Nucleic Acids–based Bionanomaterials for Drug and Gene Therapy

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Elena Stoleru and Cornelia Vasile Physical Chemistry of Polymers Department, Romanian Academy, “Petru Poni” Institute of Macromolecular Chemistry, Iasi, Romania

6.1 INTRODUCTION Deoxyribonucleic acid (DNA) plays a fundamental role in all living organisms because of its key functions in storage, duplication, and realization of genetic information; these applications occur through double-stranded DNA (dsDNA), a conclusion drawn for the first time in 1953 by Watson and Crick (Watson and Crick, 1953). The DNA molecule exists in the form of a three-dimensional double helix, produced from the hydrogen-bonded base pairing between specific nucleobases: adenine (A), thymine (T), guanine (G), and cytosine (C). This unique base-pairing can cause hairpin or loop structures in single-stranded DNA (ssDNA) or can make dsDNA from two complementary ssDNA segments. Again, dsDNA can exist in right-handed double-helical structures (B-DNA, A-DNA); among which, B-DNA is the most common form, and it can also have a left-handed double-helical form called Z-DNA (Kundu et al., 2017). The specific bonding of DNA base pairs provides the chemical foundation for genetics. This powerful molecular recognition system can be used in nanotechnology to direct the assembly of highly structured materials with specific nanoscale features, as well as in DNA computation to process complex information (Seeman, 2003). On the other hand, ribonucleic acid (RNA), which resembles DNA but contains pyrimidinederived base uracil (U) instead of thymine, is usually single-stranded (Furth et al., 1962; Hurwitz and Leis, 1972). RNA is another widely recognized biomolecule which plays several important roles: a carrier of genetic information from DNA to proteins, a messenger between DNA and protein synthesis complexes, as well as a carrier molecule of amino acids for protein synthesis (Hurwitz and Leis, 1972). Like DNA, RNA can also be designed and manipulated to produce a wide variety of nanostructures (Kundu et al., 2017). Nucleic acid
based molecules (deoxyribonucleic acid, complementary deoxyribonucleic acid, complete genes, ribonucleic acid, and oligonucleotides) are utilized as research tools within the broad borders of gene therapy and the emerging field of molecular medicine. Although most nucleic acid
based drugs are in the early stages of clinical trials, these classes of compounds have emerged in recent years to become extremely promising candidates for drug therapy for a wide range of diseases, including cancer, infectious diseases, diabetes, cardiovascular diseases, inflammatory, and neurodegenerative diseases, cystic fibrosis, hemophilia, and other genetic disorders Polymeric Nanomaterials in Nanotherapeutics. DOI: https://doi.org/10.1016/B978-0-12-813932-5.00006-6 © 2019 Elsevier Inc. All rights reserved.

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(Pushpendra et al., 2012). DNA-based therapeutics includes plasmids, oligonucleotides for antisense and antigene applications, deoxyribonucleic acid aptamers, and deoxyribonucleic acidzymes, while RNA-based therapeutics includes ribonucleic acid aptamers, ribonucleic acid decoys, antisense ribonucleic acid, ribozymes, small interfering ribonucleic acid, and microribonucleic acid (Pushpendra et al., 2012). Such genetic drugs can be clinically used, e.g., in developing gene therapies to treat most diseases by silencing pathological genes, expressing therapeutic proteins, or through gene-editing applications (Cullis and Hope, 2017).

6.2 NANOTECHNOLOGY APPLICATIONS IN NUCLEIC ACID DELIVERY AND GENE THERAPIES Gene therapy has gained widespread interest due to its potential for treatment of many genetic diseases (Ahmed and Narain, 2012). However, in order to use it at a clinical level genetic drugs need delivery systems with complex and advanced features to be developed, this represents the major impediment to successful gene therapy (Cullis and Hope, 2017; Whitehead et al., 2009; Tang et al., 2016). Gene delivery systems are becoming mandatory for gene therapy because nucleic acids are susceptible to hydrolysis in physiological conditions and have poor cell membrane permeability due to their negatively charged structure (Li et al., 2016a). In other words, therapeutic nucleic acids [TNAs; plasmid DNA (pDNA) or short interfering RNA (siRNA)] need to be shuttled and successfully transferred into the defective cells by gene carriers (vectors) (Tang et al., 2016). Fig. 6.1 schematically shows the principles of gene therapy showing that therapeutic genes of interest or growth factors that influence cellular function can be placed in viral or nonviral vectors that enter a targeted cell to significantly alter its function (Kendirci et al., 2006). Vector-assisted DNA/gene delivery systems can be classified into two types based on their origin: biological (viral DNA delivery systems) and chemical (nonviral delivery systems) (Pushpendra et al., 2012). Although viral gene carriers have shown high transfection efficiency, the clinical application based on this approach is significantly limited by several safety concerns, including immunogenicity, carcinogenicity, the immune response, and the risk of recombination with wild-type viruses. Some of these drawbacks may be solved by using nonviral carriers (Tang et al., 2016; Li et al., 2016a). The use of nonviral systems for delivery of nucleic acids is of particular interest as they can be engineered for high biocompatibility, specificity, and targeted delivery (Ahmed and Narain, 2012). Polymeric gene carriers have several unique advantages, such as inherent low immunogenicity, safety, physiological stability, easy of manufacture, as well as tunable surface and structural properties, and they are suitable for large-scale production. Nevertheless, the transfection efficiency of nonviral carriers is relatively lower than that of viral gene vectors and has largely limited their application (Tang et al., 2016; Li et al., 2016a). Gene transfection refers to the process of deliberately introducing genetic material into cells (eukaryotic or bacterial) by nonviral methods. In general, transfection can be carried out with chemical methods (using calcium phosphate, organic compounds, cationic liposomes, and polymers) and nonchemical methods (by electroporation, cell squeezing, sonoporation) (Yang et al., 2017). Generally, the complexes that the nonviral carriers form with nucleic acids for gene therapy can be classified into four categories: polyplexes, lipoplexes, micelleplexes, and others (Tang et al., 2016).

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FIGURE 6.1 Schematic representation of the principles of gene therapy. Adapted from Kendirci, M., Teloken, P., Champion, H., Hellstrom, W. and Bivalacqua, T. (2006). Gene therapy for erectile dysfunction: fact or fiction? Eur. Urol., 50(6), 1208
1222; Shasheiha, N.M., Mutalib, A., Nasuha, W.F., Nudri, W., Husna, N., Muryadi, et al. (2012). Gene Ther., https://www.slideshare.net/syeimy/gene-therapy-ppt, accessed 20 February 2018; Goodsell, D. (2010). Molecule of the month: adenovirous, PDB-101, http://pdb101.rcsb.org/motm/132, accessed 19 February 2018.

