Gene Correction For Methods And Protocols

Introduction to MicroRNAs and their Regulation

Discuss about the Gene Correction for Methods and Protocols.

MicroRNAs (miRNAs) are a decently discovered class of small non-coding RNAs of length around 22-25 nucleotides, which participate in gene regulation at a post-transcriptional level. Lin-4 in C. elegans was the first miRNA identified, which plays significant role in the temporal control of post-embryonic development in the organism (He et al. 2005). Studies have revealed that apart from temporal control of developmental stages, miRNAs have widespread functions in various aspects of development and physiology. They usually bind to 3’ Untranslated Region in mRNAs that are found to be hugely conserved over a wide range of protein-coding genes. The target recognition of miRNAs span a region of 2-7 of the nucleotide sequence, located at the 5’ end (Griffiths-Jones et al. 2006). Its crucial functions demand the biogenesis of the RNAs to be strictly regulated at several levels and any dysfunction of its regulation is often associated with cancers and various neurodegenerative disorders. MiRNA genes are widespread and abundant in the genomes of various organisms and as many as 2,588 miRNA genes are found in human.  Precursor miRNAs are produced by RNA polymerase II which are then processed by RNase III enzymes DORSHA and DICER in the nucleus and cytoplasm of the cell respectively. Various non-canonical pathways are being discovered for miRNA biogenesis. The significant role played by the miRNA in maintain the normal physiology and development of an organism has enabled scientists to use it as a diagnostic as prognostic biomarker for various cancers, heart diseases and neurological disorders. It has been widely established that miRNA act as inhibitors of protein synthesis either by translational repression or by mRNA degradation. In addition to mRNA silencing Eiring et al. in 2010 found that miRNAs can interfere with functions of regulatory proteins to affect gene transcription. Further Lytle et al. (2007) elucidated that miRNA can bind to sites on the target miRNA other than 3’ UTR such as 5’ UTR binding sites. It has been stated that miRNA can also act as translation activator at certain extreme condition in the cell (Vasudevan, Tong and Steitz 2007).

Deregulation in miRNA biogenesis and consequently in the levels of miRNA are related to the incidence of various diseases that include cancers, autoimmune diseases, cardiovascular diseases and neurodegenerative disorders.

The role of miRNA was discovered in concern to chronic lymphocytic leukaemia. The regions where miR-15 and miR-16 genes are located were found to be deleted in chronic lymphocytic leukaemia, indicating its potential role as a tumour suppressor (Calin et al. 2002). Other studies over the years have established that miRNAs can act as oncogenes or tumour suppressors in pathways that have been related to cancer prognosis. Deregulation of miRNAs can be caused by several mechanisms that influence the transcription factors controlling the biogenesis of miRNAs in a target specific manner. The mechanisms may include mutation, deletion or amplification of miRNA genes or dysregulation of transcription factors. Several lines of studies have indicated that miRNA plays a dual role in cancer metastases. It has been found to up regulate cell migration and invasion both in vitro and in vivo. On the contrary other studies show that restored levels of certain miRNA in breast cancer can suppress metastasis by overall tumour growth and decreased time of relapse. Similarly studies have shown down regulated levels of miRNA in cardiac hypertrophy and heart failure. Autoimmune diseases like the most prevalent and chronic Psoriasis, Rheumatoid arthritis and lupus erythematous have been associated with dysregulation of various miRNAs.

Deregulation of miRNA and its Relation to Diseases

The functions of miRNA in neuronal development are well-established to this date. MiRNA has been used as a biomarker for several motor and neurocognitive diseases like Alzheimer’s, Huntington’s and Parkinson’s diseases. MiRNA is also related to the pathogenesis of several psychiatric disorders like Schizophrenia, autism, Fragile X syndrome, depression and addiction. Hence, the regulatory roles of miRNA in pathogenesis and diagnosis of neurodegenerative and psychiatric disorders have paved the way for therapeutic intervention in miRNA specific manner and extensive researches are being undertaken to explore the use of miRNA as a therapeutic tool.

The initial barrier to utilize miRNA as a therapeutic toll is to identify the specific gene coding for miRNAs and its tiny regulators. The conserved nature of its sequences has been exploited for the purpose of gene identification for miRNAs. Sequence conservation between various organisms has led to the miRNA genes have been identified by computational methods.

The detection of the miRNA targets in mRNA are another barrier for in vitro experimental purposes, although computational methods have evolved and are still developing to overcome this hurdle in large-scale experiments. The fact that levels of many miRNA are deregulated in different disease has encouraged scientists to explore the therapeutic applications of miRNA. The basic hurdle for such a feat is the delivery of miRNA to targeted regions. It has been found that naked miRNAs are degraded in vivo and hence delivery systems are required to develop therapeutic applications and restore normal levels of the molecule in diseased conditions (van Rooij, Purcell and Levin 2012). In case of neurodegenerative disease and psychiatric disorders the regions affected are primarily various parts of the brain. Currently two strategies, agonistic and antagonistic approaches are employed to modify abnormal levels of miRNA in the body. In agonistic approach the decreased miRNA levels are restored by miRNA mimics and in antagonistic approach overexpressed miRNAs are suppressed by anti-miR. As these naked molecules are prone to enzyme degradation in the body, efficient and stable delivery systems need to be developed.

