The 15th Korea Scholars’ Conference for Youth

Development of a Novel Exosome-Based

Therapy Method Using Native Cells to Treat
Huntington’s Disease

2020 년 1월

서울외국인학교
공예진

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Development of a Novel Exosome-Based Therapy

Method Using Native Cells to Treat Huntington’s
Disease

서울외국인학교

공예진

1. Introduction
1.1 What are exosomes

Among many of its functions and characteristics, such as maintaining the division amongst
micro and macromolecules, this study uses exosomes because they are produced by almost every cell
in the body and are shown to impact distant cell signaling. Since they are so commonly produced and
are mobile extracellular vesicles, exosomes function as escape routes for different proteins and
microRNAs (miRNAs) to move from one cell to another even if it's far away.

Due to this property, however, the abnormal export of an exosome(s) even from one cell can
cause the death of key proteins and miRNAs eventually causing misexpression. Recently, many
studies have been conducted to find a relationship between exosomes secreted from specific cells to
their impact on certain diseases. The many functions of exosomes have led to a variety of potential
use cases for exosomes and how they can be used as therapeutic tools in an array of different diseases
like Huntington's disease.

1.2 What is miRNA and its link to exosomes

MicroRNAs are small, non-coding strings of DNA with several functions that heavily impact
gene expression which is why their specific functions in genetic diseases and the overall human
anatomy are being studied extensively. For example, one known function is how miRNAs regulate
genes by creating bonds with the three untranslated regions (UTR) of messenger RNAs (mRNAs) to
cause the deregulation of the target gene's expression, essentially giving them the power to change the
expression of multiple genes within a cell. This unique power can be linked to potential positive or
negative impacts the lack or abundance of specific miRNAs can have in a genetic disease.

Some recent studies have even proposed links between miRNAs and exosomes as there has
been speculation around miRNA secretion being controlled by vesicular/exosomal controlled
mechanisms. Either way, exosomes do have a significant role within circulatory miRNA biology
which is why exosomes have been used as transportation methods for miRNAs in some studies in
order to facilitate gene exchange amongst different cells. Since miRNAs can't move on their own,
scientists have found that by being encapsulated by exosomes, the miRNAs can be protected when
leaving or entering cells.

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1.3 What is Huntington's disease

Huntington's disease (HD) is caused by a genetic mutation in the DNA code of the Huntingtin
gene (IT15) where the C-A-G nucleotide sequence is overly repeated. This discovery has been named
the "CAG repeat expansion" where the general population has around 20 repeats in this gene while
those with clinically diagnosed HD have over 40 repeats. Everyone with this mutation will show
symptoms at some point in their lives but the reason why most patients are middle-aged when first
diagnosed is because of the impact the genetic mutation has on protein synthesis. Since genes are
instructions for proteins, a huntingtin protein also exists. The specifics of this protein’s function hasn't
been found or been verified but scientists assume that the CAG repeat expansion causes an
abnormally long chain of instructions making it difficult for the protein to function properly. These
proteins are consistently produced over the span of a lifetime and form clumps in the brain which is
why the brain cells of HD patients get damaged and eventually start to die, causing both physical and
mental symptoms which worsen over time.

Although this phenomenon affects the entire brain, the striatum is most heavily impacted. The
striatum is located deep within the brain and is in control of movement, mood, and behavior control.
So, prolonged damage to the striatum caused by the initial mutation in the huntingtin gene, and
therefore the huntingtin protein causes HD. HD doesn't have a cure at the moment partially because
there simply is not enough information on the specifics of what happens to those with the mutation.

1.4 How the relationship between miRNAs and exosomes has been used in
previou studies

In other studies that have used miRNAs and exosomes to discover more about HD, most have
used cells from another species to transfect into a miRNA vector. For example, in 2017 Lee et. al used
genetic cloning technology to replicate their target miRNA into a plasmid to create miRNA vectors.
They then transfected HEK 293 human cells with the miRNA vector and the resulting cells were put
into a medium where scientists later chose cells that overexpressed the target miRNA. The chosen
cells were cultured into an exosome-free medium where the exosomes in the cell were isolated and
the level of target miRNA left was quantified. To finish, the scientists injected the exosomes they
harvested, that now had their target miRNA, into transgenic mice using a syringe.

2. Purpose and Significance of the Study

2.1 Higher degree of accuracy

Unlike the conventional method used to insert a target miRNA into a cell, the method
proposed in this study doesn't use cultured cells nor does it use vectors. This novel method allows for
the target and origin cell for an exosome to be from the same subject allowing for a higher degree of
accuracy, a result of minimized side effects.

Much like the idea behind organ transplants, it's much more preferable and beneficial if the
original cell where an exosome was derived has a similar genetic makeup as the target cell as there's a
significantly lower possibility of the target cell rejecting the exosome. This benefit is so great that the
reason why there are so many people waiting on transplant lists is that the same species, human to
human, transplants are that much more preferable than cross-species transplants.

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However, this concept is not applied to the current, widespread method of miRNA delivery as
shown in section 1.4 which is why this study proposes a method that theoretically would work. So,
this study would be able to explore the possibility of a method that can produce remarkably more
accurate results thanks to the use of native cells.

2.2 Efficiency

Another benefit this method has is that it excludes the need for genetic cloning. In the original
method, scientists had to take their specified miRNA and genetically clone it to replicate into a
plasmid in order to create vectors, a tool used to overexpress a gene. This process not only takes time
but also resources meaning the entire experiment gets slowed down, while also allowing room for
error that can impact the entire experiment. By eliminating the need for this step, the new method
allows scientists to test more types of miRNA due to the time and resources saved.

Considering the lack of information and data on HD and its link to miRNAs and exosomes,
the experiments that take place due to the time and energy saved from removing genetic cloning can
reveal more about the disease itself and potentially lead to therapeutic advancements in the currently
somewhat mysterious disease.

