Gene therapy: Types of Gene therapy Gene transfer methods vectors for gene therapy approaches applications advantages and disadvantages. Gene therapy based drugs. Ethical considerations.

1. GENE THERAPY
Shivkumar S. Sammeta (M.Pharm)
Department of Pharmacology
Smt. Kishoritai Bhoyar College Of Pharmacy,
Kamptee.

2. GENE THERAPY
• Gene therapy is the process of transfer of a gene from one DNA molecule to
another DNA molecule. It involves the suppression or insertion of genes into
the individual’s cell and tissues to treat or prevent a disease.
• The directed desirable gene transfer from one organism to another and the
subsequent stable integration and expression of foreign gene into the
genome is referred as Genetic transformation.
• Unstable transformation occurs when DNA is not integrated into host
genome.
• Stable transformation occurs when DNA integrated into host genome and is
inherited in subsequent generation.
• In gene augmentation therapy a DNA is inserted into genome to replace the
missing gene product. In case of gene inhibition therapy, the antisense gene
inhibits the expression of dominant gene.

3. GENE THERAPY STRATEGIES
1. Gene Replacement/Correction
– Replacing a mutated gene with a healthy copy
Ex. Severe Combined Immunodeficiency
2. Gene Silencing/Gene Interference
– Inactivating or “knocking out” a mutated gene
Ex. Sickle Cell Disease
3. Gene Augmentation/Gene Addition
– Introducing a new gene to help fight disease
Ex. Parkinson’s Disease
4.“Suicide Gene”
– Can cause a cell to kill itself through apoptosis
– Makes cancer cells more vulnerable and sensitive to
anticancer drugs
Ex. Solid Tumors

4. TYPES OF GENE THERAPY
TYPES
GERM LINE
THERAPY
SOMATIC
CELL
THERAPY
EX VIVO
IN VIVO

5. Germ line therapy
• Altering a gene of an egg or a
sperm cell
or
• Altering the genetic composition of
a blastomere during an early stage
of its division
• Any zygote produced as a result of
this germ cell will have a correct
version of the defective gene and
will continue passing it on to their
offspring
• Considered unethical

6. Somatic Cell Therapy
• Involves altering the genetic code of a person’s
somatic cells
• Inserting therapeutic genes into somatic cells like:
Fibroblasts
Myoblasts
Epithelial cells
Nerve cells
Glial cells
• It is mostly performed in fully grown organisms
• Less controversial

7. APPROACHES OF GENE THERAPY
There are mainly two approaches for the transfer of genes in gene therapy:
1.Transfer of genes into patient cells outside the body (ex vivo gene therapy)
2.Transfer of genes directly to cells inside the body (in vivo gene therapy).

8. ExVivo Gene Therapy :-
➢In this mode of gene therapy genes are transferred to the cells grown in
culture, transformed cells are selected, multiplied and then introduced
into the patient.
➢The use of autologous cells avoids immune system rejection of the
introduced cells.
➢The cells are sourced initially from the patient to be treated and grown
in culture before being reintroduced into the same individual.
➢This approach can be applied to the tissues like hematopoietic cells and
skin cells which can be removed from the body, genetically corrected
outside the body and reintroduced into the patient body where they
become engrafted and survive for a long period of time.

10. InVivo Gene Therapy :-
➢In vivo method of gene transfer involves the transfer of cloned genes
directly into the tissues of the patient.
➢This is done in case of tissues whose individual cells cannot be cultured in
vitro in sufficient numbers (like brain cells) and/or where re-implantation
of the cultured cells in the patient is not efficient.
➢Liposomes and certain viral vectors are employed for this purpose
because of lack of any other mode of selection.
➢In case of viral vectors such type of cultured cells were often used which
have been infected with the recombinant retrovirus in vitro to produce
modified viral vectors regularly.These cultured cells will be called as
vector-producing cells (VPCs).The VPCs transfer the gene to
surrounding disease cells.
➢The efficiency of gene transfer and expression determines the success of
this approach, because of the lack of any way for selection and
amplification of cells which take up and express the foreign gene.

