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Background RNA interference, or RNAi, describes the set of knowledge and techniques that are based around hijacking the cell’s innate mRNA degradation machinery, namely the RISC-Dicer complex that forms outside the nucleus [1]. Much clinical research has been done that utilizes these tools, especially in diseases of the central nervous system. As with studying therapeutic development of treatment towards a specific set of tissue (like liver, cancer cells, etc.), many specific considerations must be made when working with diseases of the brain and neurological systems. RNAi Mechanisms at this Phase RNAi is an innate biological mechanism that is assumed to be evolved to interfere in RNA cell invasion, primarily against viral and bacterial transposon and transfection agents [1]. The most active endogenous form of RNAi machinery is micro RNAs (miRNAs) which are genomically encoded small fragments of RNA that contain allelic specificity, and which go through the transcriptional processing from primary miRNA transcripts containing a stem-loop, to an intermediate product called a precursor miRNA which is consequently interacts with a Dicer protein complex that cleaves the transcript. Then it reveals the region containing a single stranded antisense guide strand that will incorporate with the RISC complex to silence other target mRNA transcripts in the cell for translational inhibition via degradation or cleavage [2]. The two primary tools for RNAi synthetic modulation and clinical development comes from siRNA and shRNA therapy, which works by either mimicking the matured miRNA transcripts (sometimes referred to as artificial miRNA), or by mimicking the pre- cursor miRNAs as short-hairpin fragments that directly interact with the Dicer protein cytoplasmic pathway [2]. These can be injected and incorporated into the cells’ genome or episome via viral or non-viral mechanisms as a type of gene therapy. RNAi synthetic complexes can be focally delivered into these tissue type and section of type, making injection easy in animal models but less so in clinical models of human diseases. Virally, RNAi synthetic complexes can be injected into cells, primarily by a lentivirus (ideally for non-neuronal cells that are still dividing and proliferating) or an adeno-associated virus (AAVs), which persist episomally and can potentially attenuate the immunotoxic response in cells [1, 3]. CNS Diseases Currently in Research and Their Phases Current research in diseases of the CNS is primarily being done in genomically expressed diseases or neurodegenerative diseases [4]. These diseases and their phase of clinical development are shown in the following table:

Neurological-Specific Limitations to RNAi Use of the Most Common Tools Important in the understanding of therapeutic development of treatments is in the key limitations of what is widely used today in these disease options. It is found that the greatest limitation in technological tool design comes from stability of the siRNA complexes, endogenously present miRNA molecules, viral and non-viral immune responses to exogenously introduced RNA constructs, and off-target effects (OTEs) of out of view targets of mRNA transcripts [4]. This can be mitigated by increasing the stability of the target by increasing specificity to the antisense strand, downregulating the endogenous RNA constructs, and downregulating areas of specific off-target effects [5]. References 1. Boudreau, R.L., E. Rodríguez-Lebrón, and B.L. Davidson, RNAi medicine for the brain: progresses and challenges. Hum Mol Genet, 2011. 20(R1): p. R21-7. 2. Toro Cabrera, G. and C. Mueller, Design of shRNA and miRNA for Delivery to the CNS. Methods Mol Biol, 2016. 1382: p. 67-80. 3. Keiser, M.S., R.L. Boudreau, and B.L. Davidson, Broad therapeutic benefit after RNAi expression vector delivery to deep cerebellar nuclei: implications for spinocerebellar ataxia type 1 therapy. Mol Ther, 2014. 22(3): p. 588-595. 4. Kim, K.J.C.G.W., RNAi Therapeutic Potentials and Prospects in CNS Disease, in RNA Interference, I.Y. Abdurakhmonov, Editor. 2016: IntechOpen. 5. Aguiar, S., B. van der Gaag, and F.A.B. Cortese, RNAi mechanisms in Huntington’s disease therapy: siRNA versus shRNA. Translational Neurodegeneration, 2017. 6(1): p. 30.

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