Potential Of Gene Silencing Techniques As A Therapeutic Strategy - Research Paper Example

Potential Of Gene Silencing Techniques As A Therapeutic Strategy Introduction RNomics has been defined as the study of RNA in regard to its regulatory role in gene expression, the significance of RNA in genetic diseases and the emergence of a new technology to treat genetic diseases by silencing abnormal functions associated with aberrant gene expression by using RNAs as a novel molecular medicine to correct the defects of abnormal patterns of gene expression. RNomics specifically deals with a category of RNA called microRNA, comprised of short RNAs of approximately 19-22 bases in length. An important mechanism to achieve gene silencing in eukaryotic systems is by means of this specialized class of regulatory ribonucleotides called microRNAs (Meister, 2007). Recent research has identified these small RNA molecules as major regulators of gene expression in the eukaryotic system whose primary role is in defining gene silencing mechanisms. These short single stranded RNAs function as post-transcriptional regulators of gene expression (Moazad, 2009). The cumulative results from many research studies suggest that RNA interference represents a very important mechanism of gene silencing in the eukaryotic system (Almeida and Allshire, 2005). Molecular mechanisms of gene silencing with clinical relevance Model of miRNA biogenesis/therapeutic modalities Post-transcriptional gene silencing mechanisms are associated with a phenomenon called “RNA Interference” (RNAi) which involves an inhibition of mRNA translational activities required for the production of specific proteins in a cell (Lin and Ying, 2006). MicroRNAs play an important role in mediating these post-transcriptional gene silencing regulatory functions. MicroRNAs are single stranded RNA molecules that silence the translational capacity of mRNA by binding to mRNA by complementary base pairing to produce regions of double-stranded RNA that cannot bind to the ribosome or hybridize to the anti-codons of tRNA; the net effect is to silence gene expression of specific mRNAs by blocking their translation (Hammond, 2006). MiRNAs were first discovered in Caenorhabditis elegans (C.elegans) where they were found to play an important role in the selective targeted post-transcriptional regulation of gene expression during embryonic development (Chua et al, 2009). Subsequent research showed that miRNAs are the product of non-coding regions of the eukaryotic genome. The results of the Human Genome Project demonstrated that about 97% of the human genome consists of non-coding DNA; only 3% of the genome contains protein-encoding genes (Hammond, 2006). Recent research on comparative genomics indicates that genes encoding microRNAs may have arisen from transposable elements (TE) (Piriyapongsa et al, 2007). This work identified 55 human miRNA genes that were derived from transposable elements based on sequence homology studies. These miRNAs may be involved in the broad spectrum regulation of many human genes. It was estimated there may be close to 100 additional TE derived miRNA genes in the human genome (Piriyapongsa et al, 2007). Although heterochromatin was discovered 80 years ago, only recently has its important role in directing gene regulatory mechanisms been revealed by research that has shown that these apparently “silent” regions of the genome are actually capable of synthesizing miRNAs that play an important role in regulating the translational activity of genes transcribed from regions of active euchromation (Lippman and Martienssen, 2004). Gene silencing is a major deterministic component of gene expression profiles and is facilitated by several important molecular mechanisms (Wegel and Shaw, 2005). Chromatin structure represents a global mechanism of generalized regulation of transcriptional activation that is based on physical access to promoter elements required for the initiation of transcription by RNA polymerases and initiation factors. Active genes are generally found in regions of decondensed chromatin called euchromatin whose open configuration facilitates the formation of active promoter complexes required to initiate transcription. In contrast, regions of condensed chromatin, referred to heterochromation represent highly coiled regions of DNA and proteins largely inaccessible to promoter binding proteins and are therefore, transcriptionally silent regions of the human genome (Zaratiegui et al, 2007). The most dramatic example of gene silenced heterochromation is the inactive copy of the X chromosome found in all female cells which is silenced at 8 weeks of embryonic life in every cell of the developing embryo. Chromatin remodeling can also be a dynamic process that is responsive to epigenetic signaling pathways and