12. METHODS OF GENE TRANSFER
BIOLOGICAL CHEMICAL
PHYSICAL
VIRAL NON –VIRAL
VECTORS VECTORS

13. A. BIOLOGICAL METHODS
• Adenovirus
• Adeno associated Viruses
• Retrovirus
• Herpes Simplex Virus
1. ADENOVIRALVECTORS
Adenoviruses are large linear double-stranded DNA viruses and are known to be the second
most popular gene delivery vector for gene therapy of various diseases like cystic fibrosis
and certain types of cancer.
Following figure shows how the adenoviruses enter cells by receptor-mediated endocytosis. A
primary cellular receptor binds to viral fiber then the virus interacts with secondary receptors
which are responsible for its internalization.
Coxsackie and Adenovirus Receptor (CAR), Heparan sulphate glycosaminoglycans, sialic
acid, CD46, CD80, CD86, alpha domain of MHC I are the primary receptors which are specific
for specific strains of adenovirus. Integrins are the secondary receptors which helps in the
internalization of viral particles. Some adenovirus directly interacts with integrins like in the
case of fiber deficient Ad2 virions.
Target- non dividing human cell.

14. The adenoviral DNA has inverted terminal repeats (ITRs) and a terminal protein (TP) is
attached covalently to 5’ termini.The adenoviral genome is classified as early and late
regions based on the proteins they express. Proteins encoded by early region (E1, E2, E3, E4)
genes are involved in viral DNA replication, cell cycle modulation and defense system.The
late region genes (L1, L2, L3, L4, L5) encodes the viral structural proteins.Three classes of
adenoviral vectors namely first, second and third generation viral vectors are developed for
gene therapy purpose.

15. First generation adenoviral Vectors
These vectors are constructed by replacing the E1/E3 expression cassette and inserting our
candidate gene of 3-4kb size. E1 encodes proteins responsible for expressions of other viral
genes required for viral growth. So cell lines that can provide E1 proteins in trans are
required for the replication of the E1 deleted viral vectors

16. Second generation adenoviral Vectors
• E1/E2 or E3/E4 expression cassettes are called deleted and replaced.
• It can carry DNA insert upto 10.5kb.
Third generation adenoviral Vectors
• These vectors are called as gutless adenovirus.
• These are also known as helper dependent adenovirus as they lack all the coding sequences and
require helper virus which carries all the coding sequences.
• The size of insert DNA can be 36kb and hence called as high capacity adenoviruses.They carry
only 5’ inverted terminal repeats (ITR) and 3’ packaging signals (ψ).
2. ADENO- ASSOCIATEDVIRUS (AAV)
Adeno-associated viruses (AAVs) are a group of small, single-stranded DNA viruses which cannot
usually undergo productive infection without co-infection by a helper virus, such as an adenovirus.
• The insert size for AAV is 4.5 kb, with the advantage of long-term gene expression as they integrate
into chromosomal DNA (chromosome 19).
• AAVs are highly safe as the recombinant adeno associated vectors contains only gene of interest
and 96% viral genes are deleted.
• AAV enters host cell, becomes double stranded and gets integrated into chromosome.

17. 3. RETROVIRALVECTORS
• Retroviruses are RNA viruses which possess a reverse transcriptase activity, enabling
them to synthesize a complementary DNA. Following infection (transduction),
retroviruses deliver a nucleoprotein complex (pre-integration complex) into the
cytoplasm of infected cells.The viral RNA genome is reverse transcribed first and then
integrates into human genome at a single site of the chromosome in stable fashion.
• It can carry a DNA of size less than 3.4kb
• Target cell – dividing cells

18. 4. HERPES SIMPLEX VIRUS VECTORS:
Herpes simplex virus-1 (HSV-1) is a 150 kb double stranded DNA virus with
a broad host range that can infect both dividing and nondividing cells.
It is the virus that rarely can cause encephalitis and infect the non dividing
cells so it has the ability to transduce neurons, thus it can help in treating
neurological disorders.
The insert size is comparatively larger (>20kb)
Disadvantage
short-term expression due to its inability to integrate into the host
chromosome.

