Use of Gene Therapy in The

Treatment of Disease
What’s gene therapy?
Many medical conditions result from flaws, or mutations, in
So, if a flawed
one or more of a person's genes.

gene caused a disease, can you

"fix" it? What are your options?

• Stay silent: ignore the genetic

disorder and nothing gets fixed.

• Try to treat the disorder with drugs or

other approaches: depending on the
disorder, treatment may or may not
be good or give a long-term solution.

• Put in a normal, functioning copy of

the gene: if you can do this, it may
solve the problem!
Genetic basis of disease
• Non-genetic
– Pathogenic
– Malnutrition (vitamin/mineral)
– Overnutrition (hyperalimentation)

• Chromosomal

• Polygenic
– Metabolic
• Diabetes
• Obesity
• Cancer
• CVD

• Monogenic
– Sex-chromosomal
• Recessive
• Dominant
– Autosomal
• Recessive
• Dominant
– Mitochondrial
– Y-chromosome
Sex-chromosomal
Recessive: Hemophilia, Color blindness,
Fabry disease
Dominant: Incontinentia pigmenti;
Fragile X syndrome

Autosomal
Recessive: Cystic fibrosis, Sickle cell
disease
Dominant: Huntington disease, Marfan
syndrome
https://ghr.nlm.nih.gov/primer/inheritance/inheritancepatterns
 Background
• In the 1980s, advances in molecular biology had already linked genes to
diseases. Scientists devised method of easily producing proteins, such as
the protein deficient in diabetics — insulin. Investigated introducing human
genes to bacterial DNA. The modified bacteria then produce the
corresponding protein, which can be harvested and injected in people who
cannot produce it naturally.

• Scientists took the logical step of trying to introduce genes straight into
human cells, focusing on diseases caused by single-gene defects, such as
cystic fibrosis, hemophilia, muscular dystrophy and sickle cell anemia,
optic nerve disease, wound repair and regeneration, and cardiovascular
disease.

• However, this has been much harder than modifying simple bacteria,
primarily because of the problems involved in carrying large sections of
Direct genetic modification of cells/patients

2014
 Early Human Gene Therapy
Experiments

• Martin Cline human experiments-- 1980

• NeoR/TIL marking studies-- 1989
• ADA/peripheral blood T cells-- 1990
• LDL receptor/ex-vivo hepatocytes-- 1992
• HLA-B7 Melanoma-- 1992
• ADA/bone marrow, cystic fibrosis, cancer, HIV
First FDA approved gene therapy
 3 challenges in gene therapy

delivery delivery delivery

1)Who will deliver? Carrier molecule

2)Where will be delivered? Site!!!

3)Delivery without immune response

 How to fix it
B C A a beneficial gene
A
virus modified virus

• A virus is found which replicates by inserting its genes into the host cell's
genome. This virus has three genes - A, B and C.

• Gene A encodes a protein which allows this virus to insert itself into the
host's genome.

• Genes B and C actually cause the disease this virus is associated with.

• Replace B and C with a beneficial gene. Thus, the modified virus

could introduce your 'good gene' into the host cell's genome without causing
any disease.

• So we use the modified virus to fix the “disease”

 Gene therapy approaches

Germ line gene therapy

Somatic cell gene therapy

In vivo

Ex vivo
 Gene augmentation
most therapies simply add a useful gene into a selected cell type to
compensate for the missing or flawed version. Useful in treating loss of
function mutations such as CFTR Gene
 Gene replacement
This strategy replaces the mutant copy with a correctly
functioning copy in situ. Useful for gain of function
mutations such as retinoblastoma
 Specific inhibition of gene expression
Involves silencing of specific genes like activated oncogenes, by
using molecules that degrade RNA transcripts.
Strategies include
Antisense therapy
siRNA (small interfering RNA)
Ribozymes etc
 Antisense therapy

short stretches of
synthetic ssDNA
that target the
mRNA transcripts of
abnormal proteins
preventing its
translation
 siRNA therapy