the changing transcriptional profile of cells at different stages of differentiation. The activation of genes by chromatin remodeling involves a reorganization of nucleosomes, complexes of DNA wrapped around a basic histone protein octamer to achieve a more open configuration of expanded chromatin loops where the DNA is accessible to binding transcriptional regulatory proteins (Wegel and Shaw, 2005). Recent research has demonstrated that short RNAs regulate the methylation of DNA to effect the formation of heterochromation and the silencing of specific genes in methylated region. RNA- directed DNA methylation may play an important role in the regulation of gene expression (Bayne and Allshire, 2005). Another mechanism by which small RNAs may effect transcriptional gene silencing (TGS) is by directly binding to gene promoters to block RNA polymerase access (Hawkins et al, 2009). This process is referred to as silent state epigenetic modifications and is directed to the transcriptional activation of specific genes (Lin and Ying, 2006). Recent research on the targeted silencing of the human ubiquitin c gene (UbC) by short RNA binding to the UbC promoter for a period of three days resulted in the long term silencing of this promoter, an event which was associated with increased histone methylation followed by increased levels of DNA methylationHollister and Gaut, 2009). The promoter associated RNA was an essential component of this gene silencing complex which also included DNA methyltransferase 3a and histone deacetylase. The prolonged association of DNA methyltransferase 1 was required for the long-term maintenance of gene silencing (Hollister and Gaut, 2009). The argonaute (AGO) proteins play an important role in mediating the gene silencing activities of small regulatory RNAs and comprise an important component of the RNA-inuced silencing complex (RISC) in the eukaryotic system. These proteins function as miRNA chaperones and induce strand dissociation of double-stranded miRNA (Takanashi et al, 2009). Clinical applications: role of miRNAs in disease/therapeutic possibilities Much current research is devoted to exploiting these regulatory molecules as targets for specific therapies designed to silence the functions of dysfunctional genes. Their use is projected to be of especial importance as anti-cancer therapeutics and agents to fight virus infections. There are several documented genetic disorders that result from aberrant miRNA activity. These include fragile X syndrome and Type 2 myotonic dystrophy. These disorders result from the aberrant gene silencing activities of miRNAs encoded by the introns of these protein coding genes (Hawkins et al, 2009). This type of miRNA is called intronic miRNA as it is encoded by the noncoding spacer regions of DNA called introns or intervening sequences that separate the c protein coding exons of many eukaryotic genes (Lin and Ying, 2006). The primary transcripts encoded by this type of gene, sometimes referred to as a “split gene”, may be processed post-transcriptionally to produce small single stranded RNA from the non-coding regions of the messenger RNA. The intronic miRNAs are generated by a mechanism different from that of intergenic miRNAs located in noncoding regions of the genome. Intronic miRNAs are generated by RNA polymerase II and splioceosome complexes. Intronic miRNAs have been identified in C.elegans, mice and humans (Lin and Ying, 2006. Recent research studies suggested that intronic miRNA is capable of inducing miRNA expression from intergenic regions in numerous species of mammals, fish, chickens and mice, suggesting common evolutionary origins (Hawkins et al, 2009). Clinical research studies have provided compelling evidence that the expression and activities of microRNAs are dysfunctional in many types of human malignancies, suggesting their potential use as clinical targets in the design of novel therapeutic modalities in the treatment of cancer (Monzo et al, 2008). Many of the most common human malignancies show evidence of aberrant microRNA expression. Among the human malignancies associated with abnormal miRNA activity include breast cancer, colon cancer and cancer of the cervix (Hawkins et al, 2009). Extensive studies of colon cancer have shown that microRNAs that are active only during early stages of embrogenesis of colon are re-activated in colon tumor cells suggesting that an altered regulatory profile associating with the expression of miRNAs who activities are suppressed in normal colon tissue may contribute to the genesis of this disease (Hawkins et al, 2009). These important clinical studies implicating miRNAs in the etiology of cancer suggest a possible therapeutic application to the treatment of this disease. In the realm of preclinical therapeutic studies involving the use of miRNAs, these