Overview of Therapeutic Polynucleotides

Therapeutic nucleic acids (TNAs) and their precursors are applied to treat several pathologies and infections. TNA-based therapy has different rationales and mechanisms and can be classified into therapeutic nucleotides and nucleosides, therapeutic oligonucleotides, and therapeutic polynucleotides. Nowadays, therapeutic polynucleotides are widely used compared with less effective conventional clinical procedures for the treatment of many diseases such as cancer, inherited disorders, and viral infections. There are many genetic materials that have been investigated for nucleotide or polynucleotide delivery into cells, such as plasmid DNA (p-DNA), single-stranded DNA (ssDNA), and antisense oligonucleotides. Due to the degradation of genetic material in a biological medium, a carrier system is required to efficiently deliver a therapeutic agent into the cell. Polynucleotides are mainly employed in molecular biology, principally for the diagnosis and monitoring of hereditary diseases, in forensic science, and in parentage testing for the analysis of genetic fingerprints for DNA profiling.

Introduction of Polynucleotides

A polynucleotide is a polymer molecule composed of 13 or more ribonucleotides or deoxyribonucleotides. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are examples of polynucleotides with distinct biological functions. Both DNA and RNA are linked together by phosphodiester bonds between the ribose moiety of the nucleotides. This creates a "ribose-phosphate" backbone, a 5'-end that is phosphorylated, and a free 3'-hydroxyl at the 3'-end. Normally, DNA is a double-stranded macromolecule. DNA is a versatile macromolecule that stores genetic information in all living organisms, including eukaryotes and prokaryotes. In contrast, there are three main types of RNA molecules: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). These RNAs are single-stranded molecules that can usually form secondary structures through complementary base pairing.

Analysis of the Secondary Structure of Polynucleotides

Circular dichroism (CD) and optical rotatory dispersion (ORD) are used to study the conformation, folding, and interactions of nucleic acids. CD spectra of polynucleotides are sensitive both to their sequence and conformation and can provide a good deal of structural information. Nucleic acids are polymers of nucleotides that consist of purine or pyrimidine bases attached to a phosphorylated sugar. Monomeric sugars are asymmetric, and they induce a small amount of ellipticity in the attached planar-symmetric base. Polymerized nucleic acids, however, are rigid and highly asymmetric. In the polymerized nucleic acids, the bases stack with one another in precise orientations depending on the conformational state. Even very simple polynucleotides show multiple conformational states and complex CD and ORD spectra. Compounds as small as homodimers of (deoxy)ribonucleotides show evidence of base stacking in solution from measurements of optical activity. Theoretical calculations of the CD spectrum agree with a model where the bases are stacked in a parallel fashion in the dinucleotide. The stacking leads to hypochromicity in the absorption spectrum and the splitting of the transitions into two transitions, one perpendicular and one parallel to the helix axis, with equal strength and opposite rotatory strength. The circular dichroism spectra of nucleic acids are complex compared to those of homopolynucleotides because four different bases contribute to the CD spectra and the spectra are sequence-dependent. In addition, nucleic acids display great conformational diversity and may form single-, double-, or triple-stranded helices. The various oligomerization states of the polynucleotides, moreover, can exist in multiple conformations.

Breakdown of Polynucleotides

The polynucleotides DNA and RNA, although relatively stable in most tissues, turn rapidly in dividing cells. Both DNA and RNA must first be degraded to their constituent mononucleotides, which are themselves degraded further. A variety of enzymes capable of hydrolyzing the phosphodiester bonds have been described, including ribonucleases specific for RNA and deoxyribonucleases for DNA, as well as nonspecific nucleases, phosphorylases, and phosphomonoesterases. Purine and pyrimidine (deoxy) nucleotides are degraded to the corresponding (deoxy) nucleosides by specific 5' nucleotidases. Different purine endo- or ecto-5' nucleotidases have been identified with different substrate specificities and may be of particular importance in providing bases for nucleotide resynthesis in tissues where there is rapid cell turnover and massive cell death (e.g., thymus, spleen, bone marrow). Salvage is an active process for both pyrimidines and purines. Consequently, only a small fraction of the nucleotides turned over daily are actually degraded and lost to the body. The pyrimidine bases, uracil, and thymine, derived from nucleosides not recycled, are degraded further to the β-amino acids, and there is thus no measurable end product. However, such loss is probably comparable with that for purines, the normal end product of which in humans is uric acid, formed from the precursor purine bases xanthine and hypoxanthine by the action of xanthine dehydrogenase.

Delivery of Therapeutic Polynucleotides

Delivery of polynucleotides into patient cells is a promising strategy for the treatment of genetic disorders. Gene therapy aims to either synthesize desired proteins (DNA delivery) or suppress the expression of endogenous genes (siRNA delivery). Carriers constitute an important part of gene therapeutics due to limitations arising from the pharmacokinetics of polynucleotides. Non-viral carriers such as polymers and lipids protect polynucleotides from intracellular and extracellular threats and facilitate the formation of cell-permeable nanoparticles through shielding and/or bridging multiple polynucleotide molecules. The formation of nanoparticulate systems with optimal features, their cellular uptake, and intracellular trafficking are crucial steps for effective gene therapy.

