NUCLEIC ACID BASED THERAPEUTIC DELIVERY SYSTEM

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❖ Gene therapy

 Introduction

 Definition

 History and development

 Advantages & Disadvantages

 Types

 Vectors in the gene therapy

 Methods

❖ Gene therapy for inherited disorder and Cancer

 Conclusion

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 Gene therapy is a novel treatment method

 It utilizes genes or short Oligonucleotides sequences as therapeutic molecules,
instead of conventional drug compounds.

 This technique is widely used to treat those defective genes which
contribute to disease development.

 What Genes can do

 Genes, which are carried on chromosomes, are the basic physical and
functional units of heredity.

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 Genes are specific sequences of bases that encode instructions on how to
make proteins.

 Those proteins that perform most life functions and even make up the
majority of cellular structures.

 Why Genetic Disorders

 When genes are altered so that the encoded proteins are unable to carry out
their normal functions, genetic disorders can result.

 Definition: Gene therapy is an experimental technique for correcting
defective genes that are responsible for disease development.

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 The most common form of gene therapy involves inserting a normal gene
to replace an abnormal gene.

❖ Other approaches used:

 Replacing a mutated gene that causes disease with a healthy copy of the
gene.

 Inactivating, or “knocking out,” a mutated gene that is functioning
improperly.

 Introducing a new gene into the body to help fight a disease.

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 Researchers are studying gene therapy for a number of diseases, such as

 Severe combined immuno-deficiencies (SCID)

 Hemophilia

 Parkinson’s disease

 Cancer

 HIV

 The first approved gene therapy experiment occurred on September 14,
1990 in US, when Ashanti DeSilva was treated for ADA-SCID.

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 1850s: When Austrian monk Gregor Mendel, in a series of experiments
with green peas, described the pattern of inheritance.

 1960’s : The concepts of Gene Therapy was introduced.

 1972 : Friedman and Roblin authored a paper in Science titled “Gene
therapy for human genetic disease.”

 1984: A retrovirus vector system was designed that could efficiently insert
foreign genes into mammalian chromosomes.

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 1990: The first approved gene therapy in the US took place on 14
September 1990, at the National Institutes of Health (NIH), under the
direction of William French Anderson. Four-year-old Ashanti DeSilva
received treatment for a genetic defect that left her with ADA-SCID, a
severe immune system deficiency.

 1992: Doctor Claudio Bordignon performed the first procedure of gene
therapy using hematopoietic stem cells as vectors to deliver genes intended
to correct hereditary diseases.

 1999: A significant setback to gene therapy research in the United States.

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 2003 : A research team inserted genes into the brain for the first time. They
used liposomes which, unlike viral vectors, are small enough to cross the
blood–brain barrier.

 2006 : Successful use of gene therapy to treat two adult patients for X-
linked chronic granulomatous disease.

 2007: First gene therapy trial for inherited retinal disease

 2010 : An 18 year old male patient in France with beta-thalassemia major
had been successfully treated

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 2011: Medical community accepted that it can cure HIV as in 2008, Gero
Hutter has cured a man from HIV using gene therapy.

 2012- 2015: Research is still ongoing and the number of diseases that has
been treated successfully by gene therapy increases.

 2017: August – FDA approves Kymriah, a cell-based gene therapy, is
approved in the United States for the treatment of patients up to 25 years of
age with B-cell precursor.

 2017: December-FDA approves novel gene therapy to treat patients with a
rare form of inherited vision loss.

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 Gene therapy has the potential to eliminate and prevent hereditary diseases
such as cystic fibrosis, ADA- SCID etc.

 It is a possible cure for heart disease, AIDS and cancer.

 It gives someone born with a genetic disease a chance to life.

 It can be used to eradicate diseases from the future generations.

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 Short Lived: Long lasting therapy is not achieved by gene therapy; due to
rapid dividing of cells benefits of gene therapy is short lived.

 Immune Response: Immune response to the transferred gene stimulates a
potential risk to gene therapy.

 Viral Vectors: Viruses used as vectors for gene transfer may cause toxicity,
immune responses, and inflammatory reactions in the host.

 Multigene Disorders: Heart disease, high blood pressure, Alzheimer’s,
arthritis and diabetes are hard to treat because it need to introduce more
than one gene..

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Germ line gene therapy Somatic gene therapy

Therapeutic genes transferred into the Therapeutic genes transferred into the
germ cells. (eggs and sperms.) somatic cells.(bone marrow cells,blood

cells, skin cells etc.

It is heritable and passed on to later Will not be inherited later generations.
generations

For safety, ethical and technical reasons, At present all researches directed to
it is not being attempted at present correct genetic defects in somatic cells.

