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GENE
THERAPY
Dr. Walaa Wadie
What is Gene Therapy?
 Using nucleic acids-based molecules (genes) as
drugs.
 A key component is vehicles used to deliver
therapy to cells &/or organs.
Gene-based molecules + vehicle = gene therapy
Focus & goal of gene therapy

Inherited disorders:
(e.g. cystic fibrosis, hemophilia, adenosine deaminase deficiency).
To correct genetic defects permanently and thereby
restore normal cellular function.

Acquired diseases:
(such as cancer).
To cure disease by targeting pathogenic processes.
Types of gene therapy

Somatic gene therapy
− Exogenous
genes (= transgenes) are transferred into
somatic cell of the recipient.
− Not affect future generation.

Germ-line gene therapy
− Transgenes
are transferred into germ cells (sperm
or eggs) of the recipient.
− Affect future generation.
Gene Therapy Versus
Conventional Therapy
Gene Therapy
Conventional
Therapy
Materials
DNA, RNA; etc.
Chemicals, Peptide,
Proteins.
Mechanisms
Usually cure the causes of the
diseases
Usually relieve the
symptoms or signs
Duration of
Effect
Can be permanent and also can be
passed down to next generation in
germ-line gene therapy.
Usually stop the effect
once stop taking it.
Ethics
Major Issues (germ-line gene
therapy)
Usually Not
General guidelines in gene therapy

Disease not successfully treated by current therapies.

Genetic basis of disease must be determined.

Pathophysiology of disease must be known.

Magnitude & duration of gene expression must be
estimated.

Tools for detection of expression must be in hand.
Methods of Gene Transfer

In vivo:
Direct administration of the gene therapy formulation to
patients.

Ex vivo:
Transfection of cells in tissue culture by gene therapy
followed by administration of the transfected material into
the patient.
Cells must be capable of removal, survival outside the body
& re-implantation e.g. hepatocytes, skin fibroblasts,
lymphocytes, etc.
Gene Delivery Systems
 Non-viral
vectors:
Naked DNA, DNA–polymer conjugates & liposomes.
 Viral
vectors: DNA & RNA vectors
Retrovirus, Adenovirus,
simplex virus.
Adeno-associated
virus,
Herpes
Gene Delivery Systems
1- Non-Viral Vectors
Naked DNA
(can be administered by direct injection of the naked DNA into some
tissues, especially muscle, usually used in vitro and by electroporation)
Adv:
Ease of production.
 Safety.
 No DNA size limitation.

Disadv:
 ↓ Efficiency.
 Transient expression.
Gene Delivery Systems
Physical methods to enhance delivery:
Electroporation
is a method that uses short pulses of high voltage
to carry DNA across the cell membrane.
Gene Gun
Plasmid DNA was coated onto 1-3 µm gold or
tungsten particles and after that ‘fired’ at the
tissues via electrical or gas pulse acceleration
Sonoporation
uses ultrasonic frequencies to deliver DNA into cells
Gene Delivery Systems
Electroporation
Gene Delivery Systems
Chemical methods to enhance delivery:
a. Liposomes

Lipid molecular aggregates that can carry naked DNA
(lipoplex).
Adv:
 Ease of production.
 Low immune reactions.
 No DNA size limitation.
Disadv:
 ↓ Efficiency.
 Transient expression.
Gene Delivery Systems
b. DNA polymer conjugate (polyplex)
-
-
DNA
+
+++++
Ligand Polycation
e.g. polyethylenimine
DNA
Complex
Gene Delivery Systems
Non-viral vectors
DNA
Lipoplex
Polyplex
Production
Easy
Easy
Easy
Capacity
unlimited
unlimited
unlimited
Transfection
Efficiency
low
low
low
Integration
Non
Non
Non
Expression
Transient
Transient
Transient
Immune
Reactions



Gene Delivery Systems
2- Viral Vectors
Design focuses on efficacy & biosafety.
Rendering virus vector harmless:
 Remove disease-causing genes and replace by
therapeutic gene (gene of interest).
 The viral genes that control delivery mechanisms
are retained.
Gene Delivery Systems
Target cell
Virus may be:
Vector
uncoating
Viral
vector
Expression
Integration
&
Expression
Therapeutic mRNA
& protein
• Non-Integrating viruses
(transient expression)
– Adenovirus
– Herpes Simplex Virus
• Integrating viruses
(stable expression)
– Adeno-associated virus
– Retrovirus
– Lentivirus
Gene Delivery Systems
A. DNA-Viral Vectors
Adenovirus:
Adv:
 Ease of production.
 Efficient DNA transfer (High transfection efficiency)
 Can infect dividing and non-dividing cells.
Disadv:

Transient expression (< 10 days)(viral DNA does not integrate)