Several strategies have been discovered to overcome this hurdle. Initially chemical modification of miRNA molecules such a replacing the phosphodiester bonds with other functional groups were proposed, however, such modifications led to production of toxic metabolic by-products and reduced miRNA activity (Rupaimoole et al. 2011). In this regard viral vector systems are a relatively older strategy. Many viruses have been studied for this purpose such as recombinant adeno-associated virus, retrovirus and lentivirus. The selection criteria depend on various factors like specific tropoism, efficient transgene expression and effectively crossing the blood-brain-barrier (Wen 2016). The primary drawback of using a viral vector as a delivery system is the immunogenicity of the virus and oncogenic transformation by viruses. Further, production of high-quality and high-quantity viral vectors is another barrier to the pharmaceutical and commercial companies, leading to limited application of viral vectors.

MiRNA's Role in Neurodegenerative and Psychiatric Disorders

On the contrary non-viral vectors have certain advantages like lack of immunogenicity, high stability and easy modification. Recent researches have explored various non-viral vector systems for miRNA therapeutic purposes. Some of them include lipid-based carriers, gold nanoparticles, cationic polymer based carriers, carbon based carriers and magnetic nanoparticles. Gaining knowledge of the pharmacokinetics of these particles and the need to produce disease specific carrier systems for long term gene expression or knockdown requires a lot of future research in this domain. Further, ethical issues regarding clinical targets is another overwhelming barrier for miRNA therapeutic applications.

Sickle cell disease is a group of inherited red blood cell disorders that affects the shape of the bloods cells reducing availability of functional blood cells and consequently the oxygen carrying capacity of blood. The shape of normal red blood cells is somewhat round facilitating easy passage through small blood vessels. It is an autosomal recessive disorder affecting chromosome 11. Normal haemoglobin is classified into groups A, A2 and F. In sickle cell disease an abnormal β chain results in the formation of haemoglobin S in which the β chain contains valine instead of glutamic acid (Bender and Seibel 2014). The abnormal haemoglobin S polymerizes resulting in deformed red blood cell shape. Retardation of blood flow, mechanical vaso-occlution, and lack of oxygen tension enhances the rate of polymerization of Hb S (Dubay, Krebs and Thresh 2015). Sickle cell disease is an example of point mutation of the β- chain gene of haemoglobin. The single amino acid difference (glutamic acid to valine) results in collapse of the red blood cells, making the body to produce more red blood cells to compensate for the loss. This puts an overwhelming burden on several other organs of the body casing various clinical symptoms. Typical symptoms of the disease include pain, acute chest syndrome, pulmonary hypertension, liver disease, cardiovascular abnormalities and neurological disorders. The disease is predominant in African Americans and the population of middle-east. The disease can be prevented by genetic examination of couples prior to conceiving. The treatment of the disease includes primarily pain management and other pharmacological interventions and in extreme cases surgery and organ transplantations.

The single point mutation found in sickle cell disease can be corrected by site specific double-strand breaks produced by the CRISPR/Cas 9 system (Chu et al. 2015). To have control over the particular sequence that has undergone a double stranded break i.e. the allele with a point mutation in the β chain of haemoglobin, we can use plasmid-based systems that can recognize and but DNA at specific sites. These plasmids can be used to cut the allele in chromosome 11 that has been mutated. A high degree of homology is required to repair the damaged DNA and restore the normal form of the allele so that normal β chains of Hb are produced. Three sgRNAs were constructed that can bind to the target site in the DNA. After binding, Cas9/sg RNAs will produce a double stranded break in the DNA which then will be repaired by homology directed repair (HDR) which will result in inserting the correct nucleotide into the allele and restoring the mutation. Linearized plasmids are required for transfection. However, recent studies conclude those single stranded donor oligonucleotides (ssODNs) are more efficient for about 50bp mutations or single point mutations (Alam et al. 2014). The templates for ssODNs can be 100-200 bps in length with a Cas9 break point at the centre of the template.

Delivery Systems for Therapeutic Applications of miRNA

Non-homologous end joining pathways can also be used to repair a double strand break. Studies report than non-homologous end joining pathways are more efficient in inserting plasmid genome into the genome of target (Davis and Chen 2013). The pathway recruits a wide range of proteins that perform synapsis, preparation and ligation of the cut DNAs. Similar to homology directed repair mechanism CRISPR/Cas9 system can be utilized to make cuts at the desired site in the chromosome. After the cuts the broken ends can be recognised by Ku70/Ku80 heterodimer. Recruitment of kinase, ligase and other factors hold the DNA together to form a paired complex which is then ligated to repair the break (Bétermier, Bertrand and Lopez 2014). Hence it can be used to insert and exogenous nucleotide by Cas9 mediated break.