As already described in the first chapters of this book, nanotechnology is defined as “the science of manipulating matter at very small sizes. The matter has to be 1
100 nm in at least one dimension to qualify as nanotechnology” (Simpson, 2011). Nano-engineered materials will always be smaller than cells, meaning that there is the possibility of cell penetration by such nano-designed structures and to modify processes or genetic information (Simpson, 2011). DNA represents a natural example, having a “nano-packing” structure in a nucleus of 2- to 5-μm diameter, namely dsDNA with about 2.5 nm in width, but totals 2 m in length in the mammalian cell (Kawasaki and Player, 2005). However, normal DNA is often too large to penetrate and cross the cell membrane, so special nano-sized materials can be used to compact the DNA and allow it to enter the cell. Nanoparticulate (NP) materials hold promise for DNA and RNA delivery when a means for binding is identified that retains structure
function and provides stabilization by the nanoparticles (NPs) (DeLong et al., 2009). Gene nanocarriers generally refer to nanoparticles and nanocapsules usually produced from biocompatible materials, which are able to form gene nanocarrier complexes through wrapping or adsorbing nucleic acid molecules, such as exogenous DNA. The size of nanocarriers is generally between 10 and 100 nm (Sun et al., 2014). Nanomaterials as gene carriers (or vectors) present some important advantages, such as: (1) they can be used for wrapping,

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concentrating, and protecting nucleotides from degradation via nucleases; (2) they have a larger specific surface area and biocompatibility; (3) they easily couple to specific targeting molecules on the surface to achieve specificity of gene therapy; (4) cycle time in the circulatory system is significantly prolonged as compared with ordinary particles and, in a certain period of time; (5) they will not be rapidly cleared by phagocytic cells like ordinary particles; (6) they allow slow release of nucleotides and effectively extend the time of action and maintain an effective concentration of the product to enhance the transfection efficiency and bioavailability of the transfected products; and (7) they have fewer metabolites, fewer side effects, and no immune rejection reactions (Shi et al., 2015). The overall effectiveness of a NP-based gene delivery system is dependent on three key factors: (1) cellular uptake of NPs, (2) escape of NPs from endosomal vesicles into the cytosol, and (3) transfer of the pDNA to the nucleus (Adijanto and Naash, 2015). Despite the promise of nanotechnology for the delivery of TNAs, a major current limitation is the inability to combine therapeutic DNA and RNA with nanomaterials so that structure and function can be maintained after bioprocessing and delivery (DeLong et al., 2009).

6.3 STRATEGIES FOR OBTAINING NUCLEIC ACIDS
BASED NANOTHERAPEUTICS Programmable multitasking as well as the ability to dynamically respond to the environment makes nucleic acids an attractive material for tailormade applications in both biotechnology and personalized therapy (Halman et al., 2017). It is a common challenge to prevent degradation of DNA when it is used in nanotherapies. Degradation can occur through either mechanical shearing forces or chemical degradation by nucleases (Zelikin et al., 2007). To prevent its degradation, DNA can be condensed and/or protected by a physical barrier. There are a number of strategies employed in gene therapy to limit DNA degradation, including complexation of DNA with polycations (Putnam, 2006), blockcopolymer micelles (Kataoka et al., 2001), cationic lipids, or liposomes (Pedroso de Lima et al., 2001). Alternatively, DNA can be confined within a gel (Goh et al., 2004), micelles (Csaba et al., 2005), and polymeric microparticles (Ando et al., 1999). For applications requiring transcriptionally active nucleic acid, DNA may be encapsulated within liposomes (Tsumoto et al., 2001; Edwards and Baeumner, 2007), water-in-oil emulsions (Tawfik and Griffiths, 1998; Ghadessy et al., 2001), and polyelectrolyte capsules (Shchukin et al., 2004; Kreft et al., 2006; Zelikin et al., 2006, 2007). Fig. 6.2 shows a classification and a schematic drawing of different types of polymeric nanoparticles used for gene delivery (Vasir and Labhasetwar, 2006).

6.3.1 CONDENSATION OF DNA WITH POLYMERIC NANOMATERIALS DNA is a highly charged molecule, which cannot exist in solution without other ions. Although the abbreviation “DNA” means deoxyribonucleic acid, usually DNA comes as a salt of Na1 or other alkali metals (Teif and Bohinc, 2011). DNA is stored in vivo in a highly compact, so-called condensed phase, where gene regulatory processes are governed by the intricate interplay between different states of DNA compaction (Teif and Bohinc, 2011). DNA compaction occurs in vivo through

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Polymeric nanoparticles for gene delivery

Polymer

DNA

Condensation of DNA with polymers or complexation to the surface of nanoparticles

DNA encapsulation in polymeric matrix or reservoir

Matrix

Poly(L-Lysine); poly(ethylenimine); poly(β-amino acids); chitosan cationic dendrimers; polyamidoamine polylactide; poly(lactide-co-glycolide) polyalkylcyanoacrylate

Polyplex

Reservoir

Polylactide Poly(lactide-co-glycolide) Poly(β-amino esters) Chitosan

Cationic surfactant

FIGURE 6.2 Schematic representation of polymeric nanoparticles used as carriers for gene delivery. Adapted from Vasir, J. and Labhasetwar, V., 2006. Polymeric nanoparticles for gene delivery. Expert Opin. Drug Deliv., 3 (3), 325
344.

the interaction of DNA with low molecular weight compounds, such as spermine or spermidine, for compaction inside virus heads (Cerritelli et al., 1997), or with high molecular weight compounds, such as histones, for packaging of genetic material into chromosomes (Le Ny and Lee, 2009). DNA condensation can be induced in vitro either by applying external force to bring the double helices together, or by inducing attractive interactions between the DNA segments (Teif and Bohinc, 2011). A great number of in vivo and in vitro studies on DNA condensation, and recently focusing on DNA compaction, have been reported (Ramisetty et al., 2017; Zinchenko and Yoshikawa, 2015; Rata-Aguilar et al., 2015; Fan et al., 2017; Cherng and Lin, 2016; Li et al., 2016b). They can be roughly classified into (1) studies related to DNA compaction by cationic histone proteins as models of DNA condensation and also by positively charged polyamines and (2) DNA compaction in concentrated solutions of neutral polymers to address macromolecular crowding or depletion (Zinchenko and Yoshikawa, 2015). The main potential application of DNA condensation in medicine is its use for gene delivery in gene therapy. The main problems in gene therapy are target recognition (what in the genome should be targeted by artificial DNA or RNA constructs), target modification (the way that the drug acts on the target), and the delivery of the DNA-targeted drug to the cell (here DNA condensation comes into play) (Teif and Bohinc, 2011). DNA condensation has been shown to be essential to protect DNA from nuclease, and to allow entry of DNA into cells, mainly by the endocytic pathway (Le Ny and Lee, 2009).

