The document discusses antisense therapy as a treatment for genetic disorders, notably motor neuron diseases like Duchenne muscular dystrophy (DMD) and infections such as cytomegalovirus retinitis. Antisense oligonucleotides can inactivate specific messenger RNA to turn off disease-causing genes, with approved treatments for conditions like DMD and cytomegalovirus. It also covers the synthesis, delivery methods, and various applications of oligonucleotides in molecular biology and medicine.

1. ANTISENSE THERAPY
Under the guidance of
S.SEETHARAM SWAMY
(Assistant Professor)
Presented by:
HARIKA ENAKONDA
13GD1R0013
IVTH B-PHARMACY
CHILKUR BALAJI COLLEGE OF PHARMACY
(Affiliated to JNTU, Hyderabad-500008)

2. Antisense Therapy to motor
neuron diseases

3. Antisense therapy is a form of treatment for genetic disorders or infections.
When the genetic sequence of a particular gene is known to be causative of a
particular disease, it is possible to synthesize a strand of nucleic acid(,DNA, RNA
or a chemical analogue) that will bind to the messenger RNA (mRNA) produced by
that gene and inactivate it, effectively turning that gene "off".
This synthesized nucleic acid is termed as "anti-sense" Oligonucleotide(ASO)
because its base sequence is complementary to the gene's messenger RNA
(mRNA), which is called the "sense" sequence (so that a sense segment of mRNA
"5'-AAGGUC-3' " would be blocked by the anti-sense mRNA segment " 3'-
UUCCAG-5' ").
INTRODUCTION:

4. Antisense oligonucleotides have been researched as potential drugs for diseases
such as cancers (including lung cancer, colorectal carcinoma, pancreatic
carcinoma, malignant glioma and malignant melanoma), diabetes, Amyotrophic
lateral sclerosis (ALS), Duchenne muscular dystrophy and diseases such as
asthma, arthritis and pouchitis with an inflammatory component.
As of 2014 two antisense drugs have been approved by the U.S. Food and Drug
Administration (FDA) fomivirsen, (marketed as Vitravene) as a treatment for
cytomegalovirus and mipomersen (marketed as Kynamro) for homozygous
familial hypercholesterolemia.

5. Example antisense therapies:
Cancer
Cytomegalovirus retinitis
Familial hypercholesterolemia
Hemorrhagic fever viruses
HIV/AIDS
Spinal muscular atrophy
Duchene muscular dystrophy
Amyotrophic lateral sclerosis

6. OLIGONUCLEOTIDE SYNTHESIS:
Oligonucleotide synthesis is the chemical synthesis of relatively short
fragments of nucleic acids with defined chemical structure.
Currently, the process is implemented as solid-phase synthesis using
phosphoramidite method and phosphoramidite building blocks derived from
protected 2'-deoxynucleosides (dA, dC, dG, and T), ribonucleosides (A, C, G, and
U), or chemically modified nucleosides, e.g. LNA or BNA.
Oligonucleotides are often isolated by HPLC to obtain the desired
oligonucleotides in high purity.

8. APPLICATIONS OF OLIGONUCLEOTIDES IN MOLECULAR
BIOLOGY AND MEDICINE:
1. They are most commonly used as antisense oligonucleotides.
2. These are primers for DNA sequencing and amplification.
3. These are probes for detecting complementary DND or RNA by molecular
hybridization.
4. These are tools for the targeted introduction of mutations and restriction
sites.
5. These are used for the synthesis of artificial genes.

9. Delivery:
The nucleases that cleave the Phosphodiester linkage in DNA are expressed in
almost every cell, unmodified DNA molecules are generally degraded before they
reach their targets.
Therefore, most clinical candidates have modified DNA "backbones", or the
nucleobase or sugar moieties of the nucleotides are altered.
Additionally, other molecules may be conjugated to antisense molecules in order to
improve their ability to target certain cells or to cross barriers like cell membranes or
(BBB).

10. ANTISENSE THERAPY TO DUCHENNE MUSCULAR DYSTROPHY:
 Duchenne muscular dystrophy(DMD) is an X-linked recessive form of
muscular dystrophy, affecting around 1 in 3,600 boys, which results in muscle
degeneration and premature death. The disorder is caused by a mutation in the
gene dystrophin, located on the human X chromosome, which codes for the
protein dystrophin.
 Dystrophin is an important component within muscle tissue that provides
structural stability to the dystroglycan complex (DGC) of the cell membrane.