2.3 Possibility for success

Despite this method not being tested yet, it still has aspects that indicate its potential for
success. First, rather than using a completely new method without any scientific basis, this study
proposes a method that uses a pre-existing, standard protocol for transfection, a key part of the overall
method itself. When inserting the target miRNA into the derived exosomes, this method uses a
common transfection reagent, liposomes, to incubate the miRNA together so that the miRNA can
penetrate into the exosome's membrane which it can't do without the liposomes. Again, the miRNA +
liposome substance gets incubated with the exosome to transfect together and eventually enter a
neuron cell to observe its effects.

Also, while testing the impact of the miRNA on healthy neuron cells can be helpful data, this
study uses former knowledge of the CAG repeat expansions of HD to manipulate neuron cells so they
mimic that of Huntington's disease. Acquiring human cells with HD is not always possible nor is
animal testing which is why this method proposes the manipulation of healthy cells to replicate that of
HD-riddled ones in order to make the entire process more time and cost-efficient. If the data gathered
is significant enough, the method can later be used on animals and potentially humans but in its early
stages, manipulating isolated cells is a wiser course of action.

Lastly, with the two major benefits, this method holds over the conventional one, the success
of this method in replacing the current one will benefit most if not all future studies regarding HD and
even miRNAs and exosomes.

3. Methods of Analysis and Data Collection

3.1 Search target miRNA for HD RNAi therapy

Many bioinformatic analyses of miRNA expression profiles in HD are available.

Differentially expressed miRNAs (DEMs) are listed in previous studies. The profile of all identified
DEM from HD will be listed.

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3.2 Design In vitro model of HD

In vitro model of HD, can be developed by differentiated SH-SY5Y cell line treated with the
pro-oxidant neurotic reagent, 6-hydroxydopamine (6-ohda). First, SH-SY5Y cells can be
differentiated into neurons by retinoic acid treatment. Then, the differentiated neurons are treated with
6-ohda to mimic HD neurotoxic damage.

3.3 Isolation of exosomes from SH-SY5Y neuron cells

The neuron-derived exosomes can be isolated by the exosome isolation reagent. It enables
fast and efficient purification of exosomes from cell culture media. After the reagent is incubated
overnight at 4 oC, the exosomes are precipitated. After the centrifugation, the pure exosomes can be
resuspended in PBS (Phosphate-Buffered Saline) solution.

3.4 Transfecting target miRNAs or siRNAs to isolated neuron-derived exosomes

Liposome-based transfection reagent enables the transfection of nucleic acids directly into
isolated exosomes. The transfected siRNA or miRNA can be shuttled into the target cells by these
transfected exosomes.

3.5. Analyze cell cytotoxicity using MTT assay

MTT assay is a quantitative assay that measures cellular metabolic activity as an indicator of
cell viability and cytotoxicity. The viable cells contain NAD(P)H-dependent oxidoreductase enzymes.
This enzyme then reduces the MTT to formazan. When formazan is synthesized, it induces colored
solution, which can be quantified by measuring absorbance at 500-600 nm using a spectrophotometer.
The protective effect of isolated miRNA or siRNA transfected exosomes on 6-ohda induced
neurotoxicity can be analyzed by MTT assay.

4. Expected Results and Impacts

4.1 Expected Results (Benefits and Drawbacks)

This specific study would not be able to test every single miRNA identified to have a
relationship with HD, as there are dozens and potentially hundreds more, but it provides a more
efficient and accurate framework than the pre-existing one allowing future scientists to conduct
further experiments that may produce more precise results. Therefore, this novel method will allow
for more miRNA types to be tested, resulting in more factual information being gathered regarding
the impact of specific miRNAs on cells with HD. This information then can be used in future studies
exploring the relationship between miRNAs and how they can be used as therapeutic tools for treating
HD.

Although this study will provide a potential methodology that may significantly progress
knowledge on HD, miRNAs, and exosome-based delivery, there can also be drawbacks that follow
this study. First, even if the method proves plausible, it will still take several years before any
significant benefits provided to the actual patients. Not only will many succeeding studies have to be
performed but animal tests and clinical trials have to take place in order for a therapeutic method to be
developed and implicated. Also, this study is very focused on miRNAs and the link they have with
HD. Granted, this route was taken because of the pre-existing data regarding overexpressed and
repressed miRNA types in HD but again, this information may lead to a dead-end which is significant

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in itself but would not lead to an immediate cure.

4.2 Impacts on Future Studies and Patients

All HD studies, including ones that do not use miRNAs, can benefit from the results of this
study and those that follow that use the method developed here. Since knowledge regarding the
disease itself is quite limited, any and all information regarding the potential routes scientists can take
with the disease is considered valuable.

Key discoveries like the specific region of the brain affected (striatum) and the genetic
mutation that causes the disease has been made already, however, none have led to significant
developments in the lives of actual patients. In theory, this method will be one of the first that can lead
to results with the potential to treat patients. If this study and future ones that explore the use of
miRNAs in treating HD fail, scientists can then analyze the results and devise courses of action that
take previous failures into account. If the opposite happens, scientists will know to develop upon a
specific study or set of studies to progress their own research in creating a cure for the current chronic
disease. This research will continue and as more data is gathered, hopefully, a therapeutic solution
will be created for those struggling with HD.

Lastly, HD is a genetic disease where a child has a 50% chance of inheriting the faulty gene if
a parent has HD. More people are at risk of getting HD than those who actually show symptoms and
if treatment is not found, the disease will simply continue throughout the human gene pool. In order to
minimize further suffering, treatment must be developed and this study is one of many first steps that
need to be taken in order to reach a cure.

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Works Cited

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