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Polycations capable of condensing DNA are the core components of nonviral nanocarriers used for gene delivery (Rata-Aguilar et al., 2015; Merdan et al., 2002; Park et al., 2006; Chew et al., 2010). Generally, cationic polymer carriers are widely accepted because of their ability to efficiently condensate DNA, forming nanoparticles of sizes appropriate for intracellular entry and interaction, and allowing easy ligand attachment for cellular targeting (Gao and Huang, 1996; Mansouri et al., 2004; Mann et al., 2008). DNA interacts with cationic polymers especially by electrostatic interactions and the depletion effect. The electrostatic interactions between polycations and negatively charged DNA are influenced by the charge of the polymer, the ratio between amino groups in polymers and phosphates in DNA, as well as the complex dimensions (Kwoh et al., 1999; van de Wetering et al., 1999; Tang and Szoka, 1997; Godbey et al., 1999a; Hou et al., 2008). The depletion effect between semiflexible DNA molecules and flexible polymers is determined by the entropy changes of the mixture solution (Kojima et al., 2006). For gene vectors, the design is essential to control the number of amino groups present in the polymer structure because too many positively charged groups will increase their cell toxicity. The interaction between DNA and polymer is mostly electrostatic when the polymer presents numerous positive charges, otherwise the depletion effect will predominantly influence the DNA condensation process. To design gene delivery polymers it is very important to know the mechanism of DNA condensation induced by less charged cationic polymers (Hou et al., 2008). The interaction of positively charged polymers with the pDNA results in a large reduction of the volume occupied by the DNA as it changes from a freely moving polymer to a compact spherical structure of 30
300 nm (Bloomfield, 1997) known as a polyplex (Fig. 6.3) (Argyros et al., 2011). The interaction between DNA and cationic nanoparticles (with sizes from 10 to 100 nm) that have similar surface charge density and DNA condensation degree strongly depends on nanoparticle size, as shown in Fig. 6.4. At this moment there is a superabundance of cationic polymers that have been used to condense DNA, varying in structure, composition, molecular weight (MW), density of positive charges, hydrophobicity, biocompatibility, and cytotoxicity (Rata-Aguilar et al., 2015). It has been demonstrated that DNA/polymer complexes involving cationic polymers are more stable than those involving cationic lipids (Mansouri et al., 2004). The most often used cationic polymers for gene therapy include poly(L-lysine) (PLL), branched and linear poly(ethylenimine) (PEI), poly(amidoamine) dendrimer, chitosan, poly(β-amino esters),

FIGURE 6.3 By complexing DNA with cationic polymers polyplexes are formed with sizes in the nanometric range. Reprinted with permission from Storrie, H. and Mooney, D., 2006. Sustained delivery of plasmid DNA from polymeric scaffolds for tissue engineering. Adv. Drug Deliv. Rev., 58 (4), 500
514.

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FIGURE 6.4 Representation of the modes of the DNA interaction with cationic nanoparticles by adsorption, wrapping, and collection obtained by molecular dynamics simulations of single-chain DNA fully compact state for large, medium, and small nanoparticles and transmission electron microscopy images of the DNA complexes with nanoparticles. Reprinted with permission from Zinchenko, A., 2016. DNA conformational behavior and compaction in biomimetic systems: toward better understanding of DNA packaging in cell. Adv. Colloid Interface Sci., 232, 70
79.

helical polypeptides, and cationic aliphatic polyesters (Tang et al., 2016). These polycations have attracted widespread attention for gene delivery due to the stable complexes (termed polyplexes) they form with naked DNA (pDNA and siRNA) which have proved able to transfect cells effectively both in vitro and in vivo (Argyros et al., 2011). The inclusion of protonatable nitrogen atoms in a polymer is essential if “proton-sponge” behavior is likely to take place. This is by far the most promising method for a polyplex to avoid lysosomal degradation and it is facilitated by the fact that amines are ubiquitous in gene-delivery polymers (Argyros et al., 2011). These nitrogen atoms serve two purposes. Firstly, when protonated, they provide the cationic character needed for condensation of pDNA (or interaction with siRNA). Protonable amines are therefore an almost ubiquitous feature in all gene-delivery vectors. The second function of the protonable nitrogen atoms is that they appear to provide a means for endosomal escape, which has been explained by the “proton-sponge” mechanism (Akinc et al., 2005; Argyros et al., 2011).

6.3.1.1 Peptide-based polymers for DNA condensation Cationic poly(amino acids) like PLL are known to be efficient in condensing pDNA into compact nanostructures and have been used for in vitro and in vivo delivery of therapeutic DNA (Mann et al., 2008). PLL was one of the first polymers used for DNA condensation and continues to be widely applied for gene therapy applications, despite low transfection efficiency (Kwoh et al., 1999). Other peptide-based polymers containing cationic side chains, such as poly-L-histidine and poly-L-ornithine, have been studied as well (Plank et al., 1999). However, changing the side chain of peptide-based polymers does not appear to significantly influence the transfection efficiency (Storrie and Mooney, 2006).

