The XIST Solution for Down Syndrome

Kris Burns

Biology 1010
 All living things contain DNA, as sequenced in genes and chromosomes, which is for the
purpose of carrying out functions, defining appearance, and more. Each species is unique from
another, and within each species, each individual is unique as well. These different
characteristics are all expressed through genetics so that the individual can survive. The normal
human individual has 46 chromosomes that contain the whole blueprint of who they are and
determine what they look like. However, there can be mistakes made through the processes
nature uses that create genetic disorders. An example of this is Down syndrome or Trisomy 21.
This disorder is the most common in humans, is not preventable, occurs before fertilization,
and is not curable. However, there are possible solutions, through gene therapy, being
explored. One of these possibilities known as gene silencing using the XIST, or X-inactivation
gene.
Down syndrome, also known as DS, DNS, or trisomy 21, is one of the most common
genetic disorders in the world and occurs in one out of every one thousand babies (Weijerman,
de Winter, 2010). Individuals with Down syndrome have a well-known phenotype or physical
appearance. These characteristics include stunted growth, slanted eyes, small mouth, a flat
nose, extra space between the first and second toes, increased likelihood of obesity, extra skin
on the back of the neck, life expectancy of sixty years, and mental disability or impairment
(Hammer, McPhee, 2010). Phenotypes, the physical properties and characteristics of an
organism, are directly determined by the genotype, the genetic makeup, and its interaction
with the environment.
As briefly mentioned before, Homo sapiens, or humans, should have a total of 46
chromosomes in their karyotypes. These chromosomes are located in the nucleus of eukaryotic
cells. When cells divide, the DNA needs to be copied and replicated so that the new cell can
carry on the same work and functions of the old cell. There are two types of cell division.
Mitosis and meiosis. Mitosis is essentially creating an exact copy of the beginning cell, or
asexual reproduction (Carter, 2014). Meiosis results in four haploid cells, all unique, from two
different individuals. Haploid cells have only one set of chromosomes, in the case of humans,
23 or half of 46 (Freeman, 2011). Meiosis enables sexual reproduction and is responsible for
creating new and unique genotypes and karyotypes, introducing genetic variation and the
ability to adapt and evolve. Meiosis in humans occurs in the sex organs. Men create sperm as
haploid cells and women create eggs as haploid cells. It is a mistake during the process of
meiosis that is responsible for Down syndrome and other developmental disabilities.
It is a mistake in the process of meiosis that causes Down syndrome. In preparation for
meiosis, the cell must replicate all of the DNA so that there are identical copies that can then be
distributed to each cell. After replication, the parent cell is prepared for meiosis, which involves
prophase I and II, metaphase I and II, anaphase I and II, and telophase I and II, which includes
cytokinesis. In prophase I, the nucleus membrane breaks down while the DNA supercoils into
chromatin. They are then organized into homologous pairs while crossing over occurs. All of
the DNA was copied identically as sister chromatids, but are in similar pairs. These homologous
chromosomes are then organized in the middle, center, or equator of the cell by the meiotic
spindle fibers, known as metaphase I. The meiotic spindle fibers then separate the pairs of
chromosomes and pull them towards the poles of the cell during anaphase I. Once the DNA is
perfectly separated, ideally with the same number of chromosomes and the same karyotype,
the cell itself is ready to divide, called telophase I. In telophase I, the chromosomes are fully
gathered at the poles of the cell while the cytoplasm splits into two daughter cells. Each
daughter cell is a diploid cell that has the exact copy of the parent cell, 46 chromosomes. There
is at times, a brief period of rest called interkinesis. Meiosis II begins in the same sequence as
meiosis I, but the DNA is not replicated. In prophase II, the chromosomes are still visible after
meiosis I, but a new spindle forms in each daughter cell. In metaphase II, both cells organize
the chromosomes along the center or equator of the cell, similarly to metaphase I. The biggest
difference between meiosis I and II is anaphase I and II. Because the DNA was not replicated
after the initial division into two daughter cells, anaphase II divides the pairs from the
centromere and begins moving them towards the poles of the cells. This is where the diploid
cells become haploid cells, beginning at 46 chromosomes each in two cells and finishing with 23
chromosomes each in four cells. Telophase II is the same process as telophase I. Each daughter
cell gathers the separated chromosomes at the poles of the cell while the cytoplasm divides.
The meiosis process is finalized during cytokinesis a second time, resulting in four daughter
haploid cells (Freeman, 2011). This process occurs separately in males and females prior to
fertilization, when two haploid cells are united, generating a completely unique diploid cell.
Mitosis is then responsible for the growth from one cell into a full grown organism, or human.
Down syndrome is also known as trisomy 21, because in a 23-pair human karyotype, the
issue of Down syndrome is found in pair 21. The problem is that an individual with Down
syndrome has three copies of chromosome 21 instead of just two copies. The reason they have
three copies is because the chromosome failed to separate during meiosis while it is still within
the male or female parent individual. In this case, after meiosis II, the result would be four
daughter cells. Two of those daughter cells would be perfectly haploid with 23 chromosomes,
but the other two daughter cells would have 24 and 22 respectively (Reisner, 2013). So when a
sperm cell or egg are fertilized by one of the cells with an extra chromosome, the individual
develops differently because of the extra chromosome. Since chromosomes contain the DNA
and the genes, and the genes dictate function in an organism, having more chromosomes
results in more gene expression. There are 310 genes located at chromosome 21, and almost
half of them are overexpressed because of trisomy 21 (Lana-Elola, Watson-Scales, Fisher,
Tybulewicz 2011). This extra gene expression directly correlates to the phenotype of an
individual with Down syndrome, namely the eyes, distance between toes, and other
characteristics listed previously (Hammer, McPhee, 2010). Any individual with Down syndrome
has 47 chromosomes instead of the normal 46.
