GENE THERAPY

Gene Editing Could One Day Treat

Muscle Disorders

SUBMITTED TO: MRS. ANNALIE SOLAMO BEJOC

SUBMITTED BY: SIBI, EMMANUEL JOHN S. BSME-P4

Gene Therapy
Gene Therapy can be extensively characterized as the exchange of
characterized hereditary material to explicit target cells of a patient for a definitive
motivation behind anticipating or changing a specific sickness state.

Gene and DNA are currently being presented without the utilization of vectors
and different strategies are being utilized to change the capacity of qualities in vivo
without quality exchange.

On the off chance that one adds to this the phone treatment especially with
utilization of hereditarily changed cells, the extent of quality treatment turns out to be a
lot more extensive. Quality treatment would now be able to consolidate with antisense
systems, for example, RNA impedance (RNAi), further expanding the helpful
applications.

This report takes wide review of quality treatment and is the most modern
introduction from our report on this subject developed from a progression of quality
treatment report composed.

This report depicts the mishaps of Gene Therapy and reestablished enthusiasm for
the subject. Quality treatment innovations are portrayed in detail including viral vectors,
nonrival vectors and cell treatment with hereditarily altered vectors that Gene
treatment is an amazing strategy for medication conveyance and different courses of
organization just as focused Gene Therapy are depicted.

Therefore, there is a prologue to advances for quality concealment just as sub-

atomic diagnostics to distinguish and screen quality articulation. On behalf of Quality
altering innovations, for example, CRISPR-Cas9 and CAR-T cell treatments are
additionally included. This gives Clinical utilizations of quality treatment are broad and
spread most frameworks and their issue. Full sections have been given to hereditary
disorders, malignant growth, cardiovascular maladies, neurological disarranges and
viral diseases with accentuation on AIDS. Slanted Applications of quality treatment in
veterinary medication, especially for treating felines and canines, are incorporated.

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Gene Editing Could One Day Treat Muscle Disorders
Chengzu Long Scaling the progress and never been attempted to study and treat
Duchenne muscular dystrophy. As a PhD student in Eric Olson’s lab at the University of
Texas Southwestern Medical Center, Long had spent years knocking out genes in mice
to try to identify their role in muscle development and disease, only to find that each of
the resulting knockouts had no discernible differences from wildtype individuals. Long
and Olson make a New CRISPR method efficiently to correct Duchenne muscular
dystrophy defect in heart tissue by using these methods on a higher scale. Therefore,
Scientists have developed a CRISPR gene-editing technique that can potentially
correct a majority of the 4,000 mutations that cause Duchenne muscular dystrophy
(DMD) by making a single cut at strategic points along the patient’s DNA, according to
a new study from UT Southwestern Medical Center.

The method, was successfully tested in heart muscle cells from patients, offers an
efficient alternative to the daunting task of developing an individualized molecular
treatment for each gene mutation that causes DMD. It also opens up possible new
treatment approaches for other diseases that have thus far required more intrusive
methods to correct single-gene mutations.

"This is a significant step," said Dr. Eric Olson, Director of UT South western’s Hamon
Center for Regenerative Science and Medicine. "We're hopeful this technique will
eventually alleviate pain and suffering, perhaps even save the lives, of DMD patients
who have a wide range of mutations and, unfortunately, have had no other treatment
options to eliminate the underlying cause of the disease." But DMD is a rare disease
because this disease is affecting primarily boys and caused by defects in the gene that
makes the dystrophin protein. These defects -- which could affect any of the 79 exons
that comprise the gene -- lead to degeneration of skeletal and heart muscles, that may
force patients into wheelchairs and, due to degeneration of chest wall muscles needed
for breathing, eventually onto respirators. Most patients die by age 30. No cure has been
developed. Consequently, they need more efficient effects of developing as
researchers continued studying the best methods to maximize efficiency and reduce
the potential for unintended edits to the genome.

The method produced by Dr. Olson's lab and the other scientist was spent substantial
time developing and testing various guide RNAs (ribonucleic acids) that could lead the
Cas9 enzyme precisely to 12 designated splice sites and avoid errant edits. These sites

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were targeted because they are located at "hot spots" along the dystrophin gene
where up to 60 percent of the disease-causing mutations occur.

Therefore, Dr. Olson's lab will continue testing the method to ensure it does not
have adverse side effects, in addition to seeking ways to improve the precision of the
guide RNAs. In the meantime, the work has spawned a new biotechnology company,
Exonics Therapeutics Inc., which is working to further optimize and extend the approach
to additional DMD mutations, as well as other neuromuscular diseases. Exonics has
licensed the technology from UT Southwestern.

A major advance to them that Many different therapies have been put forward,
but this one provides real hope to extend and improve the quality of patients' lives.
Some of those patients have already seen firsthand how gene editing can correct
defects in their cells. Prolonging the method which these new gene-editing
technologies are making this vision a reality.

After successfully correcting the DMD mutation in mdx mice during embryonic
development, Long and Olson decided to use adeno-associated virus 9 (AAV9) it has
an ability to infect both nondividing and dividing cells with persistent expression have
made it an attractive vector. An additional attractive feature of the wild-type virus is the
lack of apparent pathogenicity to deliver the CRISPR system into mice after birth.