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6.3.1.2 Poly(ethylenimine)-based nanoparticles for gene delivery The principal feature of PEI that makes it useful for gene delivery is the quantity of protonable nitrogen atoms in the chain (Akinc et al., 2005; Argyros et al., 2011). PEI, formed either as a linear or branched polymer, has been one of the most often used polymers for DNA condensation because of its high buffering ability for endosomal escape of gene to be expressed, and the transfection efficiency is influenced by the geometry of the polymer (Godbey et al., 1999b; Islam et al., 2014). Linear PEI has a higher transfection efficiency than branched PEI of a similar MW, and has increased efficacy in nondividing cells (Wiseman et al., 2003; Wightman et al., 2001; Itaka et al., 2004). However, the transfection efficiency of branched PEI can be increased at low MW by decreasing the degree of branching (Storrie and Mooney, 2006). However, PEI has severe toxicity problems due to the high positive charge density and nondegradability, although the toxicity of PEI depends on its MW and structure. Therefore, considerable attention has been paid to the synthesis of degradable PEI derivatives using low MW ones, because low-MW PEI is much less toxic than high-MW PEI (Islam et al., 2014). Mimi et al. (2012) have fabricated a gelatin
PEI core
shell nanogel via a two-stage synthesis: (1) thermal treatment of gelatin to highly uniform gelatin nanoparticles, followed by covalent coupling of PEI onto the preformed gelatin nanoparticles; (2) fabrication of gelatin
PEI core
shell nanogels through repeated cycles of desolvation and drying of the PEI-conjugated gelatin nanogels. The resultant nanogels are highly uniform spherical particles and have a well-defined core
shell nanostructure with a biodegradable gelatin core and a hairy and extended PEI shell. The resultant nanogels were able to completely condense siRNA, forming stable complexes that were capable of protecting the siRNA from enzymatic degradation. The gelatin
PEI nanogels were four times less toxic than the native PEI, and were able to effectively deliver the siRNA into HeLa cells. Increasing the N/P ratio significantly improved the intracellular uptake efficiency of the siRNA.

6.3.1.3 Chitosan-based nanoparticles for DNA condensation Among polycationic polysaccharides, chitosan, derived by deacetylation of the naturally occurring polysaccharide chitin, has gained increasing interest as one of the nonviral vectors for delivery of gene materials, including oligonucleotides, pDNA, and siRNA, because of the intrinsic cationic nature, low toxicity, low immunogenicity, and excellent biocompatibility (Amaduzzi et al., 2014; Yang et al., 2017). A number of in vitro and in vivo studies showed that chitosan is a suitable material for efficient nonviral gene therapy (Mansouri et al., 2004; Corsi et al., 2003; Ozbas-Turan et al., 2003; Guang Liu and De Yao, 2002; MacLaughlin et al., 1998; Cui and Mumper, 2001; Gao et al., 2003; Kumar et al., 2002). Chitosan polycation shows charge-switching behavior because of the abundance of amine groups with a pKa value of 6.4. Hence, at pH , 6.4, chitosan is cationic and readily forms particle complexes with anionic nucleic acids which can be used as DNA and siRNA transfection vehicles (Yang et al., 2017). Different studies have indicated that the formulation parameters of chitosan are determinative for the binding affinity of chitosan to DNA as well as the transfection efficiency of DNA
chitosan complexes, these include degree of deacetylation, MW of chitosan, the charge ratio of amine (chitosan) to DNA phosphate (N/P ratio), the chitosan/DNA concentration, pH value of the transfection medium, cell type, preparation technique of DNA
chitosan nanoparticles, and so on (Yang et al., 2017; Lavertu et al., 2006; Liu et al., 2005). Mansouri et al. (2004) have

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demonstrated that chitosan
DNA complexes appear spherical with a mean size less than 100 nm and a homogeneous distribution of DNA is established within the particle. Mao et al. (2001) have prepared chitosan
DNA nanoparticles using a complex coacervation process, which have partially protected the encapsulated pDNA from nuclease degradation. It was observed also that the transfection efficiency of chitosan
DNA nanoparticles was cell-type dependent, namely higher gene expression levels were found in HEK293 cells and IB-3-1 cells, compared with 9HTEo and HeLa cells. In the Lavertu et al. (2006) study, important features were revealed regarding the factors influencing the gene transfer from chitosan/pDNA complex nanoparticles. For this purpose the authors produced chitosans with different degrees of deacetylation (DDAs) and depolymerized them to obtain different MWs. They produced 64 formulations of chitosan/pDNA complexes that were tested for gene transfection in HEK 293 cells in vitro. Maximum transgene expression occurred for DDA/MW values that run along a diagonal from high DDA/low MW to low DDA/high MW, suggesting a predominant role of particle stability, through cooperative electrostatic binding, in determining transfection efficiency.

6.3.1.4 Polyamine ester nanoparticles for DNA condensation Water-soluble cationic polyamine polyesters may be potentially useful for the delivery of DNA, since they are able to form polyionic complexes with DNA and may degrade quite rapidly (Hennink et al., 2004; Luten et al., 2008). Poly(4-hydroxy-L-proline ester) was the first watersoluble polycationic biodegradable polymer used as a gene carrier (Luten et al., 2008). Rata-Aguilar et al. (2015) have used to condense a DNA plasmid a dual-degradable polycation, composed by a linear poly(β-amino ester) chain in which ester and disulfide bonds coexist, aiming to reinforce the spontaneous hydrolysis of the ester groups with the intracellular break-up of the disulfide bonds, since these reducible bonds are degraded in the reductive intracellular environment. In this study it was noticed that the minimum condensation ratio needed in order to form stable particles was lower at pH 5 than at pH 7, due to a higher protonation of the amino groups in both polymers and positively charged particles less than 200 nm resulted regardless of the pH value used. The obtained polyplexes presented good colloidal stability and the DNA release inside the cell was caused mainly by a hydrolytic mechanism of the ester groups. However, most of the current developed nonviral gene vectors were positively charged, which were subject to nonspecific phagocytosis by the reticuloendothelium system, resulting in short blood circulation time (Romberg et al., 2008; Li et al., 2016a). To improve the properties of the cationic polymer nanocarriers, different strategies were developed as PEGylation. Poly(ethylene glycol) (PEG) is a hydrophilic polymer that has been used to modify cationic polymers to decrease polyplex selfaggregation and prevent interactions with serum proteins (Storrie and Mooney, 2006). Although PEGylation was successfully introduced to prolong the circulation, the cellular uptake, and gene release these may also be inhibited by the “PEG dilemma” (Hatakeyama et al., 2011; Li et al., 2016a). Recently, it has been demonstrated by Shevchenko et al. (2016) that the grafting of cationic polymers with propylene oxide (PEO) provides an effective approach to tuning their interactions with biological components. The introduction of single PEO units into cationic centers allows for the significant reduction of membrane-damaging and cytotoxic activities of polycations, but preserves their ability for binding, condensing, and intracellular delivery of pDNA (Shevchenko et al., 2016).