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 1st gene therapy – to correct deficiency of enzyme, Adenosine deaminase
(ADA).

 Performed on a 4yr old girl Ashanthi DeSilva.

 She was suffering from SCID- Severe Combined Immunodeficiency.

 Caused due to defect in gene coding for ADA.

 Deoxy adenosine accumulate and destroys T lymphocytes.

 Disrupts immunity , suffer from infectious diseases and die at young age.

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 Direct delivery of therapeutic gene into target cell into patients body.

 Carried out by viral or non viral vector systems.

 It can be the only possible option in patients where individual cells cannot
be cultured in vitro in sufficient numbers (e.g. brain cells).

 In vivo gene transfer is necessary when cultured cells cannot be re-
implanted in patients effectively.

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 In patients with cystic fibrosis, a protein called cystic fibrosis
transmembrane regulator (CFTR) is absent due to a gene defect.

 In the absence of CFTR chloride ions concentrate within the cells and it
draws water from surrounding.

 This leads to the accumulation of sticky mucous in respiratory tract and
lungs.

 Treated by in vivo replacement of defective gene by adenovirus vector .

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In vivo Ex vivo

Less invasive More invasive

Technically simple Technically complex

Vectors introduced directly No vectors introduced directly

Safety check not possible Safety check possible

Decreased control over target cells Close control possible

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 Gene modification
◦ Replacement therapy
◦ Corrective Gene therapy

 Gene transfer
◦ Physical
◦ Chemical
◦ Biological

 Gene transfer in specific cell line
◦ Somatic (gene therapy)
◦ Germ line gene therapy

 Eugenic approach (gene insertion)

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 Gene that is inserted directly into a cell usually does not function.

 Definition : To transfer the desired gene into a target cell, a carrier is
required. Such vehicles of gene delivery are known as vectors.

 Ideal characteristics of vector
◦ Target the right cells

◦ Integrate the gene in the cells.

◦ Activate the gene.

◦ Avoid harmful side effects.

◦ No universal vector exists.

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´
 2 main classes

´
❑ Viral vectors

´
❑ Non viral vectors

❖Viral vector:

 Viruses introduce their genetic material into the host cell as part of their
replication cycle.

 Remove the viral DNA and using the virus as a vehicle to deliver the
therapeutic DNA.

 These viruses are inactivated; they are not able to reproduce and also to
make them safe, although some risks still exist with gene therapy.

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 A number of viruses have been used for human gene therapy, including :

o Retrovirus

o Adenovirus

o Adeno-associated virus

o Herpes simplex virus

o Lentivirus

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 Used as first viruses.

 To treat X-linked severe combined immune deficiency represent the most
successful application till date.

 The recombinant retroviruses have the ability to integrate into the host
genome in a stable fashion.

 Can carry a DNA of size – less than 3.4kb

 Target cell – dividing

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 Adenoviral DNA does not integrate into the genome and is not replicated
during cell division.

 Humans commonly come in contact with adenovirus, majority of patients
have already developed neutralizing antibodies which can inactivate the
virus.

 Target- non dividing, dividing cells.

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 It is a human virus.

 It is single stranded .

 AAV enters host cell, becomes double stranded and gets integrated into
chromosome19.

 AAV is not currently known to cause disease and consequently the virus
causes a very mild immune response.

 Target- non dividing, dividing cells.

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 It is a human neurotropic virus.

 This is mostly examined for gene transfer in the nervous system.

 Viruses which have natural tendency to infect a particular type of cell.

 Lentivirus

 Subclass of Retroviruses.

 The viral genome in the form of RNA is reverse-transcribed when the virus
enters the cell to produce DNA, which is then inserted into the genome at a
random position via viral integrase enzyme.

 Target cells- dividing, non-dividing.

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 Target specific types of cells.

 They’re very good at targeting and entering cells.

 They can be modified so that they can’t replicate and destroy cells.

 Disadvantages of Viral Vector

 They can cause immune responses in patients.

 They can carry a limited amount of genetic material. Therefore, some
genes may be too big to fit into some viruses

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 Non viral approach involves the creation of an artificial lipid sphere with
an aqueous core.

 This liposome, which carries the therapeutic DNA, is capable of passing
the DNA through the target cell’s membrane.

 Methods of non-viral gene delivery have also been explored using physical
(carrier-free gene delivery) and chemical approaches (synthetic vector-
based gene delivery).