19. B. PHYSICAL METHODS
• Electroporation
• Microinjections
• Gene Gun
• Magnetofection
1. ELECTROPORATION
• In electroporation, the external electric field is applied to the protoplast,
which changes the electrical conductivity and the permeability of cell
membrane; and thus the exogenous molecules found in the medium are
taken up to either the cytoplasm (transient transfection) or into the nucleus
(stable transfection).
• 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.

20. Advantage:
• By electroporation large numbers of cells can be processed at once, and thus the amount of
time spent processing cells can be cut down.
Disadvantages:
• If the voltage applied is not calculated properly, the cells may damage.
• If electroporation does not occur in controlled environment, the potentially harmful substances
can enter the cell or the impurities from solution may enter.This is because there is no way to
control what enters the cell membrane

21. 2. MICROINJECTION
• It involves 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 neeedle
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.
• For this, glass micropipette is heated until
the glass becomes somewhat liquefied and
is quickly stretched to ressemble a
injection needle.
• The delivery of foreign DNA is done under
a powerful microscope
(micromanupulator).

22. 3. GENE GUN
This technique is also referred as Biolistics as it involves micro-projectile delivery
of DNA.
In gene gun method, the tungsten or gold particles (micro projectiles) are coated
with the foreign DNA. Micro-projectile bombardment uses high-velocity metal
particles to deliver biologically active DNA into the target cells.
The microprojectile is coated with the coated particles and is accelerated with air
pressure and shot into the target tissue. A perforated plate is used, which allows
the micro-projectiles to pass through to the cells on the other side of the plate and
stops the microprojectile.
Particle coated with the foreign gene releases the foreign gene when enters into
the target cell and integrates into the chromosomal DNA.
Mammalian cell lines like HEK 293, MCF7 showed gene expression when
transfected with luciferase and green fluorescent genes and their gene expression
was dependent on helium pressure, size and amount of gold particle and DNA load
on each particle. Cell viability depends on helium pressure.

24. 4. MAGNETOFECTION
Magnetofication is a simple and highly efficient transfection method that
uses magnetic fields to concentrate particle containing nucleic acid into the
target cell.

25. C. CHEMICAL METHODS
1. Liposomes Mediated
Liposomes are spherical vesicles which are made up of synthetic lipid bilayers which
imitate the structure of biological membranes. DNA to be transferred is packaged into the
liposome in vitro and transferred to to the targeted tissue.The lipid coating helps the
DNA to survives in vivo and enters into the cell by endocytosis. Cationic liposomes, where
the positive charge on liposomes is stabilized by binding of negatively charged DNA, are
popular vehicles for gene transfer in vivo.

26. Advantage:
The liposomes with the foreign DNA are easy to prepare.
There is no restriction in the size of DNA that is to be transferred.
Disadvantage:
Efficiency of gene transfer is low and transient expression of the foreign gene
is obtained as they are not designed to integrate into the chromosomal DNA.

27. 2. CALCIUM PHOSPHATE MEDIATED DNA
TRANSFER
• The process of transfection involves the admixture of isolated
DNA (10-100ug) with solution of calcium chloride and
potassium phosphate so precipitated of calcium phosphate to
be formed.
• Cells are then incubated with precipitated DNA either in
solution or in tissue culture dish.
• A fraction of cells will take up the calcium phosphate DNA
precipitate by endocytosis.
Disadvantage :
Transfection efficiency is quite low.