The plasmids that need to be designed for this purpose will contain no homologous sequence with our target gene. The donor will contain a promoter less ires-eGFP and a single sgRNA target site at 5’ end of the ires-eGFP (single-cut). Any possibility of frameshift mutation due to non-homologous end joining repair was avoided by using the ires element.

Non homologous end joining can be obtained by single cut vectors that cleave the DNA at a single site on the 5’ end or by double cut that cleave the DNA at two different sites and then promote repair simultaneously. In general non homologous end joining repair carries a two-third chance of producing a frame shift mutation mediated by indel error. IF double cut plasmids are used to repair the gene of our interest the efficiency of knock in will have a higher probability to be reduced. Hence, for efficient correction of the mutation found in sickle cell disease single cut plasmids should be used preferably.

References

Alam, M.R., Thazhathveetil, A.K., Li, H. and Seidman, M.M., 2014. Preparation and Application of Triple Helix Forming Oligonucleotides and Single Strand Oligonucleotide Donors for Gene Correction. Gene Correction: Methods and Protocols, pp.103-113.

Bender, M.A. and Seibel, G.D., 2014. Sickle cell disease.

Bétermier, M., Bertrand, P. and Lopez, B.S., 2014. Is non-homologous end-joining really an inherently error-prone process?. PLoS Genet, 10(1), p.e1004086

Branzei, D. and Foiani, M., 2008. Regulation of DNA repair throughout the cell cycle. Nature reviews Molecular cell biology, 9(4), pp.297-308.

Calin, G.A., Dumitru, C.D., Shimizu, M., Bichi, R., Zupo, S., Noch, E., Aldler, H., Rattan, S., Keating, M., Rai, K. and Rassenti, L., 2002. Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proceedings of the National Academy of Sciences, 99(24), pp.15524-15529.

Chu, V.T., Weber, T., Wefers, B., Wurst, W., Sander, S., Rajewsky, K. and Kühn, R., 2015. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nature biotechnology, 33(5), pp.543-548

Davis, A.J. and Chen, D.J., 2013. DNA double strand break repair via non-homologous end-joining. Translational cancer research, 2(3), p.130

Dubay, J., Krebs, D. and Thresh, L., 2015. Sickle Cell Anemia.

Eiring, A.M., Harb, J.G., Neviani, P., Garton, C., Oaks, J.J., Spizzo, R., Liu, S., Schwind, S., Santhanam, R., Hickey, C.J. and Becker, H., 2010. miR-328 functions as an RNA decoy to modulate hnRNP E2 regulation of mRNA translation in leukemic blasts. Cell, 140(5), pp.652-665.

Griffiths-Jones, S., Grocock, R.J., Van Dongen, S., Bateman, A. and Enright, A.J., 2006. miRBase: microRNA sequences, targets and gene nomenclature. Nucleic acids research, 34(suppl 1), pp.D140-D144.

He, L., Thomson, J.M., Hemann, M.T., Hernando-Monge, E., Mu, D., Goodson, S., Powers, S., Cordon-Cardo, C., Lowe, S.W., Hannon, G.J. and Hammond, S.M., 2005. A microRNA polycistron as a potential human oncogene. Nature, 435(7043), pp.828-833.

Hsu, P.D., Lander, E.S. and Zhang, F., 2014. Development and applications of CRISPR-Cas9 for genome engineering. Cell, 157(6), pp.1262-1278.

Lytle, J.R., Yario, T.A. and Steitz, J.A., 2007. Target mRNAs are repressed as efficiently by microRNA-binding sites in the 5′ UTR as in the 3′ UTR. Proceedings of the National Academy of Sciences, 104(23), pp.9667-9672.

Nicoloso, M.S., Spizzo, R., Shimizu, M., Rossi, S. and Calin, G.A., 2009. MicroRNAs—the micro steering wheel of tumour metastases. Nature Reviews Cancer, 9(4), pp.293-302.

Rupaimoole, R., Han, H.D., Lopez-Berestein, G. and Sood, A.K., 2011. MicroRNA therapeutics: principles, expectations, and challenges. Chinese journal of cancer, 30(6), p.368.

van Rooij, E., Purcell, A.L. and Levin, A.A., 2012. Developing microRNA therapeutics. Circulation research, 110(3), pp.496-507.

Vasudevan, S., Tong, Y. and Steitz, J.A., 2007. Switching from repression to activation: microRNAs can up-regulate translation. Science, 318(5858), pp.1931-1934.

Wen, M.M., 2016. Getting miRNA Therapeutics into the Target Cells for Neurodegenerative Diseases: A Mini-Review. Frontiers in Molecular Neuroscience, 9, p.129.

Yourgenome.org. (2016). What is sickle cell anaemia?. [online] Available at: https://www.yourgenome.org/facts/what-is-sickle-cell-anaemia [Accessed 9 Dec. 2016].