Immunol 121: 9-13.
65. Chuang CC, Chang CW (2015) Complexation of bioreducible cationic polymers with gold nanoparticles for improving stability in serum and application on nonviral gene delivery. ACS Appl Mater Interfaces 7: 7724–7731.
66. Lu C, Stewart DJ, Lee JJ, Ji L, Ramesh R, et al. (2012) Phase I Clinical Trial of Systemically Administered TUSC2(FUS1)-Nanoparticles Mediating Functional Gene Transfer in Humans. PLoS One 7: e34833.
67. Gascón AR, del Pozo-Rodríguez A, Solinís MÁ (2013) Non-Viral Delivery Systems in Gene Therapy. Gene Ther Tools Potential Appl 2013: 3–34.
68. Ramamoorth M, Narvekar A (2014) Non viral vectors in gene therapy- an overview. J Clin Diagn Res 9: 1-6.
69. Delalande A, Kotopoulis S, Postema M, Midoux P, Pichon C (2013) Sonoporation: Mechanistic insights and ongoing challenges for gene transfer. Gene 525: 191–199.
70. Delalande A, Leduc C, Midoux P, Postema M, Pichon C (2015) Efficient gene delivery by sonoporation is associated with microbubble entry into cells and the clathrin-dependent endocytosis pathway. Ultrasound Med Biol 41: 1913–1926.
71. Yu H, Xu L (2014) Cell experimental studies on sonoporation: State of the art and remaining problems. J Control Release 174: 151–160.
72. Kamimura K, Yokoo T, Abe H, Kobayashi Y, Ogawa K, et al. (2015) Image-guided hydrodynamic gene delivery: Current status and future directions. Pharmaceutics 7: 213–223.
73. Bonamassa B, Hai L, Liu D (2015) Hydrodynamic gene delivery and its applications in pharmaceutical research. Pharm Res 28: 694–701.
74. Suda T, Liu D (2015) Hydrodynamic delivery. Adv Genet 89: 89–111.
75. I Schwerdt J, F Goya G, Pilar Calatayud M, B Herenu C, C Reggiani P, et al. (2012) Magnetic field-assisted gene delivery: Achievements and therapeutic potential. Curr Gene Ther 12: 116–126.
76. Prosen L, Prijic S, Music B, Lavrencak J, Cemazar M, et al. (2013) Magnetofection: A reproducible method for gene delivery to melanoma cells. Biomed Res Int 2013: 209452.
77. Huang X, Schwind S, Yu B, Santhanam R, Wang H, et al. (2013) Targeted delivery of microRNA-29b by transferrin-conjugated anionic lipopolyplex nanoparticles: A novel therapeutic strategy in acute myeloid leukemia. Clin Cancer Res 19: 2355–2367.
78. Majzoub RN, Chan CL, Ewert KK, Silva BF, Liang KS, et al. (2014) Uptake and transfection efficiency of PEGylated cationic liposome-DNA complexes with and without RGD-tagging. Biomaterials 35: 4996–5005.
79. Tam YY, Chen S, Zaifman J, Tam YK, Lin PJ, et al. (2013) Small molecule ligands for enhanced intracellular delivery of lipid nanoparticle formulations of siRNA. Nanomed: Nanotechnol Biol Med 9: 665–674.
80. de Ilarduya CT, Sun Y, Düzgüneş N (2010) Gene delivery by lipoplexes and polyplexes. Eur J Pharm Sci 40: 159–170.
81. Grandinetti G, Ingle NP, Reineke TM (2011) Interaction of poly(ethylenimine)-DNA polyplexes with mitochondria: Implications for a mechanism of cytotoxicity. Mol Pharm 8: 1709–1719.
82. Vannucci L, Lai M, Chiuppesi F, Ceccherini-Nelli L, Pistello M (2013) Viral vectors: A look back and ahead on gene transfer technology. New Microbiol. 36: 1–22.
83. Collins M, Thrasher A (2015) Gene therapy: progress and predictions. Proc R Soc B 282: 20143003.
84. C Heller L, Heller R (2010) Electroporation gene therapy preclinical and clinical trials for melanoma. Curr Gene Ther 10: 312–317.
85. Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, et al. (1987) Lipofection: A highly efficient, lipid mediated DNA-transfection procedure. Proc Natl Acad Sci USA 84: 7413–7417.