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It has been reported that aminated-functionalized poly(glycidyl methacrylate)s (PGMAs) with plentiful cationic and hydroxyl groups are promising gene vectors. Taking this into account, a flexible strategy to design well-defined reducible cationic nanogels based on ethylenediamine (ED)functionalized low-molecular-weight PGMA (denoted by PGED-NGs) with friendly crosslinking reagents (α-lipoic acid) was proposed by Li et al. (2016b). PGED-NGs could effectively complex pDNA and siRNA. Compared with pristine PGED, PGED-NGs exhibited much better performance of pDNA transfection. PGED-NGs also could efficiently transport metastasis-associated lung adenocarcinoma transcript 1 siRNA into hepatoma cells and significantly suppressed cancer cell proliferation and migration (Li et al., 2016b).

6.3.1.5 Photoresponsive nanoparticles for DNA condensation Nevertheless, while the complexing agents mentioned above can increase cellular uptake as a result of DNA neutralization and compaction, the tight binding of these agents to DNA may also generally preclude or greatly reduce the interaction of DNA with intracellular molecules and the subsequent nuclear uptake, reducing transfection efficiencies relative to viral delivery methods. In this regard, photoreversible DNA complexes were proposed, which could provide a triggered release of DNA from the carrier and potentially increase transfection efficiencies (Le Ny and Lee, 2009). In a study by Le Ny and Lee (2009) it was demonstrated that DNA compaction can be reversibly and directly controlled with simple light illumination through the use of photoresponsive surfactants. The addition of azoTAB (azobenzene trimethylammonium bromide) surfactant to a T4-DNA solution under visible light, where the surfactant primarily exists as the trans form, relatively hydrophobic isomer, causes DNA to adopt a compacted conformation. Exposure to UV light causes the compacted DNA to re-expand and recover the extended-coil conformation, a result of photoisomerization of the surfactant to the relatively hydrophilic cis form. For example Nagasaki et al. (2000) synthesized and evaluated a photochromic dendrimer, which included polyazobenzenes in the branch structure knowing that polyazobenzene dendrimers are photochemically size-controllable. Polyazobenzene dendrimer (H-Lys-G2) was modified with Llysines at the periphery in order to increase the water-solubility. The authors concluded that UV light irradiation after the incorporation of DNA-complex with H-Lys-G2 into a cytoplasm significantly caused a 50% increase in the transfection efficiency. In the cytoplasm, UV irradiation promoted the dissociation of the complex and favorable transcription from a free gene was carried out.

6.3.2 ENCAPSULATING DNA IN POLYMERIC NANOSTRUCTURES The DNA encapsulated in polymers may be in a condensed or noncondensed form, depending on the nature of the polymer and the method used for formulating the vector system (Bolhassani et al., 2013). The rapidly rising demand for therapeutic-grade DNA molecules requires associated improvements in encapsulation and delivery technologies. One of the challenges for the efficient intracellular delivery of therapeutic biomolecules after their cell internalization by endocytosis is to manipulate the nonproductive trafficking from endosomes to lysosomes, where degradation may occur. The combination of the endosomal acidity with the endosomolytic capability of the nanocarrier can increase the intracellular delivery of many drugs, genes, and proteins, which, therefore, might enhance their therapeutic efficacy (Mor´an et al., 2015).

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Polyelectrolyte capsules offer the potential of encapsulating DNA within tunable, semipermeable capsules that allow small solute molecules to diffuse through the capsule membrane (Antipov and Sukhorukov, 2004), while limiting the diffusion of large peptides such as proteins (Zelikin et al., 2007). Polyelectrolyte capsules are usually obtained by using the layer-by-layer (LbL) technique. The capsules are assembled by sequential deposition of the polymers interacting via electrostatic, covalent, or hydrogen bonding forces onto colloidal particles, succeeded by the removal of the internal content of the particles. Many studies regarding DNA incorporation into thin multilayered films onto colloidal particles as one of the film components are reported (V´azquez et al., 2002; Jessel et al., 2006; Schu¨ler and Caruso, 2001; Johnston et al., 2005). However, there are a few studies about the encapsulation of uncomplexed DNA within the polyelectrolyte capsules. Some examples comprise the DNA “controlled precipitation” by spermidine action onto the colloidal particle followed by capsule formation, also called the preloading approach (Shchukin et al., 2004), and rehydration into a solution that contains DNA of the already-formed capsules (Kreft et al., 2006), referred to as the postloading approach (Zelikin et al., 2007). Mor´an et al. (2015) have used gelatin (either high or low gel strength) and protamine sulfate to form particles by interaction of oppositely charged compounds. They prepared a binary system as particles in the absence of DNA and a ternary system in the presence of DNA. DNA was effectively entrapped on the gelatin B(DNA)-protamine sulfate nanoparticles with loading efficiency values ranging between 72% and 98%, confirming the effectiveness of the encapsulation process. The authors demonstrated that these gelatin-based nanoparticles have excellent properties as highly potent and nontoxic intracellular delivery systems, rendering them promising DNA vehicles to be used as nonviral gene-delivery systems (Mor´an et al., 2015). The importance of using degradable polymers has been particularly recognized during the past years for polymeric gene-delivery vector development, with the aim of achieving sustained DNA delivery or to control the intracellular release of the encapsulated DNA and to excrete the vectors and their degradation products from the body afterward. One approach is the use of cationic polymers that degrade and thereby lose their DNA-binding properties in time. For this purpose, either polymers with (partly) degradable main chains or with degradable side chains are used. Mostly, these polymers contain hydrolytically sensitive bonds (e.g., esters), but the use of disulfide bonds which are cleaved in the reductive environment of the cytoplasm has also been reported in some papers (Luten et al., 2008). For example, Elazar et al. (2010) have encapsulated antisense genes (ASs), which were designed against osteopontin and bone sialoprotein-II, in NPs based on biodegradable and biocompatible polylactide-co-glycolide copolymer (PLGA) that have presented sustained release and stability of the ASs. The therapeutic efficacy of the AS-NP delivery system was examined in vitro, and in a breast cancer bone metastasis animal model of MDAMB-231 human breast cancer cells in nude rats. AS-NP exhibited better therapeutic efficacy than osmotic minipumps in terms of lesion ratio at later time periods (8
12 weeks), and the authors concluded that AS delivery by NP is a promising therapeutic modality providing stability of the encapsulated AS and sustained release (Elazar et al., 2010). The potential to encapsulate DNA, as opposed to retention by adsorption or charge interaction may have advantages in DNA stability and in the possibility to further surface modify the carriers (Hirosue et al., 2001). Encapsulating large hydrophilic molecules in very small nanospheres has been difficult and only a few examples exist in the literature (Hirosue et al., 2001).