Immunogenicity: life-threatening immune response.
In vivo delivery hampered by host immune response (prevents any
subsequent transfection even if a second injection of the recombinant
adenovirus is given)
Gene Delivery Systems
Adeno-associated Vectors (AAV):
Adv:
 ↓ Immunogenicity
 Transduce dividing and non-dividing cells.
 Prolonged expression (up to 9 months).
Disadv:
 Limited DNA capacity.
 Difficult production.
Gene Delivery Systems
Herpes Viral Vectors:
Adv:
 Targets CNS (brain cancer).
 ↓ Immunogenicity.
Disadv:
 Difficult to manufacture.
 Transient expression.
 Low transduction efficiency.
Gene Delivery Systems
B- RNA-Virus Vectors
Retroviruses:
Adv:
• Low immune reactions.
• Easily manufactured.
• Efficiently transferred.
• Stable expression.
Disadv:
• Targets dividing cells only.
• Small DNA capacity.
• Risk of insertional mutagenesis.
The integrase enzyme can insert the genetic material of the virus into any
arbitrary position in the host chromosome (randam DNA insertion).
If genetic material happens to be inserted in the middle of one of the
original genes of the host cell, this gene will be disrupted (insertional
mutagenesis).
If the gene happens to be one regulating cell division, uncontrolled cell
division (i.e. cancer) can occur.
Gene Delivery Systems
Lentivirus vectors (subclass of retrovirus)

Infect dividing and non-dividing cells.

Easily produced.

Low cellular immune response.

Sustained expression over six months
Viral vectors
DNA-viral vector
RNA-viral vector
Adenovirus
Production
Capacity
Transfection
Integration
Expression
Easy
limited
Efficient
Non
Transient
Immune
Response
High
Target
AAV
Lentivirus
Herpes
Retrovirus (subclass of
Simplex V
retrovirus)
difficult difficult
limited limited
Low
Efficient
Yes
Non
Stable Transient
Easy
limited
Efficient
Yes
Stable
Easy
limited
Efficient
Yes
Stable
low
low
low
low
Dividing
& nondividing cells
CNS
Dividing
cells
Dividing &
nondividing
cells
(brain cancer)
Risk of insertional
mutagenesis
Gene Delivery Systems
N.B.:
Generally, viral vector system show higher
gene transfer efficiency than non-viral gene
carrier
system,
but
viral
systems
have
potential risk of immunogenecity and cancer
formation.
Gene Therapy Approaches
N.B.:
Diseases at a genetic level result from:

Loss of expression of a gene.

Mutation of a gene.

Elevated expression of a gene.

Expression of pathogenic viral or foreign gene.
Gene Therapy Approaches
• Replacing missing or mutated gene with a healthy copy
of gene.
Scientists focused on diseases caused by single-gene defects
(monogenic disease) e.g. Cystic fibrosis, Haemophilia, SCID
• Gene silencing:
• suppress expression of undesired gene.
• Include: RNA interference, Antisense therapy.
• e.g. AIDS/HIV
• Suicide gene therapy:
• Introducing a new gene “suicide gene” into the body to help
fight a disease.
• e.g. Cancer
Gene Therapy Approaches
 Replacing missing or mutated gene
(DNA + vector)
DNA
Transcription
Translation
Gene Therapy Approaches ………Replacing
missing or mutated gene
The produced protein may:
 function intracellularly.
or
 secreted into circulation.
Evaluation of successful gene transfer (transfection)
is done by:
•
•
•
Measuring mRNA (transcription).
Measuring protein (translation).
Measuring function of target cells.
Gene Therapy Approaches
 Gene silencing:

Nucleic acids can be delivered to interrupt specific
mRNA translation & protein synthesis.
(So prevent mutated or overactive genes from directing
protein synthesis).

Two mechanisms exist to
destroying targeted mRNA:
a. Antisense therapy.
b. RNA interference.
silence
genes
by
Gene Therapy Approaches ………Gene
silencing
a. Antisense Therapy
mRNA
Transcription
Translation
DNA
Protein
Using antisense oligonucleotides (ASON) to ↓ expression
of a target gene by binding to mRNA.
ASON
mRNA
Transcription
DNA
Translation
Gene Therapy Approaches ………Gene
silencing ….Antisense Therapy
Antisense oligonucleotides (ASON):
•
are sequences of 17-30 bases of single-stranded DNA that are
complementary to a chosen sequence of target mRNA.
•
are of short length to facilitate cell internalization & ↑ hybridization
efficiency.
Mechanism of action:
 ASON bind to target mRNA sequence  DNA-RNA
duplex  activation of ribonuclease H (RNase-H).
RNase-H

ASON-mRNA
free ASON +degraded mRNA.

Net result → ↓ mRNA translation & protein synthesis.
Gene Therapy Approaches ………Gene
silencing ….Antisense Therapy
Gene Therapy Approaches ………Gene
silencing
b. RNA interference:
Mechanism of action:
 Large
double strand RNA (dsRNA)
sequence designed to target endogenous
mRNA enter the cell.

dsRNA is cut by DICER enzyme to short
interfering RNA (siRNA).

siRNA bind to a group of proteins called
RNA-induced silencing complex (RISC).
Gene Therapy Approaches ………Gene

silencing …. RNA interference
RISC performs 2 imp jobs:
– activates unwinding of siRNA to single
strand RNA that binds to target mRNA
molecule.
– cuts mRNA in the regions paired with
single strand RNA.
Gene Therapy Approaches ………Gene
silencing …. RNA interference
+
Large dsRNA
DICER
siRNA
RISC
Unwind
siRNA
Single-strand
RNA
+
RISC
mRNA degradation
Target mRNA
Gene Therapy Approaches ………Gene
silencing …. RNA interference
Gene Therapy Approaches ………Gene
silencing
Gene Therapy Approaches
 Suicide gene therapy

Introducing a new gene “suicide gene” into
the body to help fight a disease.