28. Fig. CALCIUM PHOSPHATE MEDIATED DNA TRANSFER

29. Applications of Gene Therapy
Clinically Following Diseases are treated using gene therapy :
• Cystic fibrosis
• Parkinson's Disease
• Hemophilia
• Thalassemia
• Sickle Cell Anaemia
• Familial Hypercholesterolemia
• Severe Combined Immunodeficiency

30. Applications of Gene Therapy
➢ Clinical gene transfer applications
➢ Vaccine development
➢ Production of transgenic animals
➢ Treatment of cancer and AIDS
➢ Gene discovery
➢ Genetically modified organism
(Trangenic Animals & Xenoplanters)

31. Gene Therapy Based Drugs
2004:
• First country to introduce a gene based drug was China
• Gendicine
• An recombinant adenovirus-p53 (rAd-p53) based gene seems to
act by stimulating the apoptotic pathway in tumour cells,
which increases the expression of tumour suppressor genes and
immune resposnse factors.
• Treatment of patients with head and neck squamous cell
carcinoma
• No overt adverse side effects have been reported
• Therapeutic efficacy is still controversial.

32. Gene Therapy Based Drugs
2012:
• Europe came out with first commercially available genetherapeutic
product in the Western world
• Glybera contains the human LPL gene variant LPL S447X in an
adeno-associated virus serotype 1 (AAV1) vector.
• Mechanism of action : Glybera contains alipogene tiparvovec as the active
substance.The drug works by breaking down the chylomicron particles present in
the blood. It delivers a normal LPL gene into the body for correcting the LPL
deficiency.The drug normalises the metabolism of fat in the blood and thereby
prevents episodes of pancreatitis.
• The drug is administered in the form of an injection into the leg muscle.
• Treatment of familial lipoprotein lipase deficiency (rare genetic condition that
elevates the levels of fat in the blood.The disease occurs due to alterations in LPL
genes, which are responsible for breaking down of a protein-lipid complex called
chylomicron.The chylomicron particles play a key role in transporting fat through
the blood.)
• One of the most expensive medicine in the world ($1.6 million/treatment)

33. Gene Therapy Based Drugs
2017:
Trade Name: LUXTURNA
Generic Name: voretigene neparvovec-rzyl
Indication: Confirmed bi-allelic RPE65 mutation-associated
retinal dystrophy
• An adeno-associated virus vector-based gene therapy
• Patients must have viable retinal cells as determined by the
treating physician
MOA: Transduction of some Retinal Pigment Epithelial
cells (RPE) cells with a cDNA encoding normal
human RPE65 protein, thus providing the potential
to restore the visual cycle
Side Effects:
(>5%)
Conjunctival hyperemia, Cataract,increased IOP,
Retinal tear, Eye inflammation, irritation and pain
Cost: $425,000 / eye

34. Gene Therapy Based Drugs
2017:
Trade Name: IMLYGIC
Generic Name: talimogene laherparepvec
Indication: Local treatment of melanoma with unresectable
cutaneous, subcutaneous, and nodal lesions
recurrent after initial surgery
• Genetically modified oncolytic viral therapy
MOA: Replicates within tumors to produce immune
stimulatory protein (GM-CSF) causing lysis of
tumors and promoting an antitumor response
Side Effects:
(>25%)
Fatigue, Chills, Pyrexia, Nausea, Influenza-like
illness, and Injection site pain
Cost: ~ $65,000 / patient à depends on dosing

35. Gene Therapy Based Drugs
2017:
Trade Name: KYMRIAH
Generic Name: tisagenlecleucel
Indication: Patients up to 25 years of age with B-cell precursor
acute lymphoblastic leukemia (ALL) that is
refractory or in second or later relapse.
• CD19-directed genetically modified T cell immunotherapy
• Vector used is HIV or Lentivirus
MOA: Reprograms patient’s T cells with a transgene
encoding for a chimeric antigen receptor to identify
and eliminate CD19-expressing malignant and
normal cells
Side Effects:
(>20%)
Hypogammaglobinemia, Infections, Pyrexia,
Decreased appetite, Headache, Encephalopathy,
Bleeding, Hypotension,Tachycardia, Nausea,
Diarrhea,Vomiting, Fatigue, Acute kidney injury
Cost: $475,000 for entire treatment