Small interfering RNAs

short stretches (21-23nt)
of synthetic dsRNA
Has 3’ overhangs of 2 nt
Incorporates into RISC
(RNA induced
silencing complex)
Target mRNA cleaved in
the middle
 Ribozymes
Catalytic RNAs that cleave target mRNAs in a
sequence-specific manner
e.g. hammerhead ribozymes are engineered to
recognise specific sequences and made resistant to
nucleases
 Targeted cell death
Tissue specific toxicity as a result of gene therapy. Useful
in cancer therapy

direct approach
 Targeted cell death
Indirect approach

stimulating an immune response against selected cells

or eliminating the blood supply.
 Choosing a Site For Gene Therapy

Sickle cell anemia/Thal Bone Marrow

Congenital immune deficiencies Bone Marrow
Lysosomal storage and metabolic Bone Marrow
---------------------------------------------------------------
Cystic fibrosis Lung - airways
---------------------------------------------------------------
Muscular dystrophy Muscle
---------------------------------------------------------------
Hemophilia A or B Liver
Urea cycle defects Liver
Familial hypercholesterolemia Liver
Disease Target cells Transfected gene(s)

Hemophilia A liver, muscle, Factor VIII

Hemophilia B bone marrow cells, fibroblasts Factor IX

Familial liver LDL receptor

hypercholesterolaemia

Severe combined bone marrow cells, T cells Adenosine deaminase (ADA)

immunodeficiency

Hemoglobinopathies red blood precursor cells a-globin, b-globin

Cystic fibrosis lung airway cells CFTR

Gaucher disease bone marrow cells Glucocerebrosidase

macrophages

Cancer tumor cells p53, Rb,

interleukins
growth-inhibitory genes
apoptosis genes
 Gene
Transfer
Methods
• Retroviral vectors
– Lentiviruses
• Adenovirus
• Adeno-associated virus (AAV)
• Other viral vectors
– Vaccinia – Hepatitis virus
– Herpes virus – Polio virus
– Papilloma virus – Sindbis and other RNA viruses
• Non viral methods
– Ligand-DNA conjugates Lipofection
– Direct DNA injection CaPO4 precipitation
– Ribozymes chimeric oligo/gene correction
Comparison of different types of viruses
Comparison of different types of viruses
Comparison of different types of viruses
HEK; Sf9 cell lines
 Problems
• Acute immune response to viral vectors
Viral vectors could become pathogenic

• Precision of Delivery of DNA

Genes spliced at random into the genome could upset
other genes

• Achieving high level expression

• Maintaining stable expression

• Tissue-specific expression

• Repeated treatment needed

Genes “lost” when the cell goes through mitosis

• Multigene disorders too complex to treat

 Cystic Fibrosis
• Cystic fibrosis (CF) was first described as a disease in the late
1930s by Dorothy Hansine Andersen.

• CF is an inherited autosomal recessive disease common among

whites. It is characterized by the buildup of thick, sticky mucus that
can damage many of the body's organs. 

• In 1980s, the first mutation for CF, ΔF508, was discovered on the
7th chromosome of the human genome. Subsequently, over 1000
different mutations have been identified that may cause CF.

• CFTR (cystic fibrosis transmembrane conductance regulator)

protein functions as a chloride channel across the membrane of
cells that produce mucus, sweat, saliva, tears, and digestive
enzymes.
Inheritance of Cystic Fibrosis
Gene Therapy for Cystic Fibrosis
• Cystic fibrosis should be an ideal candidate for gene therapy, for four
main reasons:

• (1) it is a single gene defect;

• (2) it is a recessive condition, with heterozygotes being phenotypically

normal (suggesting gene dosage effects are not critical);

• (3) the main pathology is in the lung, which is accessible for treatment;

• (4) it is a progressive disease with a virtually normal phenotype at birth,

offering a therapeutic window.
 Choices of Vectors
• Viral vectors:
• Vetrovirus Non-viral vectors:
Liposome
• Adenovirus
DNA–polymer conjugates
• Adeno-associated virus Naked DNA
• Herpes Simplex Virus

The ideal vector system would have the following characteristics:

(1) an adequate carrying capacity;
(2) to be undetectable by the immune system;
(3) to be non-inflammatory;
(4) to be safe to the patients with pre-existing lung inflammation;
(5) to have an efficiency sufficient to correct the cystic fibrosis phenotype;
(6) to have long duration of expression and/or the ability to be safely re-
administered.
 1993 vector used: Adenovirus
• The first cystic fibrosis gene therapy clinical
trials used an adenovirus vector to deliver the
full-length CFTR gene to cells.