This approach is used in cancer gene therapy.
1.
2.
Tumor cells are transfected with a gene coding for an enzyme such as
herpes simplex virus-1 thymidine kinas.
Systemic administration of ganciclovir.
ganciclovir (nontoxic) --thymidine kinase its active cytotoxic form 
death of tumor cells.
Therefore, cancer cells become more vulnerable, more sensitive to
chemotherapy
Gene Therapy Approaches ………
Suicide gene therapy
Applications of Gene Therapy
Obstacles facing gene therapy:






Immunogenicity.
↓ Efficiency.
Transient transgene expression.
Mutagenicity or oncogenesis
Ethical factors (germ-line gene therapy): DNA could
accidentally be introduced into reproductive cells
Gene product toxicity: expression is higher than
normal endogenous levels & concentrated within localized
population of cells.
Applications of Gene Therapy
Gene Therapy
Human Application Considerations
Specificity
Effective
Safety
Only desired cells or tissue
The delivery efficiency dependent on the disease
requirements
Biocompatible
Non-cytotoxicity
Non-immunogenecity
Non-inflammation
Non-Tumor Generation
Applications of Gene Therapy
(AIDS)
Cancer
Applications of Gene Therapy
Cancers:
1) Addition of a tumor suppressor gene (genes encoding
P53).
2) Expression
of immunomodulating
expressing IL-2).
gene
(genes
3) Deliver
genes expressing co-stimulatory molecules
necessary for activation of T-lymphocytes.
4) Use ASON to turn-off expression of an oncogene (bcl-2
oncogene).
5) Inhibition of tumor angiogenesis (suppress angiogenic
growth factor VEGF).
6) Transfer of a gene leading to tumor-specific cell killing
(suicide gene therapy).
Applications of Gene Therapy
Infectious diseases:
HIV/AIDS
produce HIV-infected cells that express
thymidine kinase that activate non-toxic prodrug ganciclovir
into cytotoxic one (suicide gene therapy).
– Trials
to
– Decrease HIV replication by modification of CD4 T cells ex
vivo to express proteins that interfere with HIV
transcription.
Cytomegalovirus retinitis
Fomivirsen (marketed as Vitravene)-
21-base pair
ASON- 1st ASON agent approved by FDA – used for
CMV retinitis in HIV patients.
Applications of Gene Therapy
Monogenic diseases
Disease
Defect
Target Cells
Severe combined
Adenosine
deaminase Bone-marrow
immunodeficiency (ADA) deficiency in 25% cells or T
of SCID patients
lymphocytes
(SCID)
Cystic fibrosis
(CF)
Defect in Cystic fibrosis
transmembrane
conductance
regulator Airways in the
(CFTR) 
lungs
Faulty transport of Cl- in lung
epithelium
Hemophilia
-- A (80%) Factor VIII deficiency
-- B (20%) Factor IX deficiency
Liver, muscle,
fibroblasts or
bone marrow
cells
Applications of Gene Therapy
Adenosine deaminase deficiency


Severe combined
immunodeficency
syndrome
(SCID)
(Patients cannot withstand
infection  die if untreated)
Applications of Gene Therapy
Progress in SCID treatment:
 The baby is kept in a bubble-like structure (isolated in a germfree environment). David Vetter ‘Bubble boy’ (1971 - 1984).
 Bone marrow transplantation: the most common
However, a proper 'bone marrow match' is necessary for
transplantation.
 Gene therapy:
()
- 1st clinical gene therapy trial (1990): T cells were removed
from body → ADA gene was inserted into these T cells →
again injected into body → normal immune system.
Only worked for a few months  and therefore process
(replacement gene therapy of T cells) should be repeated.
-Recently, Strimvelis is the first ex-vivo stem cell gene therapy.
Applications of Gene Therapy
Applications of Gene Therapy
Cystic fibrosis
Applications of Gene Therapy
Gene therapy in CF
usually in form of
aerosol spray
Insert transgene
into liposome or
viral vector
Applications of Gene Therapy
Gene therapy in hemophilia
Applications of Gene Therapy
Multifactorial diseases:

Delivery of genes coding for angiogenic
growth factors e.g. VEGF ➙ stimulate
vascular proliferation in coronary artery
disease (CAD) & peripheral vascular diseases.

Inflammatory diseases (RA, IBD, asthma):
• Delivery of genes encoded with immuno-modulatory or
anti-inflammatory cytokines.
• ASON to silence pro-inflammatory cytokines.
Thank you