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Types of Therapeutics for Trinucleotide Repeat Expansion

Trinucleotide repeat expansion, also known as triplet repeat expansion is a DNA mutation that is responsible for causing any type of disorder classified as a trinucleotide repeat disorder. These disorders are progressive and frequently tend to occur within the nervous system, available therapeutics only have modest results at best. The most advanced available therapies aim to target mutated gene expression by using antisense oligonucleotides (ASO) or RNA interference (RNAi) to target the messenger RNA (mRNA). While solutions for the interventions of this disease is a priority, RNAi and ASO have only reached clinical trial stages.

RNA Interference (RNAi)

RNA interference is a mechanism that can be used to silence the expression of genes, RNAi is a naturally occurring process that is leveraged using synthetic small interfering RNAs (siRNAs) that are used to change the action and duration of the natural RNAi process. Another synthetic RNA is the short hairpin RNAs (shRNA) these can also be used to monitor the action and predictability of the RNAi process.

RNAi begins with RNase Dicer cleaving a 21-25 nucleotide long stand of double stranded RNA substrates into small fragments. This process results in the creation of the siRNA duplexes that will be used by the complex RNA induced silencing complex (RISC). The RISC contains the antisense that will bind to complementary mRNA strands, once they are bound they are cleaved by the protein found within the RISC complex called Argonaute 2 (Ago2) between the bases 10 and 11 relative to the 5’ end. Before the cleavage of the mRNA strand the double stranded antisense of the siRNA is also cleaved by the Ago2 complex, this leaves a single stranded guide within the RISC compound that will be used to find the wanted mRNA strand resulting in this process to have specificity. Some problems that may occur is if the guide single strand siRNA within the RISC complex may become unstable when cleaved and begin to unwind, resulting in binding to an unfavorable mRNA strand. The perfect complementary guides for the targeted RNAs are easily recognized and will be cleaved within the RISC complex ;if there is only partial complementary pairing between the guide strand and the targeted mRNA may cause the incorrect translation or destabilization at the target sites.

Antisense Oligonucleotides (ASO)

Antisense Oligonucleotides (ASO) are small strand single stranded oligodeoxynucleotides approximately 15-20 nuceleic acid length that can alter the expression of a protein. The goal of using these antisense oligonucleotides are the decrease in protein expression of a specific target usually by the inhibition of the RNase H endonuclease as well as inhibition of the 5’ cap formation or alteration of the splicing process. In the native state ASOs are rapidly digested, this requires the use of phosphorthiation in order for the ASO to go through the cell membranes.

Despite the obvious benefits that antisense therapeutics can bring to the world with their ability to silence neural disease, there are many issues with the development of this therapy. One problem is the ASOs are highly susceptible to degradation by the nucleases within the body resulting in high amount of chemical modification when altering the chemistry to allow for the nucleases to surpass the degradation of these synthetic nucleic acids. Native ASOs have a very short half-life even before being filtered throughout the body especially in the kidney and with the a high negative charge makes the crossing through the vascular system or membranes very difficult when trying to reach the targeted DNA or mRNA strands. With all these barriers the chemical modifications may lead to devastating effects when being introduced into the body making each problem develop more and more side effects.

The synthetic oligonucleotides are negatively charged molecules that are chemically modified in order for the molecule to regulate the gene expression within the cell. Some issues that come about this process is the toxicity and variability that can come about with chemical modification. The goal of the ASO is to modulate the gene expression through proteins which can be done in 2 complex ways, a)the RNase H-dependent oligonucleotides, which induce the degradation of mRNA, and (b) the steric-blocker oligonucleotides, which physically prevent or inhibit the progression of splicing or the translational machinery. The majority of investigated ASOs utilize the first mechanism with the Rnase H  enzyme that hydrolyzes an RNA strand, when this enzyme is assisted using the oligonucleotides the reduction of RNA expression is efficiently reduced by 80-95% and can still inhibit expression on any region of the mRNA

The effects on the mechanism after the threshold has been reached

In trinucleotide repeat expansion there is a certain threshold or minimum amount of repeats that can occur before a sequence becomes unstable. Once this threshold is reached the repeats will start to rapidly expand causing longer and longer expansions in future generations. Once it hits this minimum allele size which is normally around 30-40 repeats, diseases and instability can be contracted, but if the number of repeats found within a sequence are below the threshold it will remain relatively stable. There is still not enough research found to understand the molecular nature that causes thresholds but researchers are continuing to study that the possibility could lie with the formation of the secondary structure when these repeats occur. It was found that diseases associated with trinucleotide repeat expansions contained secondary structures with hairpins, triplexes, and slipped-strand duplexes. These observation has led to the hypothesis that the threshold is determined by the number of repeats that must occur to stabilize the formation of these unwanted secondary structures, due to the fact that when these structures form the there is an increase number of mutations that will form in the sequence resulting in more trinucleotide expansion.