86. Yingyongnarongkul BE, Howarth M, Elliott T, Bradley M (2004) Solid-Phase Synthesis of 89 Polyamine-Based Cationic Lipids for DNA Delivery to Mammalian Cells. Chem: A Eur J 10 463–473.
87. Savarala S, Brailoiu E, Wunder SL, Ilies MA (2013) Tuning the self-assembling of pyridinium cationic lipids for efficient gene delivery into neuronal cells. Biomacromolecules 14: 2750–2764.
88. Chen C, Han D, Cai C, Tang X (2010) An overview of liposome lyophilization and its future potential. J Control Release 142: 299-311.
89. Thermo Fisher Scientific (2015) How cationic lipid mediated transfection works. Thermo Fisher Scientific Inc.
90. Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, et al. (2014) Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol 33: 73–80.
91. Rao NM (2010) Cationic lipid-mediated nucleic acid delivery: beyond being cationic. Chem Phys Lipids 163: 245–252.
92. Yamano S, Dai J, Yuvienco C, Khapli S, Moursi AM, et al. (2011) Modified Tat peptide with cationic lipids enhances gene transfection efficiency via temperature-dependent and caveolae-mediated endocytosis. J Control Release 152: 278–285.
93. Ojeda E, Puras G, Agirre M, Zarate J, Grijalvo S, et al. (2016) The influence of the polar head-group of synthetic cationic lipids on the transfection efficiency mediated by niosomes in rat retina and brain. Biomaterials 77: 267–279.
94. Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, et al. (2015) Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol 33: 73–80.
95. Buschmann MD, Merzouki A, Lavertu M, Thibault M, Jean M, et al. (2013) Chitosans for delivery of nucleic acids. Adv Drug Deliv Rev 65: 1234–1270.
96. Grunwald T, Ulbert S (2015) Improvement of DNA vaccination by adjuvants and sophisticated delivery devices: Vaccine-platforms for the battle against infectious diseases. Clin Exp Vaccine Res 4: 1-10.
97. Huang YW, Lee HJ, Tolliver LM, Aronstam RS (2015) Delivery of nucleic acids and nanomaterials by cell-penetrating peptides: Opportunities and challenges. BioMed Res Int 2015: 834079.
98. Yamano S, Dai J, Hanatani S, Haku K, Yamanaka T, et al. (2014) Long-term efficient gene delivery using polyethylenimine with modified Tat peptide. Biomaterials 35: 1705-1715.
99. V Ngwa VM, Axford DS, Healey AN, Nowak SJ, Chrestensen CA, et al. (2017) A versatile cell-penetrating peptide-adaptor system for efficient delivery of molecular cargos to subcellular destinations. PLoS One 12: e0178648.
100. Yue ZG, Wei W, Lv PP, Yue H, Wang LY, et al. (2011) Surface charge affects cellular uptake and intracellular trafficking of chitosan-based nanoparticles. Biomacromolecules 12: 2440–2446.
101. Ikeda Y , Nagasaki Y (2014) Impacts of PEGylation on the gene and oligonucleotide delivery system. J Appl Polym 131.
102. Ge Z (2014) Targeted gene delivery by polyplex micelles with crowded PEG palisade and cRGD moiety for systemic treatment of pancreatic tumors. Biomaterials 35: 3416–3426.
103. Tian H, Chen J, Chen X (2013) Nanoparticles for gene delivery. Small 9: 2034–2044.
104. Ballarín-González B, Howard KA (2012) Polycation-based nanoparticle delivery of RNAi therapeutics: Adverse effects and solutions. Adv Drug Deliv Rev 64: 1717–1729.
105. Hu SH, Hsieh TY, Chiang CS, Chen PJ, Chen YY, et al. (2014) Surfactant-free, lipo-polymersomes stabilized by iron oxide nanoparticles/polymer interlayer for synergistically targeted and magnetically guided gene delivery. Adv Healthc Mater 3: 273–282.
106. Xiang B, Dong DW, Shi NQ, Gao W, Yang ZZ, et al. ( 2013) PSA-responsive and PSMA-mediated multifunctional liposomes for targeted therapy of prostate cancer. Biomaterials 34: 6976–6991.