Down syndrome cannot be prevented because there is nothing we can do to properly
control the developmental error of sperm or eggs through meiosis. Two parents with normal
karyotypes can have a child with Down syndrome, simply because chromosome 21 was unable
to separate during anaphase II in meiosis before the sperm even comes close to fertilizing the
egg, regardless of which haploid cell contains the extra chromosome, the sperm or the egg.
Also, because Down syndrome is a genetic disorder, there is no known cure. Scientists have
made strides in possible solutions to Down syndrome and other genetic diseases through gene
therapy. Gene therapy, also known as human gene transfer, is treatment through delivering a
functional gene to a patient’s cells in order to replace or disrupt the defective gene. Two
different types of cells are targeted. Somatic cells are all the normal diploid cells in the body
which, if treated through gene therapy, would only affect the patient. The other targeted cells
are the germ cells, the sperm, eggs, gametes, etc. Targeting these haploid cells means
preventing the passing of the defective gene and thereby affecting the offspring (Kaji, Leiden
2001). Gene therapy is used for cancers, blood diseases, central nervous system disorders,
immune system disorders, as well as trisomy 21.
The delivery of the DNA is done by either viral vectors, or non-viral methods. The
purpose is to replace or disrupt the dysfunctional gene with a properly functioning, therapeutic
gene in order for the cells to have the correct protein synthesis and carry out proper functions.
Viral vectors utilize the abilities of a virus to spread and transfer their DNA to cells. By
modifying and engineering safe viruses by replacing the viral genome with the functional or
therapeutic gene, the DNA is taken to a large percentage of cells through the efficient
transportation of viruses (Kaji, Leiden 2001). Non-viral methods are sometimes favored
because they are safer and you can produce a large amount of the DNA. The simplest non-viral
method is injecting naked DNA into the host cells. There are different physical and chemical
enhancements used to facilitate the delivery of the functional gene. Some of these are
electroporation, sonoporation, gene gun, magnetofection, oligonucleotides, lipoplexes,
polymeresomes, polyplexes, dendrimers, and more.
There have been two main attempts at therapy or editing the trisomy 21 genome. One
complicated way was trying to remove the extra chromosome (Saey 2013). The most promising
solution of therapy for Down’s syndrome, or at least what is helping scientists learn about the
disorder itself, is called gene silencing. Scientists Jun Jiang and Jeanne Lawrence at the
University of Massachusetts Medical School in Worcester are working on using the natural X-
inactivation gene and applying it to the extra chromosome 21. The X-inactivation gene is also
referred to as XIST. The XIST gene is activated in women due to females having two X
chromosomes, where only one is needed, as is proven in all males that have one X
chromosome. When the XIST is activated, it produces RNA that coats a chromosome,
essentially “boarding it up” with proteins and preventing the expression of genes on that
chromosome. In women, this means that only one of the X chromosomes is doing the protein
synthesis and carrying out those functions, essentially making them similar to men (Saey, 2013;
Jiang, Lawrence 2013). Jiang and Lawrence decided to use this natural biological silencing
process and apply it to the extra chromosome in trisomy 21. The goal is that the XIST will be
able to shut down any chromosome it is activated on and prevent the expression of those
genes. Overexpression is a main problem of Down’s syndrome due to the other chromosome
21, so if successful, XIST chromosome therapy could be a step forward into knowledge about
trisomy 21 and possibly make some problems associated with Down’s syndrome correctable
(Jiang, Lawrence 2013).
Jiang and Lawrence used a non-viral technique that is more genome editing than just
gene therapy. Using zinc finger nucleases to splice the DNA at chromosome 21, they insert a
large, record-breaking strand of XIST DNA along with an antibiotic doxycycline switch to
activate the XIST gene and silence the expression of the whole chromosome. Once activated,
the extra chromosome 21 was successfully silenced (Jiang, Lawrence 2013). When silenced, the
cells developed normally and even neural rosettes or clusters of cells that make up the central
nervous system (Coghlan, 2013; Jiang, Lawrence 2013). None of the untreated trisomy 21 cells
had made that progress. One of the impacts of these tests is the knowledge gained about
Down’s syndrome and where some of the gene expression is made. Most notably, the gene
and protein linked to Alzheimer’s, hastened by Down’s syndrome, was silenced (Coghlan 2013).
 One problem with any type of “cure” or altering of trisomy 21 is the necessity to target
all cells in the patient because the DNA is in every cell. The XIST solution is promising because
of the rapid reversals of expression when the extra chromosome is silenced, but it is not yet
possible to silence all of the extra 21 chromosomes of all cells in the patient. All of the viral and
non-viral methods may be enhanced and improved, but all of the sources found were clear that
a cure for Down’s syndrome is in the very distant future. Although trisomy 21 is the most
common genetic disorder among humans, there is extensive research being done to improve
quality of life, learn about the disorder, and move towards possible solutions and cures for
Down’s syndrome. One of those most promising paths is the XIST gene silencing method to
silence the extra chromosomal expression of trisomy 21.
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