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Incorporation of hydrophilic macromolecules, such as DNA, into nanospheres made of hydrophobic polymers is difficult, especially when it is desirable to limit the diameters within the nanosize ranges of interest for targeting and intracellular trafficking. The commonly used emulsion method requires sonication steps that may shear and degrade the nucleic acid. In contrast, particle formations based on solvent diffusion produce nanospheres under nonshearing conditions. These formulations are attractive for biological molecule encapsulation, especially for double stranded and other forms of nucleic acids. Hirosue et al. (2001) have thus explored such formulations using PLGA in the phase inversion nanosphere formulation of pDNA. The nanospheres are made by a modified phase inversion/solvent diffusion method using a cationic lipid dimethyldioctadecylammonium bromide (DDAB) as an excipient. They have demonstrated that a charged macromolecule, such as a plasmid, can be incorporated in and released from phase inversion/solvent diffusion nanospheres. Cohen et al. (2000) have efficiently encapsulated pDNA [alkaline phosphatase (AP), a reporter gene] in submicron-size particles (600 nm size range) composed of poly(DL-lactide-co-glycolide) biodegradable and biocompatible polymer. They have assessed in vitro and in vivo gene expression mediated by NPs comparing with the cationic-liposome delivery. The nanoparticulate system exhibited sustained release of pDNA of over a month and maintained its structural and functional integrity, though gene expression was significantly lower in comparison with standard liposomal transfection. Therapeutic applications of PLGA nanoparticles also include sustained gene delivery, protein delivery, vaccine adjuvant, and intracellular targeting (Panyam and Labhasetwar, 2003). Zelikin et al. (2007) had developed a polycation-free method for the encapsulation of DNA within polymer capsules, resulting in uncomplexed oligonucleotide DNA in the interior of capsules. The encapsulation method involves the adsorption of DNA onto positively charged silica particles, followed by the LbL deposition of poly(methacrylic acid) (PMA) and poly(vinylpyrrolidone) multilayers, multilayer film crosslinking, and removal of the template silica particles. A signature feature of this hydrogen-bonded multilayer film is its instability above pH 7 when the PMA becomes ionized. To impart stability at neutral pH, they used thiol-modified PMA (PMASH). When oxidized within the multilayer, disulfide bridges between PMASH chains provide stabilization to the film/ capsules. These capsules are stable at physiological pH but degrade, releasing their cargo once the disulfide linkages are destroyed in a reducing environment. The capsules formed are monodisperse in size and exhibit uniform DNA loading. The functional integrity of the encapsulated DNA, both ssDNA and dsDNA, is largely maintained (Zelikin et al., 2007). Many papers are reported dealing with the controlled release of molecules, which are encapsulated in microspheres, by simple diffusion or under action of the external stimuli (Morimoto et al., 2015; Mor´an et al., 2015; Zelikin et al., 2007; Elazar et al., 2010). Still, the efficiency of the transport of encapsulated molecules between microspheres is expected to be low because of the simple, uncontrollable diffusion of the released molecules. In addition, the released molecules could be encapsulated into other microspheres under the same conditions with release in the absence of receptors. Therefore, construction of a contact-dependent transport system would improve the effective transport of molecules between microspheres (Morimoto et al., 2015). Morimoto et al. (2015) have prepared microspheres through self-assembly of PEG-block-poly(3-dimethyl(methacryloyloxyethyl)ammonium propane sulfonate) (PEG-b-PDMAPS) by the intermolecular dipole
dipole interaction of sulfobetaine side chains in water. Into the PEG-b-PDMAPS microsphere they encapsulated by thermal treatment a 30 -tetramethylrhodamine-labeled ssDNA oligomer (ssDNA) to obtain

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FIGURE 6.5 Schematic representation of interaction, encapsulation, and contact-dependent transport/double-strand formation of ssDNA encapsulated in PEG-b-PDMAPS microsphere. Reprinted with permission from Morimoto, N., Muramatsu, K., Nomura, S. and Suzuki, M., 2015. Trading polymeric microspheres: exchanging DNA molecules via microsphere interaction. Colloids Surf. B Biointerfaces, 128, 94
99.

vehicles for contact-dependent transport (Fig. 6.5). Below the upper critical solution temperature (UCST) of PEG-b-PDMAPS, the microspheres (B1 μm) interact with other microspheres by partial and transit fusion and the rate of double-strand formation in the microsphere was controllable by the composition of PEG-b-PDMAPS and the UCST.

6.3.3 BINDING OF NUCLEIC ACIDS TO POLYMERIC NANOPARTICLES For binding to nanoparticle supports, most esearch has focused on DNA, not RNA. Typical modes of DNA binding involve: (1) alkylthiol- or disulfide-terminated oligonucleotides on a metal nanoparticle surface; (2) covalent binding of oligonucleotides to a preactivated nanoparticle surface; and (3) absorption of biotinylated oligonucleotides on surfaces coated with avidin or streptavidin (Li et al., 2002; Demers et al., 2000; DeLong et al., 2009).

6.3.4 SELF-ASSEMBLY OF NUCLEIC ACIDS Drugs derived from nucleic acids are beginning to make an impact on the nanomedicine scene (Kawasaki and Player, 2005). It is a demonstrated fact that the features of nucleic acids are not limited only to properties providing template-dependent biosynthetic processes. Studies of DNA and RNA unveiled unique features of these polymers able to make various self-assembled three-dimensional structures using the complementarity principle (Fig. 6.6) (Rudchenko and Zamyatnin, 2015). The self-assembly of nucleic acids to obtain therapeutic nanocarriers represents an alternative strategy to overcome the drawbacks of the conventional delivery nanoparticles, such as liposomes and polymeric systems, which are heterogeneous in size, composition, and surface chemistry,