There are new methods to the assembly of Gene Editing of genetics by Charles
Gersbach. Because the genetics of the disease are well understood, researchers could
theoretically replace the mutated version of DMD with a healthy copy to cure the
disease. Unfortunately, the gene for dystrophin is massive, with 2.6 million base pairs. As
a result, it’s not feasible to insert the entire gene, or even just the 11,000 coding base
pairs (introns excluded), into a viral vector that could deliver the therapeutic package
to the muscle. “Gene editing therefore was a great opportunity to correct the
endogenous gene rather than trying to deliver” a nonmutated version of it, says Charles
Gersbach, a biomedical engineer at Duke University. Therefore, by Correcting
endogenous genres are better than trying to deliver a nonmutated version of the said
gene.

There are a few reasons why successful gene edits, once made this method, were
so efficient in these examples. Muscle cells are multinucleated, with each cell having
hundreds of nuclei. “If you can correct just a few of them you can protect the whole

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muscle fiber,” says Gersbach. And just a little dystrophin can go a long way toward
improving muscle function, Olson adds. “It’s estimated that as little as 15 percent of
normal dystrophin levels could be curative, or at least highly beneficial.”. Consequently,
Dystrophin protein is made up of redundant coils that acts like a shock absorber during
contraction. The idea here is that these coils are redundant and if some of these coils
are removed, the rest can still function. This is why Gersbach theorized that as little as
15% of the normal dystrophin levels can at least mitigate or for the better be a curative
for those that lack these proteins due to the DMD mutation.

By restoring the defective parts of the gene in the first place. Doing so relies on a
template-driven DNA repair process called homology-directed repair (HDR), which
occurs infrequently in nondividing cells such as those of skeletal and heart muscle. “The
problem with inserting stuff is that it’s very inefficient and makes the drug more
complicated,” says Gersbach. “We’re exploring a number of ways by which we might
increase that efficiency, but for the time being that’s not really an option.”

In Duchenne, DMD mutations disrupt the gene’s reading frame, causing

translation to terminate prematurely and leading to a complete lack of a functional
dystrophin protein. In a closely related disease called Becker muscular dystrophy (BMD),
patients carry mutations in the DMD gene that are in-frame, typically deletions that result
in a smaller but still partially functional dystrophin. As a result, patients with BMD generally
suffer less-severe symptoms and survive considerably longer than Duchenne muscular
dystrophy patients. “The dystrophin protein is built like a shock absorber with a series of
redundant coils in the center,” says Olson. “You can delete several of those coils and
still retain function.”

The key idea of these are the characteristics of the BMD mutation to give the
scientists ideas that as long as there are amounts of dystrophin present, as shown in the
BMD mutation where there are still dystrophin proteins but in a smaller and partially
functional version that the patients of this mutation are shown to perform better that
those with the DMD mutation. This is because of the presence of a little amount of
dystrophin protein that still retains a portion of functionality.

To improve Duchenne patients’ prognoses, then, researchers can provide them

with smaller versions of the dystrophin protein. One promising approach is to scale down
gene therapy so that the DNA encoding a functional, pared-down protein can fit into
a viral vector. A recent trial testing the implantation of such a “microdystrophin” showed
that the therapy increased levels of the small protein in muscles and reduced levels of
a Duchenne-associated enzyme, called creatine kinase, in three patients. The results

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are promising, although it’s still too early to know what their clinical significance will be.
“There are a number of these types of trials that are ongoing that look really exciting,”
says Gersbach.

Basing from the method of using the fundamentals of BMD mutation, the
researchers provide the patients with smaller versions of dystrophins to somewhat
improve their muscle conditions. The results are promising and Gersbach hopes that this
will lead them to curing the DMD mutation completely.

A Method has been attached for good editing genes was CRISPR also known for
(Clustered Regularly Interspaced Short Palindromic Repeat). This name refers to the
unique organization of short, partially palindromic repeated DNA sequences found in
the genomes of bacteria and other microorganisms. While seemingly innocuous, CRISPR
sequences are a crucial component of the immune systems of these simple life forms.
The immune system is responsible for protecting an organism’s health and well-being.
Just like us, bacterial cells can be invaded by viruses, which are small, infectious agents.
If a viral infection threatens a bacterial cell, the CRISPR immune system can thwart the
attack by destroying the genome of the invading virus.

Seemingly the genome of the virus includes genetic material that is necessary for
the virus to continue replicating. Thus, by destroying the viral genome, the CRISPR
immune system protects bacteria from ongoing viral infection.

The CRISPR immune system works to protect bacteria from repeated viral attack
via three basic steps.

Step 1) Adaptation – DNA from an invading virus is processed into short segments that
are inserted into the CRISPR sequence as new spacers.

Step 2) Production of CRISPR RNA – CRISPR repeats and spacers in the bacterial DNA
undergo transcription, the process of copying DNA into RNA (ribonucleic acid). Unlike
the double-chain helix structure of DNA, the resulting RNA is a single-chain molecule.
This RNA chain is cut into short pieces called CRISPR RNAs.

