Sickle Cell Disease (SCD) is a hereditary genetic blood disorder which

leads to the production of erythrocyte derivatives in the body. Erythrocytes

start their life cycle in the bone marrow and are produced with the function
of oxygen distribution. When deoxygenatedproduced, these derivatives take
on the form of sickles, which unlike a normal erythrocytes oval form, is
inefficient and harmful. APrimarily, a sickled erythrocytes life cycle is
typically around 10 days, unlike the 120 days of a normal erythrocyte. Early
death is a consequence of these cells unusual formation, which leads to
them accumulating in the spleens filter and macrophages removing them. It
is important good to note that the liver and bone marrow also play a role in
removing erythrocytes. A patient with SCD is unable to produce erythrocytes
at the same rate the cells die off causing anemia. Sickled cells also have a
tendency to scrape of scrapping capillaries, which leads to various infections,
or ruptures. Typical symptoms vary for each individual vary but are not
limited to pneumonia, strokes, spleen damage, and bone and joint problems
to name a few. (Powers, 2013).
To combat the side effects of SCD, supportive measures and modern
medicine are utilized to minimize the damage produced by the erythrocytes
form. Prevalent treatments, such as those involving the use of penicillin and
hHydroxyurea, are becoming more prevalent as a standard. (Segal, 2008)
Yet, minimizing the pain caused by either infections, or perhaps a crisis, is

not what a patient wants to hear for the rest of their his/her lifeves when
selecting medicine which attempt to alleviate their his/her condition. Thus,
as on an optimistic approach, scientists believe they are on their way to fully
curing this genetic disease with the help of genetic medicine. Considering
that SCD is a genetic disorder, it is only seems natural as well to treat this
disease at a genetic level. This research will cover more details regarding
what is defined as genetic medicine today. Following,is an overview of
erythrocytes and their its corresponding target subunits for gene therapy.
ThenUltimately, a fully detailled analysis of a promising experiment
whichwith involves correcting the mentioned subunits is discussed.
Genetic medicine (gene therapy) is a practice revolving the
identification of mutated genetic sequences and the utilization of modern
technology in order to isolate, remove, or replace, harmful sequences from
an individuals beings DNA. The ultimate goal is to be able to alleviate an
individual from genetic abnormalities in such a way that no excess harm is
done to the individual by agitation, or progression, of the disease caused by
the genetic therapy. Modern gene therapy revolves around engineered
nucleases in which the nuclease is a protein component which breaks
segments of DNA. Engineered nucleases are composed in such a way that
they may only attach to a specific part of a host cells DNA which contains
specific complimentary bases to itself. Once attached, the nuclease will
create a double stranded break in the area containing the target sequence.
The break causes the host cell to attempt to fix its DNA, usually it

accomplished s this by utilizing a donor template inside its sister

chromatid(?). This process is called Non- Homologous End Joining (NHEJ),
which the end result leads to original gene deletion, replacement through
gene inversion(?), and overall gene disruption. To elaborate, NHEJ leads to
the termination of unwanted genetic sequences via silent mutations, or
mutations of a specific gene that do not negatively impact an individual.
(silent mutations are defined as those that dont change the coding capacity
of a gene) Although, Mmore commonly, gene therapy is utilized to remove
and replace an unwanted genetic sequence with a more preferred one.
Homology Directed Repair (HDR) is the process of utilizing a nuclease as well
as a composed DNA sequence (codelivering). The DNA sequence is contained
inside of an Integrase dDeficient lLentivirus (IDLV), or as an oligonucleotide.
Currently, there are four types of gene therapy that are widely
reportedgarded. These involve engineeedr mega nucleases with reengineered homing endonucleases, zinc finger nucleases, transcription
activator-like effector nucleases (Talons), and clustered regularly interspaced
short palindromic repeats (CRISPR-CAS9 System). (Hoban, 2015)
An IDLV is a former retrovirus which has a vector containing an
engineered therapeutic gene. The vector is composed of former parts of the
virus, including GAG (proteins which line the envelope, protect the core, and
protect the genome), POL (contains integration information and reverse
transcriptase but no integrase), and ENV genes (for self-reproduction). This
means that an IDLV is unable to reproduce, or integrate DNA, without the