studies have involved the use of high affinity antisense RNAs to bind and inactivate specific targeted microPNAs in rodent and primates have resulted in high level silencing of microRNA activites, suggesting potential therapeutic applications for these ologonucleotides as a cancer therapeutic (Petri et al, 2009). Protocols have also been developed for producing miRNAs to explore gene function by knock-out of specific ell targeted mRNAs as a first step in developing miRNAs for therapeutic purposes in molecular medicine (Heninger and Bucholz, 2007). E.coli RNAse III is used to digest long dsRNA into endoribonuclease-prepared short interfering RNAs (esiRNAs). This can be used for high throughput loss of function analyses. One can also synthesize pre-designed miRNAs from the database RiDDLE. PCR products with P7 promoters are transcribed and then hybridized to generate long double stranded RNA which can then be digested to generate overlapping esiRNAs which can be purified using spin columns (Heninger and Bucholz, 2007) (see diagram). . Another recently developed technology involves the use of “locked” nucleic acid which is a structural analogue of RNA that can bind its complement with extremely high affinity (Stenvang et al, 2008). These locked RNAs can therefore be used to screen tumors and other abnormal cells for the presence of specific miRNAs that may be important in the etiology of pathogenesis. Once identified, the microRNAs may also be used as a therapeutic target for locked complementary RNAs. Recent studies involving the use of locked miRNA to silence specific miRNAs in vivo and in vitro have produced excellent results suggesting these structural analogues may have useful application in the treatment of cancers with abnormal miRNA profiles (Stenvang et al, 2008). Technology Critique While researchers are intrigued by the potential applications of gene silencing technologies to the treatment of human diseases, there are many challenges that must be addressed to achieve a successful implementation of this novel targeted therapeutic approach. The significance of miRNAs in the etiology of cancer and other diseases with a genetic component is indisputable; however, there is a long road between discovery and application. Successful gene targeted therapeutics are very difficult to achieve and require a detailed understanding of the biological system. There has been much progress in this area. Since the discovery if miRNAs in 1993, there has been much progress in understanding their biogenesis and how they achieve post-transcriptional silencing. The study of microRNAs has also furthered our understanding of chromatin remodeling and gene regulatory mechanisms as the two regulatory modes are linked. The study of miRNAs has afforded a greater understanding of the human genome, particularly the non-gene coding component which is so vast and so poorly understood. This understanding of genomics and gene regulation will certainly aid in the appreciation of the structure and function of the human genome and the disorders which can arise from its dysfunction. There are many roadblocks that must be overcome, however, before these advances in understanding produce a clinical therapeutic application. Some of the current areas of concern with this developing technology include problems with efficacy, delivery methodologies, potential side effects and consistency of effect (Stenvang et al, 2008). One of the most important issues relates to the transient nature of the effect of microRNAs and MRNA silencing. These must be a continued application of the therapeutic to achieve sustained repression. This is a problem in general with post-transcriptional targets; they are continuously synthesized and require ongoing constitutive repression Stenvang et al, 2008). The most promising area for the development of novel miRNA therapeutics may be in the design of promoter blocking miRNAs that may mediate long ternm chromatin remodeling to an inactive configuration to control and silence the expression of dysfunctional genes Stenvag et al, 2008). The study of gene silencing pathways in the living system has opened the door to understanding basic biological phenomena and also has increased our understanding of the molecular basis of human disease. A combination of these approaches may be important in developing targeted therapeutics that employs miRNA-based technologies. References Almeida, R. and Allshire, R. C. (2005). Rna silencing and genome regulation. Trends in Cell Biology, 15(5):251-258. Azorsa, D., Mousses, S., and Caplen, N. (2003). Gene silencing through rna interference: Potential for therapeutics and functional genomics. Letters in Peptide Science, 10(3):361-372. Azuma-Mukai, A., Oguri, H., Mituyama, T., Qian, Z. 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