• Adequate doses of adenovirus vector cause an

immune response. If the adenovirus is to be
useful, researchers need to find ways to both
improve the virus’s ability to enter cells and
reduce the chances of immune response.
 1995 liposome
• Trials using liposome-mediated CFTR gene transfer began in 1995.

• Non-viral vectors have the potential to avoid some of the critical

problems observed with viral vectors, such as the immune response,
limited packaging capacity, and random integration.

• Liposomes may be mildly effective, but their activity does not last.
For this approach to work, researchers need to figure out how to
improve delivery, make the effects more permanent.

• To date, only cationic liposome-based systems have been tested in

clinical trials in cystic fibrosis subjects.
 1998 adeno-associated virus

• Trials using adeno-associated virus to deliver the

CFTR gene began in 1998.

• The adeno-associated virus is a good way to

deliver the CFTR gene to patients’ airway cells.

• But researchers need to learn more about how

the virus infects cells in order to make it an
effective delivery method.
 Mode of delivery
• The majority of experience in terms of vector delivery
to the lungs has involved the instillation of large
volumes of vector-containing fluid into the airway
surface, via the nose.

• However,
I. this mode of delivery poses safety problems because
of the potential for aspiration.

II. In addition, large volumes of fluid leads to enhanced

alveolar exposure, as a result of bulk flow into the lung
parenchyma and may induce adverse reactions.

III. At the same time, it is likely that airway epithelial cells,

rather than alveolar epithelial cells, are the appropriate
target for CFTR gene transfer.
 Mode of delivery
• Another mode of lung delivery for vector-containing fluid
is by oral inhalation of aerosolized vectors.

• However, aerosolization of a fluid is typically achieved by

means of a nebulizer, and most nebulizers have been
designed to generate small particles. This is because
most nebulizers have been developed to deliver drugs to
treat patients with asthma.

• One way to avoid alveolar deposition is to generate an

aerosol that is composed primarily of large droplets.
Delivery of the vector by means of a spray device that is
inserted into a bronchoscope may have another
advantage over nebulization.
 Current treatment
• Modern treatment now includes

① the intake of digestion enzymes,

nutritional supplements,
② percussion and postural drainage of
the lungs, improved antibiotics
③ inhalation of aerosols containing
medication.
A typical breathing treatment for
Cystic Fibrosis, using a nebulizer
• The most visible gene therapy drug and the ThAIRapy Vest

under development is inhaled

complementary DNA to treat CF.
 Challenges
• The goal of developing an effective genetic therapy for CF lung disease has led to
the attainment of several milestones in the larger field of gene therapy. These
include:

• the first published in vivo gene transfers with adenovirus (Ad), and with recombinant
adeno-associated virus (rAAV), and

• the first phase I clinical trials using each of these vector systems.

• Choice of vector, mode of delivery to the airways, translocation of genetic

information, and expression of normalized CFTR in sufficient amounts to correct the
CF phenotype in the lungs of CF patients continue to be hurdles in the development
of gene therapy for CF.

• A few attempts at gene therapy were initially successful, but failed to produce
acceptable long-term results.
 Adenosine Deaminase Gene (ADA) Deficiency and
Severe Combined Immunodeficiency (SCID) Disease
32,213 bp Gene
Chromosome 20
12 Exons
1,092 bp mRNA
323 aa protein

Degradation of Purine

David Vetter-Died at Age 12

SCID Destroys T & B Cells
 Immune System
 Symptoms
– ADA is involved in purine
degradation
– Accumulation of nucleotide
metabolites = TOXIC to
developing T lymphocytes
– B cells don’t mature because
they require T cell help
– Patients cannot withstand
infection  die if untreated