assistance of a nuclease. Thus, an IDLVs function is to implant the

therapeutic gene in a host cells DNA and die off. Inflammation due to
antibody response is minimalized due to this preventive method (?). (Vigue,
2013)
Oligonucleotides are composed of a specific sequence of nucleic acids,
which are hydrophilic. Due to a cell membranes outer hydrophobic qualities,
transfection of these nucleic acids may not occur under normal
circumstances. Thus, oligonucleotides may be coded with lipid base
reagents, in which the cell membrane will absorb the substance. Another
method is to utilize calcium phosphate, which creates an opening in the cell
membrane. Ultimately, electroporation may be used to make a cell
membrane permeable via electricity. It is important good to note that
oligonucleotides are cheaper to produce than IDLVs and produce no
inflammation. In contrast, a vital drawback is the lack of amount in DNA
capacity an oligonucleotide possesses in comparison to an IDLV.
(Pengpumkiat, 2016)
SCD occurs due to a genetic abnormality in chromosome 11, at the
bBeta gGlobin gGene cCluster. This cluster is associated with the production
of hemoglobin-A, including derivatives, in an erythrocyte. Hemoglobin is
composed of heme which is a porphyrin ring, a carbon ring molecule which
contains iron in the middle, and globin, that consists of amino acids which
provide structure for the erythrocyte. Hemoglobin A B, a derivative, is

produced in a healthy individual, unlike Hemoglobin S which is produced in a

patient possessing SCD. The mutation causes a beta globin substrate
sequence change in which vValine is usedproduced instead of glutamic acid
at position 6 of beta globin. . This change leads tofor the hemoglobin to
becominge sticky and hard when deoxygenated. Ultimately when a mutated
erythrocyte expels its oxygen particles, the hemoglobins attaches to
themselves and the inner membrane of the erythrocyte, distorting its overall
shape. (Powers, 2013)
The focus experiment concentrates on the usage of zinc fingers along
with either an IDLV, or oligonucleotide, in order to remove the mutated
sequence in hematopoietic progenitor cells. Hematopoietic progenitor cells
are stem cells found in bone marrow that lead to the production of
erythrocytes, along with myeloid and lymphoid cells. By targeting the
hematopoietic progenitor cells, hemoglobin can be rectified before an
erythrocyte is fully developed. The experimented cells originated from
donated bone marrow samples from patients with SCD, umbilical cord blood
from patients with SCD, and cells from mice with SCD. The mice served as an
experimental buffer before mimicking the procedure on humans. To assure
experimental success, hematopoietic cells were primarily experimented in
vitro, or outside the body, to verify that healthy erythrocytes were being
produced. Modified cells were then transferred into immunosuppressedant
mice in order to maximize engraftment percentage. For comparison, mock
cells were utilized as a control group. Mock cells were not treated with zinc

fingers, or an IDLV/oligonucleotide. Each resulting data corresponds to a

different batch of sickled hematopoietic progenitor cells. Note that the usage
of zinc fingers is regarded as a more popular approach when using gene
therapy. This experiment took place in December of 2014 for relative
relevancy. (Hoban, 2015)
It should be noted the nature of zinc fingers, mutations, and silent
mutations in this experiment. A silent mutation occurs due to a break in a
genetic sequence in which the host expresses a novel gene after DNA repairs
itself. Usually these mutations are harmless when mitigating the specific
target area (see above note about silent mutations; also,I dont know what
mitigating means here). As mentioned, zinc fingers function by locating a
specific sequence in the DNA and binding to it before commencing a double
strand break. Once the zinc finger breaks the strand, and a homologous
donor template fills the created gap, the zinc fingerer may again cut the
sequence further if it detects similar remaining complementary bases. This
would lead to an unmitigated potential mutation. This is the reason
engineered zinc fingers in this experiment were designed to attack longer
sequences. Zinc fingers which attack longer sequences require a more
specific base to base pattern. This means two things, the chance of a zinc
finger being able to bond to a separate sequence site in the DNA is lowered
drastically, and that the gap left by the zinc fingers is longer. (Caroll, 2011)
Lentivirus vectors possess enough space in which enough RNA can be
packed to cover the gap quickly. If the gap is left untreated, NHJEJ will