11. Signs and Symptoms:
 Pseudo hypertrophy.
Difficulty in standing.
Fibrosis.
Neuromuscular disorder i.e., muscle weakness.
Hips, pelvic area, thighs, shoulders and calves are affected.
Awkward manner of walking, stepping or running.
Fatigue.
Skeletal deformities.
Cardio myopathy.
Congestive heart failure.
Loss of muscle mass.
Respiratory disorder.

12. Diagnosis:
Genetic counselling is advised for people with a family history of the disorder.
Duchenne muscular dystrophy can be detected with about 95% accuracy by genetic
studies performed during pregnancy.
DNA test:
• The muscle-specific dystrophin gene is composed of 79 exons.
• DNA testing and analysis can usually identify the specific type of mutation of the
exon or exons that are affected.
Muscle biopsy:
•If DNA testing fails to find the mutation, a muscle biopsy test may be performed. A
small sample of muscle tissue is extracted using a biopsy needle.
•The key tests performed on the biopsy sample for DMD are immunocytochemistry
and immunoblotting for Dystrophin.

13. These tests provide information on the presence or absence of the protein. Where the protein
is absent, this is a positive test for DMD. Where Dystrophin is present, the tests will indicate
the amount and molecular size of Dystrophin, helping to distinguish DMD from milder
dystrophinopathy phenotypes.
Prenatal tests:
 DMD is carried by an X-linked recessive gene. Males have only one X chromosome, so
one copy of the mutated gene will cause DMD. Fathers cannot pass X-linked traits on to
their sons, so the mutation is transmitted by the mother.
 If the mother is a carrier, and therefore one of her two X chromosomes has a DMD
mutation, there is a 50% chance that a female child will inherit that mutation as one of her
two X chromosomes, and be a carrier. There is a 50% chance that a male child will inherit
that mutation as his one X chromosome, and therefore have DMD.

14. Prenatal tests can tell whether their unborn child has the most common
mutations.
Prior to invasive testing, determination of the foetal sex is important; while
males are sometimes affected by this X-linked disease, female DMD is
extremely rare. This can be achieved by ultrasound scan at 16 weeks or more
recently by free foetal DNA testing.
Chorion villus sampling (CVS) can be done at 11–14 weeks, and has a 1%
risk of miscarriage. Amniocentesis can be done after 15 weeks, and has a 0.5%
risk of miscarriage. Foetal blood sampling can be done at about 18 weeks.
Another option in the case of unclear genetic test results is foetal muscle
biopsy.

15. TREATMENT:
There is no cure for DMD, and an on-going medical need has been recognized by
regulatory authorities. Phase 1-2a trials with exon skipping treatment for certain
mutations have halted decline and produced small clinical improvements in walking.
Corticosteroids
• Ex: prednisolone and deflazacort.
• Randomized control trials have shown that beta2-agonists increase muscle strength
but do not modify disease progression.
• Mild, non-jarring physical activity such as swimming is encouraged. Physical
therapy is helpful to maintain muscle strength, flexibility, and function. Orthopedic
appliances (such as braces and wheelchairs) may improve mobility and the ability for
self-care.

16. Stem cell replacement:
 Though stem cells isolated from the muscle (satellite cells) have the ability to
differentiate into myotubes when injected directly into the muscle of animals, they
lack the ability to spread systemically throughout.
 This problem was circumvented by using another multipotent stem cell, termed
pericytes that are located within the blood vessels of skeletal muscle.
 Once pass the vasculature, pericytes have the ability to fuse and form myotubes.
This means that they can be injected arterially, crossing through arterial walls into
muscle, where they can differentiate into potentially functional muscle.

17. Gene therapy:
 In 2014 and 2015, researchers used a new gene editing method to correct a mutation that
leads to Duchenne muscular dystrophy (DMD) in a mouse model of the condition.
 Researchers used a technique called CRISPR/Cas9-mediated genome editing, which can
precisely remove a mutation in the Dystrophin gene in DNA.
 The benefit of this over other gene therapy techniques is that it can permanently correct
the “defect” in a gene rather than just transiently adding a “functional” one.
 Biostrophin is a delivery vector for gene therapy in the treatment of Duchenne muscular
dystrophy and Becker muscular dystrophy.

