As described in the introduction to this chapter, nucleic acid-based therapeutics are attracting increasing interest. Gene replacement therapies and gene vaccines are no longer the only targets, as antisense oligonucleotides, which suppress or block expression of certain proteins, are now considered efficient and promising future therapeutics for clinical development. Currently, a number of DNA vaccines and oligonucleotides are in clinical trials for treatment of infectious diseases including AIDS/HIV infections, different stages and types of cancer, cardiovascular and neurological diseases, as well as genetic deficiency disorders, and autoimmune and metabolic diseases (1,166). To date, however, few products have been approved, one being Vitravene® (Ciba Vision/Isis Pharmaceuticals), an injectable that was approved by Food and Drug Administration (FDA) in 1998 for the treatment of cytomegalovirus retinitis in patients with AIDS (1,2). However, the drug has currently the marketing status of discontinued (167). Another product is Macugen® (Osi Eyetech), which is an aptamer RNA oligonucleotide, which binds to extracellular vascular endothelial growth factor (VEGF)(2,168). Other products are Gendicine® (Shenzhen SiBono GenTech) and HlOl/Oncorine® (Shanghai Sunway Biotech), which are both approved by the Chinese State FDA with the indication of cancer treatment supplementing chemotherapy. The latter two products are adenovirus vector-based p53 gene therapeutics (2,169).
Because of the high-sequence specificity of nucleic acid-based therapeutics, and thereby high potencies and low levels of off-target effects, the use of gene medicine seems promising. However, as for peptide and protein therapeutics, one of the major challenges is to efficiently deliver the nucleic acid-based drug to the target organ as well as to the intracellular target.
By nature, the nucleic acid therapeutics have a high negative charge density and are enzymatically labile hydrophilic biomacromolecules; thus, efficient delivery of these therapeutics faces similar challenges as the peptide and protein therapeutics. Irrespective of whether the nucleus (in the case of DNA delivery) or the cytoplasm (e.g., for siRNA delivery) of the cell is the intracellular target site, protection and targeting approaches are required to efficiently deliver the drug.
Although the siRNA duplexes used in gene silencing therapy are more resilient toward degradation by nucleases than single-stranded DNA, unmodified sequences of nucleic acids are inherently unstable in biological systems and need protection against degradation by either chemical modification or by use of a protecting delivery system. Chemical modifications to increase half-lives as well as binding affinities include sugar and backbone modifications, resulting in morpholino and peptide nucleic acid (PNA) analogs, sugar modifications by 2'-position modifications of the sugar moieties, resulting in formation of locked nucleic acids (LNA), heterocyclic modification as well as conjugations of fatty acids and cholesterol (170). Example of promising therapeutics is the HIF-1 a-antagonist developed from the LNA-platform and currently in clinical trials for treatment of solid tumors and lymphoma.
Parenteral administration is the primary route of testing delivery for nucleic acid therapeutics irrespective of whether systemic or local effects are desired. However, to some extent, pulmonary and oral routes are also investigated as potential routes for local targeting to treat cystic fibrosis or colonic tissue (171-173). For nonparenteral delivery, the use of pharmaceutical excipients in the formulation is critical. In addition, the production costs of nucleic acid therapeutic-containing drug delivery systems should be minimized. Even for intravenously or subcutaneously injected nucleic acid-based therapeutics, the use of protective carriers is most likely necessary, and advantageous as compared to injection of the naked RNA or DNA. Carriers can be divided into viral or nonviral carriers, with the viral vectors based on adeno- or retrovirus. As a result of the overall negative charge of the nucleotides, the nonviral carriers are most often based on positive carriers that complex with, encapsulate, or are even covalently conjugated to the siRNA. One of the most-studied carrier systems is liposomes, containing cationic lipids in which the biomacromolecule is encapsulated and which facilitate interaction with the cellular plasma membrane components. The liposomes may be modified by PEG. Thereby, a prolonged systemic circulation and passive targeting to inflammatory and cancer tissue are obtained due to the enhanced retention and permeability effect. In addition, active targeting moieties as for example folate or the vasculature targeting arginine-glycine-aspartate (RGD) peptide may be incorporated into the liposomal structure to achieve better delivery efficiencies (174). One of the main concerns with positively charged lipids and positively charged polymers such as polyethyleneimine (PEI) is their toxicity. However, the cationic charge provides the advantage of high loading efficiency but the disadvantage of tissue toxicity, thus surface modifications are necessary. Other approaches to deliver nucleic acids to the appropriate cellular interior are the use of cationic peptides conjugated or complexed with the nucleic acid (16,175), fusogenic lipids or protein sequences (176178), and localizing sequences, such as the nuclear localizing signal (NLS) sequences that mediate nuclear entrance from the cytoplasm (179).
Since the chemistry used in nucleic acid drug research has reached the current level, the major limiting step is delivery of the therapeutic, and several technological challenges need to be overcome in terms of stability, efficient and controlled delivery as well as addressing safety. So the limited number of currently registered products is by no means indicative of failure of these potentially revolutionary types of drugs.
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