commence leaving the cut area to have a possibility of being filled with
mutations. This would occur with oligonucleotides, which pack a relatively
small amount of nucleic information. To compensate, engineered zinc fingers
only cut in one area of the DNA instead of two or more , which would drop
the entire sequence. Oligonucleotides are designed to create an Avrll (see
below) RFLP, a restriction enzyme, on the target area which is cut. A
restriction enzyme has the role of breaking DNA at nearby locations, based
on base to base compatibility. Thus a zinc finger makes the initial cut, and
the Avrll (see below) RFLP makes corrections inside to out. The donor
template is also introduced with pre-engineered silent mutations. Typically,
any unwanted mutation is avoided, yet these silent mutations play a role in
changing the composition of the target sequence in which the zinc finger
may not bind again. This makes utilizing oligonucleotides possible inside a
host while also decreasing the chance of mutation to a minimum. Also noted
is that oligonucleotides are both faster and cheaper to reproduce.

Figure 1
Results from figure 1 (make this more prominent in the figure) were
accomplished by electroporation of in vitro hematopoietic progenitor cells
under the influence of Zinc fingers and IDLV donor templates. The beta
globin locus experienced cleavage by the zinc fingers in which allelic
disruption became present. The underlined portion of section A portrays the
zinc finger target site (cleaved segments), in bold the start codon for the new
sequence (note that the sequence will be shorter when rectified (do you
mean corrected instead of rectified?) ), and in bold italic (I dont see any
italics) the future dropped (I dont know what dropped means here.) mutated
segment. Section B takes place after 3 days of electroporation. The black
arrows, portrayed with the use of a Surveyor Nuclease, signify cleavage
confirmation at the beta globin locus after exposuree to the zinc fingers.
Section C corresponds to an overview of the removal of the mutated genetic
sequence and replacement with the donor template. A Hha Hhal RFLP, which
detects changes in genetic sequence due to restriction enzymes, was used to

confirm genetic variation. The arrows ion section D portray overall gene
modification. This section represents the outcome of combinations involving
using zinc fingers plus the IDLV, using only the restriction enzyme/IDLV by
itself, to not using any at all. These cells were a separate group which was
introduced to transducedtion for 4 days. Section E shows gene modification
success rate in the overall pool of zinc finger/IDLV experimented cells. The
cells were then harvested after 3 days of electroporation to test for any
possible mutations produced by the zinc fingers. (Hoban, 2015)

Figure 2
Results from figure 2 were derived from the usage of oligonucleotides
as the donor template. Section A portrays were (?) the Avrll (fix this) RFLP
changed the genetic makeup of the mutated sequence. The underlined
bases in italic (again I dont see italics) represent the inserted sequence,
which changes the expressed gene for the entire sequence. In section B the

arrows point to Avrll(this too) cleavage success percentage when using both
zinc fingers and oligonucleotides. No electroporation was required. Section C
shows all silent mutations around the sickled base. The zinc finger binding
site ledt to 6 possible mutations. Section D shows an average of gene
modification and gene with oligonucleotide derivatives. White bars indicate
overall successful gene modification. Gray bars are associated with random
insertion and probable silent mutation. Section E shows overall NHEJ double
strand break corrections. Section F aids in establishing that an optimal zinc
finger threshold exists. In the experiment the threshold is at 15 micrograms
per milliliter. Passing this threshold signifies a higher possibility for zinc
fingers to re attach themselves to the target area, lowering gene
modification.

Figure 3
This data correlates to the engraftment of hematopoietic progenitor
cells in immunosuppressedant mice. Section A portrays the percentage of

successful gene modification in cells treated with both zinc fingers and an
IDLV, compared to the mock cell. These cells were primarily treated with
electroporation and were then transferred into the test candidate (?).
Section B indicates the amount of erythrocytes produced which contain
either complete gene modification (Wild Type Hemoglobin), or gene
disruption in the sickled site. Percentage of total sickle change due to zinc
fingers and IDLVs varied from 10-15% success rate. Section C mimics Section
B except that zinc fingers are paired with oligonucleotides. Percentage of
total sickle change due to zinc fingers and oligonucleotides varied from 1520% success rate. Section D portrays engraftment in the peripheral blood of
mice after 8 weeks. Closed diamonds signify that the cells treated were still
expressing healthy erythrocytes after 8 weeks. Open Diamond signifies the
mock cells after 8 weeks. Section E mimics section D except that closed
diamonds represent hematopoietic progenitor cells treated with
oligonucleotide. Hematopoietic progenitor cells treated with only zinc fingers
represent closed triangles and those treated with only oligonucleotides
represented as closed circles.