107. Wang Y, Su HH, Yang Y, Hu Y, Zhang L, et al. (2013) Systemic delivery of modified mRNA encoding herpes simplex virus 1 thymidine kinase for targeted cancer gene therapy. Mol Ther 21: 358–367.
108. Chen W, Li H, Liu Z, Yuan W (2016) Lipopolyplex for therapeutic gene delivery and its application for the treatment of Parkinson’s disease. Front Aging Neurosci 8: 1–10.
109. Saleh T, Bolhassani A, Shojaosadati SA, Aghasadeghi MR (2015) MPG-based nanoparticle: An efficient delivery system for enhancing the potency of DNA vaccine expressing HPV16E7. Vaccine 33: 3164–3170.
110. Arukuusk P, Pärnaste L, Oskolkov N, Copolovici DM, Margus H, et al. (2013) New generation of efficient peptide-based vectors, NickFects, for the delivery of nucleic acids. Biochim Biophys Acta – Biomembr 1828: 1365–1373.
111. Liu Q, Su RC, Yi WJ, Zheng LT, Lu SS, et al. (2017) pH and reduction dual-responsive dipeptide cationic lipids with α–tocopherol hydrophobic tail for efficient gene delivery. Eur J Med Chem 129: 1–11.
112. Martens TF, Remaut K, Deschout H, Engbersen JF, Hennink WE, et al. (2015) Coating nanocarriers with hyaluronic acid facilitates intravitreal drug delivery for retinal gene therapy. J Control Release 202: 83–92.
113. Apaolaza PS, Del Pozo-Rodríguez A, Solinís MA, Rodríguez JM, Friedrich U, et al. (2016) Structural recovery of the retina in a retinoschisin-deficient mouse after gene replacement therapy by solid lipid nanoparticles. Biomaterials 90: 40–49.
114. Jiang Q, Yue D, Nie Y, Xu X, He Y, et al. (2016) Specially-made lipid-based assemblies for improving transmembrane gene delivery: comparison of basic amino acid residue rich periphery. Mol Pharm 13: 1809–1821.
115. Jiang G, Min SH, Oh EJ, Hahn SK (2007) DNA / PEI / Alginate polyplex as an efficient in vivo gene delivery system. Biotechnol Bioprocess Eng 12: 684–689.
116. Patnaik S, Arif M, Pathak A, Singh N, Gupta KC (2010) PEI-alginate nanocomposites: Efficient non-viral vectors for nucleic acids. Int J Pharm 385: 194–202.
117. Soininen SK, Vellonen KS, Heikkinen AT, Auriola S, Ranta VP, et al. (2016) Intracellular PK/PD relationships of free and liposomal doxorubicin: Quantitative analyses and PK/PD modeling. Mol Pharm 13: 1358–1365.
118. Hanzlíková M, Ruponen M, Galli E, Raasmaja A, Aseyev V, et al. (2011) Mechanisms of polyethylenimine-mediated DNA delivery: Free carrier helps to overcome the barrier of cell-surface glycosaminoglycans. J Gene Med 13: 402–409.
119. Hattori Y, Yamasaku H, Maitani Y (2013) Anionic polymer-coated lipoplex for safe gene delivery into tumor by systemic injection. J Drug Target 21: 639–647.
120. Villate-Beitia I, Puras G, Soto-Sánchez C, Agirre M, Ojeda E, et al. (2017) Non-viral vectors based on magnetoplexes, lipoplexes and polyplexes for VEGF gene delivery into central nervous system cells. Int J Pharm 521: 130–140.
121. Patnaik S, Gupta KC (2013) Novel polyethylenimine-derived nanoparticles for in vivo gene delivery. Expert Opin Drug Deliv 10: 215–228.
122. Shen J, Zhao DJ, Li W, Hu QL, Wang QW, et al. (2013) A polyethylenimine-mimetic biodegradable polycation gene vector and the effect of amine composition in transfection efficiency. Biomaterials 34: 4520–4531.
123. Liu K, Wang X, Fan W, Zhu Q, Yang J, et al. (2012) Degradable polyethylenimine derivate coupled to a bifunctional peptide R13 as a new gene-delivery vector. Int J Nanomedicine 7: 1149–1162.