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FIGURE 6.6 Construction of DNA-based 3D structures. (A) Self-assembly of oligonucleotide into a structure containing vertices and three parts of cube faces; (B) diagram of a cube assembled from deoxyribo-oligonucleotides; (C) diagram of a triangle assembled from deoxyribo-oligonucleotides; (D) different tiling elements assembled from deoxyribo-oligonucleotides; (E) diagram of microtubule assembled from DNA molecules. DNA nucleotide sequences are designated by long broad lines; hydrogen bonds connecting nucleotides according to the principle of complementarity are designated by short perpendicular lines. Reprinted with permission from Rudchenko, M. and Zamyatnin, A. (2015). Prospects for using self-assembled nucleic acid structures. Biochemistry (Moscow), 80(4), pp. 391
399.

leading to suboptimal performance, lack of tissue specificity, and potential toxicity (Lee et al., 2012a; Whitehead et al., 2009; Oh and Park, 2009; Lv et al., 2006). The rapidly emerging DNA nanotechnology began with the pioneer Seeman’s hypothesis that DNA can not only carry genetic information but also can be used as a molecular organizer to create well-designed and controllable nanomaterials for applications in materials science, nanotechnology, and biology (Tam and Lo, 2015). Seeman (1982) demonstrated the importance of the DNA nanotechnology research domain, proposing to construct ordered arrays using branched DNA-building blocks. He stated that “it is possible to generate sequences of oligomeric nucleic acids which will preferentially associate to form migrationally immobile junctions, rather than linear duplexes, as they usually do” (Seeman, 1982). Later, his group designed and constructed modified Holliday junctions to convert one-dimensional DNA strands into branched DNA tiles with sticky ends at the edges that can work as anchoring sites for further assembly of 2D structures (Seeman, 2003; Kallenbach et al., 1983; Ma et al., 1986; Winfree et al., 1998; Tam and Lo., 2015). However, the major revolution in “structural DNA nanotechnology” came in 2006 when Rothemund (2006) introduced the concept of “DNA origami” (Fig. 6.7), a new programmed DNA assembly system based on the folding of a long ssDNA (named as a “scaffold strand”), with the help of hundreds of sequence-designed complementary short strands (called “staple strands”) (Kundu et al., 2017). The structural features of nucleic acids form the basis of constructing a wide variety of DNA nanoarchitectures with well-defined shapes and sizes, in addition to controllable permeability and flexibility. More importantly, self-assembled DNA nanostructures can be easily functionalized to construct artificial functional systems with nanometer-scale precision for multiple purposes (Tam and Lo, 2015).

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FIGURE 6.7 Rothemund’s DNA origami constructed using a single-stranded circular phage M13 DNA is shown. The nucleotide sequence of phage M13 DNA is designated by the long broad line used as the basis for constructing DNA origami. Nucleotides that make folds in phage DNA after interacting with it are designated by short arrows (50 -30 ). Reprinted with permission from Rudchenko, M. and Zamyatnin, A., 2015. Prospects for using self-assembled nucleic acid structures. Biochemistry (Moscow), 80 (4), 391
399.

Zheng et al. (2013) have obtained a new multifunctional spherical nucleic acids (SNAs) platform based on self-assembly of DNA biopolymer to be used in cancer therapy. They have engineered a nanoparticle-conjugated initiator that triggered a cascade of hybridization reaction resulting in the formation of a long DNA polymer as the nanoparticle shell. Then, by employing different DNA fragments, self-assembled multifunctional SNAs were constructed (Zheng et al., 2013). An interesting approach for DNA delivery was developed by Kim et al. (2015) using rolling circle amplification (RCA), an enzymatic process that produces an ssDNA from a circular template, obtaining an oligonucleotide [two antisense oligonucleotides (ASOs)] delivery system. For loading ASO to the delivery system they used sequence-specific hybridization. In this study cationic Mu peptides derived from the adenovirus core complex were used to condense ASO-hybridized RCA products and produce DNA nanoballs. The system obtained by Kim et al. (2015) was intended to be used in tumor-targeted delivery so that the surface of ASO-loaded DNA nanoballs was coated with hyaluronic acid (HA), which is a ligand for CD44 receptors overexpressed by tumor cells (Lee et al., 2012a). The notion of RNA nanotechnology gained attention at the end of the 20th century, and in 1998 the first confirmation for the RNA nanostructures construction through the self-assembly of several re-engineered natural RNA molecules was reported (Guo et al., 1998). Even if the

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folding properties of RNA and DNA differ in certain features regarding the base-pairing complementarity, in RNA nanotechnology, the fundamental principles found in DNA nanotechnology are applicable. Furthermore, the emerging field of RNA nanotechnology (Guo, 2005, 2010) appears to be more promising since RNA can easily be transcribed into a single strand, which can be directly folded into a programmed nanostructure in cells (Kundu et al., 2017). In addition to being carriers of genetic information, RNAs are now recognized to function as natural scaffolds, enzymes, switches, aptamers, and regulators of gene expression and editing. The emerging field of RNA nanotechnology applies the current knowledge related to the structure and function of natural and artificial RNAs to further address specific biomedical challenges by engineering nanodevices that can interact with cellular machinery (Guo, 2010; Afonin et al., 2013; Halman et al., 2017). A few reports have been found concerning self-assembled DNA/RNA nanoparticles. These are usually administered by injection directly into the target tissue or systemically into the circulation via intravenous injection. In addition to optimizing DNA/RNA nanoparticle structural complexity, introducing chemical modifications to the backbone and individual nucleotides are commonly used to improve the stability of DNA and RNA oligonucleotides in biological matrices and to increase their circulation time following systemic administration. Despite the increased oligonucleotide stability and prolonged circulation time, self-assembled DNA/RNA nanoparticle delivery to target tissues and cells requires additional approaches. These approaches are similar to those used for traditional TNAs (Dobrovolskaia, 2016). Lee et al. (2012b) prepared monodisperse nanoparticles through the self-assembly of complementary DNA strands obtaining DNA tetrahedral nanoparticles with a well-defined size that can deliver siRNAs into cells and silence target genes in tumors.