Movie: The Boy in the Plastic Bubble is a 1976

 Symptoms
> 8 new ear infections per year

> 2 serious sinus infections per year

> 2 pneumonias per year

> 2 month on antibiotics with little effect

lymphopenia (absolute lymphocyte count less than 200)

-- failure to gain weight and grow
-- recurrent deep skin and organ abscesses
 SCID treatments
Life in germ-free envinronment

Histocompatible bone-marrow transplantations

(with potential graft vs host disease)

Enzyme replacement therapy

with weekly injections of the PEG-ADA (ADAGEN)
VERY expensive; not a cure; temporary effect
An excess of adenosine deaminase leads to hemolytic anemia

GENE THERAPY
 Treatment trails
• September 14, 1990 @ NIH, French
Anderson and R. Michael Blaese perform
the first GT Trial
– Ashanti (4 year old girl)
• Her lymphocytes were gene-altered (~109) ex vivo
 used as a vehicle for gene introduction using a
retrovirus vector to carry ADA gene (billions of
retroviruses used)

– Cynthia (9 year old girl) treated in same year

 Humans Have Been Genetically Engineered To
Cure a Lethal Genetic Disease (SCID)

Several Teenagers
Are Alive Because They
Have Been Engineered
With an ADA Gene That
They Were Not Born
The Age of Human Genetic With!!!
Engineering Began Almost
Twenty Years Ago Treating
SCID With Normal
ADA Genes!!!

Adenosine Deaminase Gene (ADA)

 Ex Vivo Gene Therapy for Severe Combined
Immunodeficiency (SCID)

1.

2.

3.

4.
Gene Therapy for Hemophilia A

Good justification for using this ex vivo gene therapy approach for
hemophilia A (factor VIII):

- Factor VIII production is not regulated in response to bleeding

- Only need to raise levels a little bit, not to 100%, as low levels of the
Factor VIII can be beneficial to the patient

- Broad therapeutic index of factor VIII minimizes risk of overdose

- Delivery of factor VIII into the bloodstream does not require cell-
specific expression

-
NEJM
(2001)
341:1735-1742
Roth et al. NEJM 344:1735, 2001.
Pro-drug activation GT
Suicide gene therapy

Thymidine kinase
Pro-drug: Ganciclovir
Bystander Effect
 Other suicide GT scheme
Suicide gene Prodrug Active drug

Viral thymidine kinase Ganciclovir Ganciclovir triphosphate

Cytosine deaminase 5-fluorocytosine 5-fluorouracil

Linamarase Amygdalin cyanide

nitroreductase CB 1954 nitrobenzamidine

Examples of suicide gene/prodrug combinations and the active

cytotoxic drug
selectively produced in the target cell.
Cautions: Somatic Gene Therapy

• Early trials often limited to desperate situations

(fatal childhood illnesses, cancer); patients/parents
will “try anything”
• Media “hype” may lead to false hopes and fears
• Future long term benefits and unanticipated risks
difficult to judge from animal experiments, especially
in healthy individuals
• Truly informed consent may be difficult to obtain
given the lack of general genetic knowledge in the
public and gravity of some situations
 9/17/1999
Jesse Gelsinger, 18 yo
High school graduate with OTC deficiency, died
participating in a gene therapy experiment at the
University of Pennsylvania in Philadelphia,
 Alternatives to Gene Therapy
• Conventional transplantation
– Bone marrow
– Other tissues (islets)
• Implantable bioreactors
• Infusion of recombinant proteins
– Continuous pump
– Implantable devices
• Other novel pharmaceuticals
• Prenatal diagnosis and prevention
 Pharmaceuticals Produced by
Recombinant DNA Technology
Recombinant Product Disease Target
• Human insulin • Diabetes
• Growth hormone • Growth hormone deficiency
• Recombinant factor VIII • Hemophilia A
• Tissue plasminogen activator • MI and stroke
• Erythropoietin • Anemia
• G-CSF • Neutropenia following chemotherapy
• Hepatitis B vaccine • Prevention of hepatitis B
• a interferon • Hairy cell leukemia,chronic hepatitis
• b interferon • Multiple sclerosis
• Infections in chronic granulomatous
•  interferon
disease patients
Non-viral methods of gene delivery