124. Fan W, Wu X, Ding B, Gao J, Cai Z, et al. (2012) Degradable gene delivery systems based on Pluronics-modified low-molecular-weight polyethylenimine: Preparation, characterization, intracellular trafficking, and cellular distribution. Int J Nanomedicine 7: 1127–1138.
125. Nicolas J, Mura S, Brambilla D, Mackiewicz N, Couvreur P (2013) Design, functionalization strategies and biomedical applications of targeted biodegradable/biocompatible polymer-based nanocarriers for drug delivery. Chem Soc Rev 42: 1147–1235.
126. Nam K, Jung S, Nam JP, Kim SW (2015) Poly(ethylenimine) conjugated bioreducible dendrimer for efficient gene delivery. J Control Release 220: 447–455.
127. Wang C, Bao X, Ding X, Ding Y, Abbad S, et al. (2015) A multifunctional self-dissociative polyethyleneimine derivative coating polymer for enhancing the gene transfection efficiency of DNA/polyethyleneimine polyplexes in vitro and in vivo. Polym Chem 6: 780–796.
128. Needham CJ, Williams AK, Chew SA, Kasper FK, Mikos AG (2012) Engineering a polymeric gene delivery vector based on poly(ethylenimine) and hyaluronic acid. Biomacromolecules 13: 1429–1437.
129. Yin H, Kanasty RL, Eltoukhy AA, Vegas AJ, Dorkin JR (2014) Non-viral vectors for gene-based therapy. Nat Rev Genet 15: 541–555.
130. Wegman F, Öner FC, Dhert WJ, Alblas J (2013) Non-viral gene therapy for bone tissue engineering. Biotechnol Genet Eng Rev 29: 206–220.
131. Grosse S, Thévenot G, Aron Y, Duverger E, Abdelkarim M, et al. (2008) In vivo gene delivery in the mouse lung with lactosylated polyethylenimine, questioning the relevance of in vitro experiments. J Control Release 132: 105–112.
132. Lee YS, Kim SW (2014) Bioreducible polymers for therapeutic gene delivery. J Control Release 190: 424–439.
133. Teo PY, Yang C, Hedrick JL, Engler AC, Coady DJ, et al. (2013) Hydrophobic modification of low molecular weight polyethylenimine for improved gene transfection. Biomaterials 34: 7971–7979.
134. Parhiz H, Shier WT, Ramezani M (2013) From rationally designed polymeric and peptidic systems to sophisticated gene delivery nano-vectors. Int J Pharm 457: 237–259.
135. Kim J, Lee YM, Kim H, Park D, Kim J, et al. (2016) Phenylboronic acid-sugar grafted polymer architecture as a dual stimuli-responsive gene carrier for targeted anti-angiogenic tumor therapy. Biomaterials 75: 102–111.
136. Pan S, Cao D, Huang H, Yi W, Qin L, et al. (2013) A Serum-resistant low-generation polyamidoamine with PEI 423 outer layer for gene delivery vector. Macromol Biosci 13: 422–436.
137. Bechara C, Sagan S (2013) Cell-penetrating peptides: 20 years later, where do we stand? FEBS Letters 587: 1693–1702.
138. Parhiz H, Hashemi M, Ramezani M (2015) Non-biological gene carriers designed for overcoming the major extra-and intracellular hurdles in gene delivery, an updated review. Nanomedicine J 2: 1–20.
139. Rehman ZU, Hoekstra D, Zuhorn IS (2013) Mechanism of polyplex- and lipoplex-mediated delivery of nucleic acids: Real-time visualization of transient membrane destabilization without endosomal lysis. ACS Nano 7: 3767–3777.
140. Kodama Y, Shiokawa Y, Nakamura T, Kurosaki T, Aki K, et al. (2014) Novel siRNA Delivery System Using a Ternary Polymer Complex with Strong Silencing Effect and No Cytotoxicity. Biol Pharm Bull 37: 1274–1281.
141. Peng SF, Hsu HK, Lin CC, Cheng YM, Hsu KH (2017) Novel PEI/Poly-γ-gutamic acid nanoparticles for high efficient siRNA and plasmid DNA co-delivery. Molecules 22: 1–16.
142. Vázquez E, Ferrer-Miralles N, Villaverde A (2008) Peptide-assisted traffic engineering for nonviral gene therapy. Drug Discov Today 13: 1067–1074.