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 It employ a physical force that permeates the cell membrane and facilitates
intracellular gene transfer

 Direct injection

 Micro injection

 Electroporation

 Gene gun/particle bombardment

 Ultrasound/ Sonoporation

 Magnetofection

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 Direct introduction of pure DNA into target tissue by parenterally.

 Consequently, large quantities of DNA have to be injected periodically
since low efficiency of DNA uptake by cells.

❖Micro injection :

 The delivery of foreign DNA, by the help of

glass micropipette into a living cell.

 The cell is held against a solid

support or holding pipette and micro needle

containing the desired DNA is inserted into the cell.

 The tip of the pipette used is about 0.5 to 5 micro meter diameter which
resembles an injection needle.

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 The external electric field is applied to the protoplast

 Changes the electrical conductivity and the permeability of cell membrane.

 The efficiency of electroporation can be increased by giving the cell a heat
shock, prior to the application of electric field or by using small quantity of
PEG while doing electroporation.

 However, a high rate of cell death following electroporation has limited its
use, including clinical applications.

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 An alternative approach uses particle bombardment (‘gene gun’) technique.

 DNA is coated on to metal micro particles and fired from a ballistic gun
into cells/tissues.

 It can cross the physical barriers like skin, muscle layer for which it is used
for vaccination.

 Particle bombardment is used to deliver drugs, fluorescent dyes, antigenic
proteins etc.

Disadvantage:

 If the DNA is integrated in the wrong place in the genome, for example in a
tumour suppressor gene, it could induce a tumour.

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 Ultrasonic frequencies to deliver DNA into cells.

 The process of acoustic cavitations is thought to disrupt the cell membrane
and allow DNA to move into cells.

Magnetofection:

 DNA is complexed to magnetic particles.

 A magnet is placed underneath the tissue culture dish to bring DNA
complexes into contact with a cell monolayer.

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 Oligonucleotides

 Hybrid methods

 Dendrimers

 Using detergent mixtures

 Lipofection

Oligonucleotides

 To inactivate the genes involved in the disease process.

 Uses antisense specific to the target gene to disrupt the transcription of the
faulty gene.

 Small molecules of RNA called siRNA to signal the cell to cleave specific
unique sequences in the mRNA transcript of the faulty gene, disrupting
translation of the faulty mRNA.

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 Virosomes are one example; they combine liposomes with an inactivated
HIV or influenza virus.

 This has been shown to have more efficient gene transfer in respiratory
epithelial cells than either viral or liposomal methods alone.

 Other methods involve mixing other viral vectors with cationic lipids or
hybridising viruses.

Dendrimers:

 A highly branched macromolecule with a spherical shape.

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 Certain charged chemical compounds like Calcium phosphates are mixed
with functional cDNA of desired function.

 The mixture is introduced near the vicinity of recipient cells.

 The chemical disturb the cell membrane, widen the pore size and allows
cDNA to pass through the cell.

Lipofection:
 It is a technique used to inject genetic materials into a cell by means of

liposomes.

 Liposomes are artificial phospholipid vesicles used to deliver a variety of
molecules including DNA into the cells.

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❖ Severe Combined Immunodeficiency Syndrome (SCID)

❖ Muscular Dystrophy

❖ Respiratory Diseases

➢Emphysema

➢Cystic fibrosis

❖Parkinson’s diseases

❖Blindness

❖Diabetic polyneuropathy

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 Caused by a defective enzyme adenosine deaminase (ADA)

 Encoded by a gene on chromosome 20. (autosome)

 Necessary for the breakdown of purines salvage pathway.

 Lack of ADA causes accumulation of dATP.

 Deoxyadenosine triphosphate (dATP) is a nucleotide used in cells
for DNA synthesis (or replication).

 Without functional ribonucleotide reductase, lymphocyte proliferation is
inhibited and the immune system is compromised

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 The ADA gene therapy involves four steps:

 Cloning of normal ADA gene into a retroviral vector.

 Transfection of ADA recombinant into cultured ADA- T lymphocytes
derived from patient.

 Identification of resulting ADA+ T cells and multiplication via cell culture.

 Re-implantation of these cells in the patient.

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 A group of inherited disorders .

 Characterized by progressive muscle weakening.

 Often occurring in early childhood.

 Nine main categories of muscular dystrophy .

 Most common is Duchene muscular dystrophy (DMD)-severe X-linked
recessive disorder.

 MD arise from defects in the dystrophin gene .

 Which encodes a large cytoskeletal protein called dystrophin.

 Important for membrane stability and force transduction from muscle
fibres.

 Replacement or modification of a gene, is emerging as a promising
approach for treatment.

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❖ Emphysema

 Emphysema is a form of Chronic obstructive pulmonary disorder (COPD).

