INVESTIGATION ON NUCLEIC ACID INTERACTION WITH VARIOUS CATIONIC LIGANDS FOR GENE DELIVERY APPLICATIONS

Abstract

A new paradigm in medicine called gene therapy involves altering of genes entailed in disease by delivering therapeutic gene (nucleic acid based drugs). Delivering such therapeutic gene to the specific target site is an irresistible challenge to drug delivery scientists. However, the polyanionic nature of DNA limits the cellular membrane interactions. Hence, successful therapeutic DNA based gene delivery highly relies on the efficient extracellular and intracellular delivery of DNA molecules for empowering target interaction.(1-5) Various Carrier molecules have been designed specifically to enter cells & deposit therapeutic genes. Vectors can be viral or non-viral. Viral vectors are one of the successful gene delivery systems available, such as retrovirus, adenovirus (types 2 and 5), adeno-associated virus, herpes virus, pox virus, human foamy virus (HFV), and lentivirus. The genomes of the viral vector have been modified by deleting some areas of their genomic sequences so that their replication becomes deranged and it makes them more safe, but these viral systems has accompanied with some problems, such as their marked immunogenicity that causes induction of inflammatory system leading to degeneration of transducted tissue; and toxin production, including mortality, the insertional mutagenesis; and their limitation in transgenic capacity size. These limitations could be overcome by using synthetic non-viral gene carrier systems. (6) Non-viral vectors are being used in improving potential therapeutic effect of DNA because they are safe and non-immunogenic as compared to viral vectors. Even though lower transfection efficiency of non-viral vectors are proven compared to viral-mediated gene transfection, the observed shortcomings could be overcome by appropriate structural designing of carriers like cationic polymers or lipids. For example, non-viral vectors can easily be targeted to a target tissue or cell by coupling of cell- or tissue-specific targeting moieties on the carrier. Additionally, by controlling Synopsis iv the size of DNA-cationic vector complex the bio-distribution, cellular internalization and intracellular trafficking of the micro- or nanoparticle can be influenced. Most importantly, the success of the non-viral gene therapy is dependent on the various extra- and intracellular barriers that affect the efficiency of all gene delivery systems, including cellular uptake, endosomal escape, nuclear uptake and gene expression.(7-9) Poly cationic vector such as poly (amidoamine) (PAMAM) dendrimer is one of the versatile non-viral based gene delivery systems for efficient targeted delivery of DNA amongst numerous other cationic vectors.(10-12) In particular, PAMAM dendrimer is shown as a potential vehicle especially through several routes of administration, including oral, ocular, parenteral and transdermal.(10,13,14) The uniqueness of these dendrimers are that they are monodisperse, hyperbranched and of controlled size and molecular weight, possessing number of terminal groups with the increasing generation number.(15) More interestingly, PAMAM-DNA complexes have been found to be highly soluble and stable in almost all the physiological conditions and also observed to be resistant to nuclease digestion by Bielinska et al.(16) These distinctive characteristics of PAMAM dendrimer coupled with their architecture has attracted attention of several research groups and their applications in biology and medicine have been explored.(10,17-19)Additionally, various research groups also commenced to determine dendrimer toxicity and immunogenicity and reported that cytotoxicity increases with increase in generation number i.e. smaller the generation of PAMAM dendrimer less the chances of cytotoxicity.(20,21)In spite of high cost and high cytotoxicity, there exist enormous successful targeted transfection reports using unmodified and modified dendrimers of higher generations.(11,21-23) It is pertinent to mention that even though PAMAM dendrimer-based gene transfection reagents such as Superfect and Priofect are already commercially available, these products are based on toxic high generation dendrimers and also not cost effective.