6.4 U.S. FOOD & DRUG ADMINISTRATION
APPROVED CELLULAR AND GENE THERAPY PRODUCTS Table 6.1 lists the licensed products from the Office of Tissues and Advanced Therapies (OTAT) (U.S. Food & Drug Administration, n.d.). During a press briefing, Gottlieb heralded Luxturna as the FDA’s third gene therapy approval in 2017. In August 2017, the agency cleared Novartis’ Kymriah, a CAR T-cell immunotherapy in which a patient’s immune cells are removed, injected with DNA outside the body, and returned to the bloodstream to fight leukemia. A second CAR T-cell therapy, Gilead Sciences’ Yescarta, was approved in October 2017 (InChemistry, 2017). In Europe only one gene therapy was approved by the European Medicines Agency, namely Glybera (alipogene tiparvovec), which is a gene therapy indicated for the treatment of lipoprotein lipase deficiency (LPLD). It was developed by Amsterdam Molecular Therapeutics (AMT), which was acquired by uniQure in April 2012. In October 2012, uniQure received marketing authorization approval for Glybera from the European Commission (EC) for treating patients with LPLD who have chronic and acute pancreatitis attacks. Glybera was the first gene therapy to be licensed in Europe, but the license expired October 25, 2017 and was not renewed (Drug Development Technology, n.d.; Science Business, 2017).

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Table 6.1 The List of Cellular and Gene Therapy Products Approved by the U.S. Food & Drug Administration (FDA) Trade Name/Company

Developer

Description

ALLOCORD (HPC cord blood)

SSM Cardinal Glennon Children’s Medical Center Cleveland Cord Blood Center New York Blood Center

An allogeneic cord blood hematopoietic progenitor cell therapy indicated for use in unrelated donor hematopoietic progenitor cell transplantation procedures in conjunction with an appropriate preparative regimen for hematopoietic and immunologic reconstitution in patients with disorders affecting the hematopoietic system that are inherited, acquired, or result from myeloablative treatment

CLEVECORD (HPC cord blood) HEMACORD (HPC, cord blood) Ducord, HPC cord blood HPC, cord blood

HPC, cord blood—LifeSouth HPC, cord blood—Bloodworks LAVIV (Azficel-T)

Duke University School of Medicine Clinimmune Labs, University of Colorado Cord Blood Bank LifeSouth Community Blood Centers, Inc. Bloodworks Fibrocell Technologies

MACI (autologous cultured chondrocytes on a porcine collagen membrane)

Vericel Corp.

GINTUIT (allogeneic cultured keratinocytes and fibroblasts in bovine collagen)

Organogenesis Incorporated

IMLYGIC (talimogene laherparepvec)

BioVex, Inc., a subsidiary of Amgen Inc.

KYMRIAH (tisagenlecleucel)

Novartis Pharmaceuticals Corporation

An autologous fibroblast product indicated for improvement of the appearance of moderate to severe nasolabial fold wrinkles in adults Autologous cellularized scaffold product based on cultured chondrocytes on a resorbable porcine type I/III collagen membrane indicated for the repair of symptomatic, single or multiple fullthickness cartilage defects of the knee with or without bone involvement in adults Allogeneic cellularized scaffold product, consisting of human keratinocyte and fibroblast cells, human extracellular matrix proteins, and bovine collagen, indicated for topical (nonsubmerged) application to a surgically created vascular wound bed in the treatment of mucogingival conditions in adults IMLYGIC is a genetically modified oncolytic viral therapy indicated for the local treatment of unresectable cutaneous, subcutaneous, and nodal lesions in patients with melanoma recurrent after initial surgery KYMRIAH is a CD19-directed genetically modified autologous T-cell immunotherapy indicated for the treatment of patients up to 25 years of age with B-cell precursor acute lymphoblastic leukemia (ALL) that is refractory or in second or later relapse (Continued)

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Table 6.1 The List of Cellular and Gene Therapy Products Approved by the U.S. Food & Drug Administration (FDA) Continued Trade Name/Company

Developer

Description

LUXTURNA/Spark Therapeutics—DNA-filled viruses injected directly into the eye is now the first gene therapy approved (December 20, 2017) in the US to treat a genetic disease. First gene therapy for genetic disease approved PROVENGE (sipuleucel-T)

Spark Therapeutics Inc

LUXTURNA is an adeno-associated virus vectorbased gene therapy indicated for the treatment of patients with confirmed biallelic RPE65 mutationassociated retinal dystrophy. Restores vision in patients with an inherited form of blindness

Dendreon Corp.

YESCARTA (axicabtagene ciloleucel)

Kite Pharma, Incorporated

PROVENGE is an autologous cellular immunotherapy, containing autologous CD54 1 cells activated with PAP-GM-CSF, indicated for the treatment of asymptomatic or minimally symptomatic metastatic castrate-resistant (hormone-refractory) prostate cancer YESCARTA is a CD19-directed genetically modified autologous T-cell immunotherapy indicated for the treatment of adult patients with relapsed or refractory large B-cell lymphoma after two or more lines of systemic therapy, including diffuse large B-cell lymphoma (DLBCL) not otherwise specified, primary mediastinal large B -cell lymphoma, high-grade B-cell lymphoma, and DLBCL arising from follicular lymphoma

U.S. Food & Drug Administration. (n.d.) Approved cellular and gene therapy products, ,https://www.fda.gov/ BiologicsBloodVaccines/CellularGeneTherapyProducts/ApprovedProducts/default.htm. (accessed 05.02.2018).

6.5 CONCLUSIONS AND FUTURE TRENDS Impressive efforts have been made in the last few decades to develop new carriers for gene delivery. Even though viral vectors dominate in therapeutic clinical trials, synthetic vectors have gained attention because of the advantages they have demonstrated. The use of biocompatible polymeric nanoparticles in gene delivery has brought considerable development to this area. Polymeric nanoparticles can be a great alternative to conventional gene-delivery carriers, as it has been demonstrated that they have less side effects and better targeting. It is a common challenge to prevent degradation of DNA when used in nanotherapies. To prevent degradation, DNA can be condensed and/or protected by a physical barrier. There are different strategies employed in gene therapy to limit DNA degradation, including complexation of DNA with polycations or by encapsulation. Although various synthetic vectors for gene delivery have been developed over time, there is not one that can be used in different disease conditions and there are still several problems for the

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clinical application of nanoparticle-based gene therapy. Accordingly, depending on the final use, a suitable nonviral carrier for gene delivery can be selected; however, in-depth in vivo studies are necessary. The use of polymer nanoparticles in gene therapy is a relatively recent strategy that requires particular attention in the future, mainly regarding the need for clinical evaluation of these synthetic vectors.

ACKNOWLEDGMENT The authors acknowledge the financial support given by the Romanian Academy and UEFISCDI through research projects.

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FURTHER READING Giacca, M., 2010. Gene Therapy. Springer, Milano, pp. 139
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