The triblock copolymer of poly(2-methyl-2-oxazoline-b-tetrahydrofuran-2-methyl-2-oxazoline) has been synthesized, suggesting that, as compared with neutral block copolymers, charged (positively charged) polymers are a better vector for gene delivery. But the positive charge of the vector causes cytotoxicity and instability in body fluid. To overcome this problem, a star-shaped carrier system with an ethylenediamine moiety at the center has been designed and attached to four PEO/PPO blocks. DNA interacts with the copolymer through hydrogen bonding, electrostatic force, and hydrophobic interaction, and DNA present at the surface develops a negative charge. This system is effective for gene transfer in heart muscle and skeletal muscle-related diseases.

Additionally, methods have been developed to deliver aerosolized formulations containing naked or formulated and concentrated polynucleotides to specific regions of the respiratory tract. The different areas of the respiratory tract can be targeted by 1) adjusting the size of particles of aerosol and/or 2) adjusting the volume inhaled during delivery. Aerosols may be used to transport naked or condensed and formulated polynucleotides via the lung into the lymph, blood, macrophages, or other cells of the body. Several conventional pharmaceutical therapies for pulmonary diseases could be supplanted by gene transfer therapies. Delivery to the lung of polynucleotide therapeutics has proved more difficult than delivery of small molecule therapeutics, in part due to the larger size of polynucleotides and their greater susceptibility to physical disruption from the forces required to generate an aerosol, thereby hindering or preventing efficient therapy. In vivo, systemic expression of genetic material introduced into the respiratory tract has also been used to provide therapeutically effective levels of a secreted cytokine. Another therapeutic approach involving polynucleotide administration is the generation of an immune response in the absence of a viral vaccine. The introduction of expression vectors into animals generates an immune response to the expressed protein. This technique is useful, for example, when a viral vaccine is difficult to produce, or a nonpathogenic strain of the virus is not available. Administration of such expression vectors to the lung can yield immune responses without the disadvantages associated with injections and may be directed to pathogens affecting the respiratory tract such as influenza virus, respiratory syncytial virus, hantavirus or adenovirus, and respiratory tract disorders such as asthma.

Roles of Therapeutic Polynucleotides in Disease Treatment

The DNA polymeric molecules polydeoxynucleotide (PDRN) and polynucleotide (PN) can be used as a new alternative treatment for osteoarthritis (OA). Arthritis can be triggered by several factors, such as aging, genetic factors, stress, and inflammation, and presents either inflammatory or non-inflammatory manifestations. OA, a degenerative condition and one of the most prevalent causes of disability among elderly populations, is involved in non-inflammatory arthritis. PDRN is a mixture of deoxyribonucleotide polymers with chain lengths ranging from 50 to 2000 bp that result from enzyme degradation and comprise simple nucleotides, nucleosides, and bases. PDRN-related mechanisms are mediated through the activation of the adenosine A2A receptor, which is involved in the modulation of the oxygen supply/demand ratio by vasodilation, ischemic post-conditioning, anti-inflammation, and angiogenesis. PN consists of higher-order polymerized molecules relative to those of PDRN and is capable of promoting physiological repair related to intra-articular OA treatment. Proinflammatory cytokines (IL-1β and IL-6) and/or chemokines (IL-8 and CCL3) are capable of inducing cartilage destruction and contributing to OA progression through inflammation. It has been found that treatment of an OA cell model with PDRN and PN suppressed inflammation by attenuating the expression of these molecules.

Nowadays, approaches based on polynucleotide ocular delivery hold great promise since they may alter gene expression without affecting the structure and sequence of the gene. The eye is an attractive organ for the development of polynucleotide-based therapies due to the fact that the target tissues are accessible without the need for systemic administration. However, apart from this, the eye is protected by extraordinary barriers that are very difficult to circumvent, especially in the case of hydrophilic and high-molecular-weight molecules such as polynucleotides. Although therapies involving polynucleotides for treating ocular diseases are in an early development stage, preliminary human clinical trials are beginning to show promising results. Currently, there are two FDA-approved nucleic acid-based drugs for eye conditions: Vitravene®, an antisense oligonucleotide antisense oligonucleotide (ASO) for cytomegalovirus-induced retinitis treatment in immunocompromised patients, and Macugen®, an aptamer designed to treat wet age-related macular degeneration (AMD). In principle, polynucleotide-based drugs do not comply with the best physicochemical properties to be used as effective drugs. However, their chemical modification and the development of easy-to-produce nanocarriers offer a great window of opportunity for the exploitation of these new therapies. Currently, several ocular diseases of the anterior and posterior segments of the eye, such as glaucoma, AMD, ocular pain associated with dry eye, and choroidal neovascularization (CNV), are under gene-based clinical trial evaluation. The development of effective future ocular treatments will be a combination of understanding the disease's genetic basis as well as improving and developing long-term and nontoxic ocular drug delivery systems for both segments of the eye. It is believed that AS-ODN and RNA-based therapeutics, especially siRNA (small interfering RNA)-based as it is a potent inhibitor of protein expression, will continue further development, reaching the market in a reasonable time frame being the major task to achieve their nano-system delivery along with investigating alternative routes of administration.

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