18. ANTISENSE THERAPY TO CYTOMEGALOVIRUS RETINITIS:
Cytomegalovirus retinitis, also known as CMV retinitis, is an inflammation of the retina of
the eye that can lead to blindness. Caused by human cytomegalovirus, it occurs
predominantly in people whose immune system has been compromised, 15-40% of those
infected with AIDS. There are different types of retinitis, such as retinitis pigmentosa (causes
tunnel vision).
Fundus photograph of CMV retinitis

19. Signs/symptoms
 Blurred vision
 Blind spots
 Specks in your vision
Cause:
Cytomegalovirus (herpes viruses) causes cytomegalovirus retinitis. These viruses include
herpes simplex viruses and Epstein-Barr virus. Once an individual is infected it stays in the
body for life.
Triggers for reactivation of CMV virus
 Leukaemia:
 AIDS
 Immunosuppressive chemotherapy
Leukaemia

20. MECHANISM:
Human cytomegalovirus (HCMV or CMV) is a DNA virus in the family Herpesviridae
known for producing large cells with nuclear and cytoplasmic inclusions,CMV infects
around 40% of the population worldwide.
Those areas infected by cytomegalovirus have cells evolve to necrosis,though
inflammation within the retina is not great. Rhegmatogenous retinal detachments can occur
following the development of holes in areas of healed retinitis (retina may be
atrophic).[Proliferative vitreoretinopathy has been observed in cases of retinal detachment.

21. Diagnosis:
The diagnosis of CMV retinitis can be done by the following:
•Ophthalmic screening frequency is based on CD4 count.
•BUN
•CD8+ T-lymphocyte count
•CMV DNA capture ( polymerase chain reaction (PCR) test)
•DNA PCR ( ocular fluids)
•Viral load
•Complete blood count

22. Treatment:
Structure of ganciclovir
1.Ganciclovir:
oral valganciclovir,
 intravenous ganciclovir,
IV foscarnet, and
IV cidofovir
 Intravitreal injections:
 An injection of medicine into the vitreous near the retina, of foscarnet in concomitance with
oral valganciclovir can be used for treatment as well.
Often individuals with CMV retinitis will need surgery for either retinal detachment or
intravitreal instillation of ganciclovir.
 Retinal detachment occurs in up to 29% of affected eyes, repair being most effective with
endolaser and silicone oilendotamponade.

23.  Intravitreal ganciclovir implant has the benefit of less systemic toxicity.
Adverse effect:
• vitreous hemorrhage
2.Fomivirsen:
Fomivirsen (brand name Vitravene) is an antiviral drug. It is used in the treatment of
cytomegalovirus retinitis (CMV) in immunocompromised patients, including those with
AIDS. It was licensed by the FDA for CMV in Aug 1998.
Mechanism:
It is an oligonucleotide that blocks translation of viral mRNA by binding to the
complementary sequence of the mRNA transcribed from the template segment of a key
CMV gene UL123, which encodes the CMV protein IE2. It was the first antisense
antiviral approved by the FDA.
Dose:
6.6mg/ml through intraocular injection

24. CONCLUSION:
As discussed throughout this report, human health is determined by the interaction of
several factors, including the social environment, genetic inheritance and personal
behaviors. Socioeconomic status, race or ethnicity, social networks or social support, and
the psychosocial work environment all have been shown to affect health outcomes. These
social determinants influence health at multiple levels through out the life course. In
addition to the vast array of social determinants that influence health, a person inherits
complete set of genes from each parent that contributes both directly and indirectly to the
pathogenesis of disease. Genes have been identified for relatively uncommon, simple
Mendelian patterns of disease inheritance such as cystic fibrosis and recently research has
begun to explore genetic susceptibility to disease as the consequence of the joint effects of
many genes. Behaviors also have been shown to affect health for example tobacco use,
obesity and physical inactivity further more complex traits such as sex or gender and race.
As this report demonstrates, research has documented association's between social factors
and health, behaviors and health, genetics and health.

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