143. Lam AP, Dean DA (2010) Progress and prospects: nuclear import of nonviral vectors. Gene Ther 17: 39–447.
144. Parhiz H, Hashemi M, Hatefi A, Shier WT, Farzad SA, et al. (2013) Molecular weight-dependent genetic information transfer with disulfide-linked polyethylenimine-based nonviral vectors. J Biomater Appl 28: 112–24.
145. Jaeger M, Schubert S, Ochrimenko S, Fischer D, Schubert US (2012) Branched and linear poly(ethylene imine)-based conjugates: synthetic modification, characterization, and application. Chem Soc Rev 41: 4755.
146. Chen J, Tian B, Yin X, Zhang Y, Hu D (2014) Preparation, characterization and transfection efficiency of nanoparticles composed of alkane-modified polyallylamine Alkane-modified polyallylamine as gene delivery vector. Nanomed J 23: 111–120.
147. Gillies ER, Frechet JM (2005) Dendrimers and dendritic polymers in drug delivery. Drug Discov Today 10: 35–43.
148. Quadir MA, Haag R (2012) Biofunctional nanosystems based on dendritic polymers. J Control Release 161: 484–495.
149. Lakshminarayanan A, Ravi VK, Tatineni R, Rajesh YB, Maingi V, et al. (2013) Efficient dendrimer-DNA complexation and gene delivery vector properties of nitrogen-core poly(propyl ether imine) dendrimer in mammalian cells. Bioconjug Chem 24: 1612–1623.
150. Jin L, Zeng X, Liu M, Deng Y, He N (2014) Current progress in gene delivery technology based on chemical methods and nano-carriers. Theranostics 4: 240–255.
151. Zhou J, Patel TR, Fu M, Bertram JP, Saltzman WM (2012) Octa-functional PLGA nanoparticles for targeted and efficient siRNA delivery to tumors. Biomaterials 33: 583–591.
152. Yuan X, Naguib S, Wu Z (2011) Recent advances of siRNA delivery by nanoparticles. Expert Opin Drug Deliv 8: 521–536.
153. Yuan X, Shah BA, Kotadia NK, Li J, Gu H (2010) The development and mechanism studies of cationic chitosan-modified biodegradable PLGA nanoparticles for efficient siRNA drug delivery. Pharm Res 27: 1285–1295.
154. Erbetta CD, Alves RJ, Resende JM, de Souza Freitas RF, de Sousa RG (2012) Synthesis and Characterization of Poly(D,L-Lactide-co-Glycolide) Copolymer. J Biomater Nanobiotechnol 3: 208–225.
155. Layek B, Singh J (2013) Caproic acid grafted chitosan cationic nanocomplexes for enhanced gene delivery: Effect of degree of substitution. Int J Pharm 447: 182–191.
156. Layek B, Singh J (2013) Amino acid grafted chitosan for high performance gene delivery: Comparison of amino acid hydrophobicity on vector and polyplex characteristics. Biomacromolecules 14: 485–494.
157. Castillo G, Spinella K, Poturnayová A, Šnejdárková M, Mosiello L, et al. (2015) Detection of aflatoxin B1 by aptamer-based biosensor using PAMAM dendrimers as immobilization platform. Food Control 52: 9–18.
158. Hosseini SM, Dufort I, Nieminen J, Moulavi F, Ghanaei HR, et al. (2016) Epigenetic modification with trichostatin A does not correct specific errors of somatic cell nuclear transfer at the transcriptomic level; highlighting the non-random nature of oocyte-mediated reprogramming errors. BMC Genomics 17: 16.
159. Gopal V (2013) Bioinspired peptides as versatile nucleic acid delivery platforms. J Control Release 167: 323–332.
160. Schellinger JG, Pahang JA, Johnson RN, Chu DS, Sellers DL, et al. (2013) Melittin-grafted HPMA-oligolysine based copolymers for gene delivery. Biomaterials 34: 2318–2326.
161. Soltani F, Sankian M, Hatefi A, Ramezani M (2013) Development of a novel histone H1-based recombinant fusion peptide for targeted non-viral gene delivery. Int J Pharm 441: 307–315.
162. Liashkovich I, Meyring A, Oberleithner H, Shahin V (2012) Structural organization of the nuclear pore permeability barrier. J Control Release 160: 601–608.