 The disease has two forms; familial and non-familial.

 Familial emphysema is a result of a Genetic mutation of a common
respiratory protein Alpha-1-antitrypsin (AAT) .

 Lack of AAT protein gene, leads to lung tissue degradation .

 Cationic liposomes are microscopic lipid (fat-based) vesicles which bind to
DNA and facilitate cellular uptake.

 .

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 Caused by a presence of mutation in both copies of the gene cystic fibrosis
transmembrane conductance regulator (CFTR).

 The gene that encodes the human CFTR protein is found on chromosome 7

 The most common mutation,ΔF508, is a deletion of three nucleotides

 Results in a loss of the amino acid phenylalanine (F)at the 508th position on
the protein.

 Transferring the normal CFTR gene into the affected epithelium cells would
result in the production of functional CFTR protein in all target cells, without
adverse reactions or an inflammation response.

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 A long-term degenerative disorder of the central nervous system that
mainly affects the motor system

 Transfer a gene for producing a chemical called glutamic acid
decarboxylase (GAD) into the basal ganglia.

 GAD gene being introduced is involved in increasing levels of a signalling
chemical called GABA.

 Significantly improved the weakness of the symptoms such as tremors,
motor skill problems, and rigidity done with local anesthesia, used a
harmless, inactive virus [AAV-2 ]

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 Leber’s congenital amaurosis (LCA) is a rare inherited eye disease that
appears at birth or in the first few months of life.

 Caused by an abnormality in a gene called RPE65. located at chromsome
1 Retinal pigment epithelium enzyme

 AAV vector was used to deliver a functional copy of the RPE65 gene,
which restored vision in children suffering from LCA.

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 Nerve damaging disorders associated with diabetes mellitus who’ve had the
disease for many years.

 Intramuscular injections of vascular endothelial growth factor (VEGF)
gene may help patients with diabetic neuropathy.

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 Cancer is a group of two hundred or more diseases

 Which results due to uncontrolled growth and proliferation of normal cells.

 Sometimes change or mutation in the genetic material of a cell may lead to
abnormal cell growth and proliferation.

 Cancer gene therapy can be divided into three broad categories:

❖ Immunotherapy,

❖ Oncolytic virotherapy and

❖ Gene transfer

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 Boosting immune system to target and destroy cancer cells.

 Gene therapy is used to create recombinant cancer vaccines.

 Cancer vaccines helps in cancer cell recognition by presenting them with
highly antigenic and immuno stimulatory cellular components.

 For example, administration of reovirus to cancer cells resulted in a
specific antitumor activity , which could be enhanced by combination with
chemotherapy and immunosuppressive drugs.

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 Cancer is a group of two hundred or more diseases which results due to
uncontrolled growth and proliferation of normal cells.

 Sometimes change or mutation in the genetic material of a cell may lead to
abnormal cell growth and proliferation.

 Cancer gene therapy can be divided into three broad categories:

 Immunotherapy,

 Oncolytic virotherapy and

 Gene transfer

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 Based on the concept of boosting immune system to target and destroy
cancer cells.

 Gene therapy is used to create recombinant cancer vaccines.

 Cancer vaccines helps in cancer cell recognition by presenting them with
highly antigenic and immuno stimulatory cellular components.

For example, administration of reovirus to cancer cells resulted in a
specific antitumor activity which could be enhanced by combination with
chemotherapy and immunosuppressive drugs.

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 In this technique genetically engineered viruses for example vaccine,
adenovirus, herpes simplex virus type I, reovirus are used to kill the cancer
cells.

 The potential of this technique is popularized after the initial phase I trials
for several vectors.

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 Introduction of candidate genes using viral or non- viral vectors into a
cancerous cell or the surrounding tissue to cause cell death or slows down
the growth of the cancer cells while remaining innocuous to the rest of the
body.

 Viruses used for this purpose, include vaccinia, adenovirus, herpes simplex
virus type I, reovirus and Newcastle disease virus.

 Example: Gendicine is a modified adenovirus that delivers the p53 gene
to cancer cells and has been approved for the treatment of head and neck
squamous cell carcinoma in certain countries.

 Non-viral methods include transfer of naked DNA and oligo-dendromer
DNA coatings using electroporation as a mode of gene delivery.

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1. PPT- Gene therapy presented by Nurul Miza Shasheiha binti Abdul
Mutalib

2. PPT – Gene therapy presented by Damaris Benny Daniel

3. PPT- Gene therapy basics Dr.T.V.Rao MD

4. Gene therapy: Introduction and methodsby NPTEL – Bio Technology –
Genetic Engineering & Applications

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