36. Gene Therapy Based Drugs
2019:
Trade Name:
Zolgensma
Generic Name: Onasemnogene abeparvovec
Indication: It was approved for children less than two years old in 2019
to treat spinal muscular atrophy, a disease linked to
a mutation in the SMN1 gene on chromosome 5q.It is used as
a one-time injection into a vein with at least two months
of corticosteroids.
• Vector used is Adeno-associated virus serotype 9 i.e. AAV9
MOA: Onasemnogene abeparvovec is a biologic drug consisting
of AAV9 virus capsids that contains a SMN1 transgene along
with synthetic promoters.
Upon administration, the AAV9 viral vector delivers
the SMN1 transgene to the affected motor neurons, where it
leads to an increase in SMN protein.
Side Effects: Common adverse reactions may include nausea and
increased liver enzymes.Serious adverse reactions may
include liver problems and low platelets.
Cost: 18 Crores per dose or US$2.125 million per treatment

37. Recent Developments & Ongoing Trials
Recent gene therapy projects are targeted at conditions such as:
•Cancers - Phase I and II clinical trials for brain,
skin, liver, colon, breast and kidney
cancer
•Heart Disease - A phase I clinical trial just completed
showing SDF-1 gene therapy
improved Heart Failure symptoms
•Parkinson’s Disease - Phase I completed- Modified virus
delivers 3 genes to striatum to boost
production of dopamine
•Diabetes Mellitus - Studying approaches of transferring
the insulin gene into other cells
•Arthritis - Delivery to synovium achieved with a
retrovirus
•Alzheimer’s Disease - Switching off Alzheimer’s gene and
using exosomes to deliver drugs to brain

38. Adverse effects, contraindications and hurdles for
use:
Some of the unsolved problems include:
▪ Short-lived nature – Before gene therapy can become a permanent cure for a
condition, the therapeutic DNA introduced into target cells must remain
functional and the cells containing the therapeutic DNA must be stable. Problems
with integrating therapeutic DNA into the genome and the rapidly dividing
nature of many cells prevent it from achieving long-term benefits. Patients
require multiple treatments.
▪ Immune response – Any time a foreign object is introduced into human tissues,
the immune system is stimulated to attack the invader. Stimulating the immune
system in a way that reduces gene therapy effectiveness is possible.The immune
system's enhanced response to viruses that it has seen before reduces the
effectiveness to repeated treatments.
▪ Problems with viral vectors – Viral vectors carry the risks of toxicity,
inflammatory responses, and gene control and targeting issues.
▪ Multigene disorders – Some commonly occurring disorders, such as heart
disease, high blood pressure, Alzheimer's disease, arthritis, and diabetes, are
affected by variations in multiple genes, which complicate gene therapy.

39. Adverse effects, contraindications and hurdles for
use:
Some of the unsolved problems include:
▪ Some therapies may breach the Weismann barrier (between soma and germ-
line) protecting the testes, potentially modifying the germline, falling afoul of
regulations in countries that prohibit the latter practice.
▪ Insertional mutagenesis – If the DNA is integrated in a sensitive spot in the
genome, for example in a tumor suppressor gene, the therapy could induce a
tumor.This has occurred in clinical trials for X-linked severe combined
immunodeficiency (X-SCID) patients, in which hematopoietic stem cells were
transduced with a corrective transgene using a retrovirus, and this led to the
development of T cell leukemia in 3 of 20 patients. One possible solution is to
add a functional tumor suppressor gene to the DNA to be integrated.This may
be problematic since the longer the DNA is, the harder it is to integrate into cell
genomes. CRISPR technology allows researchers to make much more precise
genome changes at exact locations.
▪ Cost – Alipogene tiparvovec or Glybera, for example, at a cost of $1.6 million
per patient, was reported in 2013 to be the world's most expensive drug.

40. ▪Ethical & Social Considerations
• High cost makes this “promising” therapy available only to the
wealthy
• Can the widespread use of gene therapy make society less
accepting of people who are different?
• Does it interfere with God’s plan?
• Should people be allowed to use gene therapy to enhance basic
human traits such as height, intelligence, or athletic ability?