Figure 4
This result involves gene-modified hematopoietic progenitor cells
persistence after 16 weeks inside the mice. Section A portrays continuous
gene modification rates in both the bone marrow and spleen of the mice.
Closed diamonds represent hematopoietic progenitor cells modified by zinc
fingers and IDLV. Open diamonds represents mock cells. The majority of
hematopoietic progenitor cells inserted have modification rate drops. This is
due to the fact that mature progenitor cells have a higher chance of being
corrected than younger ones. A balance exists between which hematopoietic
progenitor cell will derive into erythrocytes, megakaryocytes/platelets,
myeloid cells (monocyte/macrophage and granulocytes), mast cells, T- and
B- lymphocytes, natural killer (NK) cells, or dendritic cells (DCs). () Before
commencement of interphase, based on the surrounding environment and
influence due to neighboring cell, a hematopoietic progenitor cells will decide
if it will self-proliferate (clone itself), or if it will differentiate. Commonly,
mature hematopoietic progenitor cells tend to differentiate at a higher rate

than younger ones, in contrast to self-proliferation which continuoues the

cycle. (Seita, 2010) Thus, it would be more advantageous to correct younger
hematopoietic progenitor cell, yet isolating that task is difficult. Section B
represents insertion and deletion of sequence at the zinc finger cut site.
Closer to the line means proper modification area was reached. Section C
mimics section C (?) except that closed diamonds represents zinc fingers and
oligonucleotides, closed circles represent zinc fingers, and closed triangles
represent oligonucleotides. Zinc fingers with oligonucleotides portrayed the
highest amount of gene modification at the desired target site. Zinc fingers
and oligonucleotides as seldom portrayed a low level of gene modification at
cut site(?). Section D mimics part B with similar representations as part C.(?)
Anything under the not signifiicant area implies that gene modification
occurred, yet the results were not significant in causing mutations. Asterisks
signify percent modification variance, one asterisk equals below .05 percent
and two asterisk equals below .01 percent.

Figure 5
Data from figure 5 signifies the overall functional correction at the
gene site from zinc finger and IDLV gene therapy. Section A corresponds to
the total percent of hematopoietic progenitor cells which possessed a cut in
the proper target base and proper correction in the target sequence.
Engraftment rates were as high as 20% for mice after 16 weeks. Section B is
a comparison between wild type hemoglobin which is corrected in the mock
cells and the corrected hematopoietic progenitor cells. Hematopoietic
progenitor cells had a drastic decrease in hemoglobin S, and a drastic
increase in fetal hemoglobin and hemoglobin A. Section C represents a chart
of all the total hemoglobin A expressed under the total amount of peaks.
Results from this experiment help in concluding the current state of
gene therapy as a possible cure for SCD. Prior to these results, what should
be addressed is the cost of gene therapy for an individual. Due to the fact
that gene therapy would be personal for each individual, (Isolating host cells,

arranging gene modification in vitro at labs, inserting modified cells back into
the host, and monitoring engraftment), treatment would be relatively very
expensive. Most likely this sort of procedure would be for the relatively
wealthy as insurance companies would most likely not cover the expenses.
This is due to the rarity of this disease in the overall population and the fact
that some patients with SCD wouldnt even be strong enough to handle the
procedure (ref?). As for the results, although engraftment is possible with a
20% of gene correction and engraftment success, the possibility of causing a
mutation in the host due to the zinc fingers, or improper HDR, is still
relatively high. Considering the whole process of engraftment in itself takes a
huge toll oin the body due to the immunosuppressing process, the possibility
of making an individual worse should never be an option. (Mosesso, 2015)
Gene therapy has certainly seen a lot of progress in the last couple of
years specially. In 2013, a new system for gene therapy was introduced as
the CRISPR-CAS System (CAS-9). Unlike the usage of zinc fingers, CAS-9 is
very precise in isolating the genetic mutated area and removing it without
creating an excess gap. Thus, the chance of creating an unwanted mutation
becomes extremely low. Of course the CAS-9 system is still being researched
on, yet the future of gene therapy is still looking a bit brighter. However,
challenges of making gene therapy available which do not involve the
possibility of creating more mutations are still relevant. Regardless, with the
rapid progression of science, one never knows if the future of medicine might
just be right around the corner. (Jing, 2015)

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