Step 3) Targeting – CRISPR RNAs guide bacterial molecular machinery to destroy the
viral material. Because CRISPR RNA sequences are copied from the viral DNA sequences
acquired during adaptation, they are exact matches to the viral genome and thus
serve as excellent guides.

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 Duchenne Muscular Dystrophy was a Genetic disorder characterized by
progressive muscle degeneration and weakness that caused by an absence of
dystrophin, muscle cells become fragile and easily damaged. There are symptoms can
be found between ages 3 and 5 most especially primarily affected by boys.
Consequently,

A French neurologist Guillaume Benjamin Amand Duchenne in 1860’s. There are

MDA-supported researchers identified a particular gene on the X chromosome that
leads to DMD when mutated (1986). Dystrophin was identified as the protein affected
by the mutation in (1987).

The method engaging the disease and by typically appear in early childhood of
Duchenne Muscular Dystrophy are the examples of Frequent falls, Difficulty rising from a
lying or sitting up position, trouble running and jumping, large calf muscles, muscle pain,
and heart failure. Therefore, possibly because the gene involved may subject to sudden
abnormal changes.
Duchenne is caused by mutations (changes) within the dystrophin gene. A gene
is made up of coding regions called exons, and the areas in between exons are
called introns. Dystrophin has 79 exons, which makes it one of the largest genes in the
body.

Making the dystrophin protein from the gene involves several steps. One of the
first steps is removing the introns and fitting the exons together, 1-79, like puzzle pieces.
Therefore, if there is a missing piece within the dystrophin gene (deletion) or an extra
piece (duplication), your body can have difficulty making dystrophin.

These methods identify each type of mutations. Large deletions caused 60-70%
cases of how often mutation causes Duchenne and 80-85% cases of mutation cause
Becker. Deletions occur when pieces of the gene (called exons) are missing. Deletions
of one or more exons are the most common type of mutation. Since there are a total of
79 exons in the dystrophin gene, there are many different deletions that can occur.
However, there are certain areas of the gene that are more likely to have a deletion,
and these areas are called “hot spots”.

Becker Muscular dystrophy is similar to Duchenne muscular dystrophy, tend to be

milder and progress more slowly, A Large duplication intended 10% cases of mutation
of causes Duchenne and 5-10% cases mutation of causes Becker.

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 Duplications occur when one or more exons within the gene are doubled.
Duplications are not as common as deletions. Like deletions, duplications can occur
throughout all 79 exons of the dystrophin gene.

Point mutations and other small changes (including ‘nonsense’ mutations)

intended between 15-30% cases of mutation cause Duchenne and 10-15% cases of
mutation cause Becker.

Point mutations are smaller changes in the gene that do not involve an entire
exon. Sometimes just one letter in the DNA code is missing (deleted), doubled
(duplicated), or changed. One of the most common point mutations is called
a nonsense mutation. Nonsense mutations cause a premature stop in the gene which
results in little or no dystrophin protein production.

There are new method Readings about the difference between In-frame vs out-
frame errors and refers to Duchenne and Becker Dystrophy. Seemingly, if you or your
child have a deletion mutation.

The first new method is In-Frame, the reading frame of the gene is preserved and
not disrupted, so some dystrophin protein can be made. The protein may be shorter
than normal, but it is still functional. In-frame deletions typically result in Becker muscular
dystrophy, which usually has a milder presentation (compared to Duchenne) because
there is some dystrophin protein present in the cells.

The second method is Out-Frame, the reading frame is completely disrupted, so

that no dystrophin protein can be made. Out-of-frame deletions typically result in
Duchenne muscular dystrophy, which usually has a more severe presentation
(compared to Becker) because there is no dystrophin protein present in the cells.

Therefore, it is important to remember that this reading frame rule is not always
perfect. There are some out-of-frame deletions that cause Becker, and some in-frame
deletions that cause Duchenne.

Methods on determined your mutations through Genetic testing, gold standard

for the diagnosis of Duchenne and Becker. Genetic testing determined what type of
mutation is present, as well as the specific details of the mutation. Genetic testing can
predict if someone is more likely to have Duchenne or Becker. However, as stated
previously, predictions of severity based on the mutation are not perfect and a person’s
symptoms and disease progression must be considered. Therefore, families must consult

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with their doctor for their final diagnosis. there are individuals who are intermediate
because their features fall in between Duchenne and Becker.

A fundamental why is it important to know your mutation? There are three

reasons to know your genetic mutation.

The first method is to confirm your diagnosis: Genetic testing will confirm if you
have Duchenne or Becker, or you may have another type of muscular dystrophy that
shares some of the same features as Duchenne or Becker.

The second method is to enable testing of family members: Once the mutation
in a family is known, other family members can be tested to determine if they
are carriers of the gene mutation. Genetic testing is the best method to performed
accurate carrier testing.

The third method is to determine what mutation-specific therapies may benefit

you: many therapies in development and/or approved for Duchenne are mutation-
specific, meaning they will only benefit individuals with certain mutations. You must
know your mutation in order to participate in a clinical trial and to access any current
or future mutation-specific therapies.

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