Gene therapy

1. Gene Therapy – Lecture 1
Dr. Aysha karim kiani

2. Gene Therapy Course
General Information
1. Course code: BBS-602 Title: Gene Therapy Credit hours: 3(3+0)
2. Description of Course:
Gene therapy is likely to be key in the combat against cancer, inherited disorders and many other diseases. Decades worth of
advances in this field have resulted in a growing number of successful clinical trials to develop safe and effective treatments. In
near past, a number of new nucleic acid-based therapies have been developed, which continue to improve the versatility of
these genetic-based treatment approaches. This course will start by building a fundamental understanding of gene therapy,
then with an in-depth look at important trends, research and advances in gene therapy. Students will gain a clear
understanding of how gene therapy works in different diseases, how it has developed and advanced, and its potential
applications.
Program in which the course is offered:
B.S Biological Sciences with major in Genetics
Name of faculty member responsible for the course:
Year/Semester in which this course is offered:
Location if not on main campus:

3. 1. Course Objectives:
Lectures comprises the knowledge on
● Introduction
● Germ line gene therapy, somatic gene therapy
● Viral and non-viral systems used for therapy
● Clinical applications of gene therapy in certain diseases and human trials of gene therapy.
● Ethical and regulatory considerations and future prospects.
2. Learning Outcomes:
At the end of the course students will be able to:
● Describe the techniques used for gene therapy.
● Know about diseases that might be treatable by gene therapy.
● Differentiate the differences between somatic and germline gene therapy and some of the problems
involved in these potential treatments.
● Describe human trials of gene therapy.
● Know the ethical and regulatory considerations and future prospects.

4. ´ Learning Sources:
´ James Wolfe, Genetic testing and gene therapy, New York: Britannica Educational Publishing in association with Rosen
Educational Services, 2016.
´ John Allen, How gene therapy is changing society, San Diego, CA: Point Press, Inc, 2016.
´ Joseph P. P. 2014. Gene therapy: treating disease by repairing genes. Info Base Publishing. United Kingdom.
´ Mingjie Li, B Joy Snider, Gene therapy in neurological disorders, London: Academic Press, 2018.
´ Nancy, S. T. 2015. Gene and cell therapy: Therapeutic Mechanisms and strategies, 4th Edition. CRC Press, United States of
America.
´ Perin, E. C., Miller, L. W., Taylor, D. A., & Willerson, J. T. 2016. Stem cell and gene therapy for cardiovascular disease.
Waltham, MA : Academic Press
´ Sherman, D. 2014. Gene Transfer, Gene therapy and genetic pharmacology: Principles, delivery and pharmacological and
biomedical applications, National Scientific Research Centre (CNSR) ICP Text Books France
´ Shuji Terai, Takeshi Suda, Gene therapy and cell therapy through the liver: current aspects and future prospects, Tokyo:
Springer, 2016.

5. Molecular Bases Of Gene Therapy
´ Genetic therapies aim to treat or cure conditions by correcting problems in your DNA.
´ Your DNA, including specific gene, contains instructions for making proteins that are
essential for good health.
´ Mutations, or changes in your DNA, can lead to proteins that do not work properly or
that are missing altogether.
´ These changes can cause genetic, or inherited, disorders such as cystic fibrosis,
thalassemia, hemophilia, and sickle cell disease.
´ Gene therapy involves the direct genetic modification of the cells of a person (or animal
disease model) in order to achieve a therapeutic goal.

7. ´ Genetic therapies are approaches that treat genetic disorders by providing new
DNA to certain cells or correcting the DNA.
´ Gene transfer approaches, also called gene addition, restore the missing
function of a faulty or missing gene by adding a new gene to affected cells.
´ The new gene may be a normal version of the faulty gene or a different gene
that bypasses the problem and improves the way the cell works.
´ Gene transfer or genome editing treatments can directly modify the cells in
your body, or your cells can be collected and treated outside of your body and
then returned to you.
´ For example, a doctor can remove immune system cells, cells that are part of
your body’s natural defense system, or bone marrow cells from your body,
modify their DNA, and then re-introduce them to your body.

8. ´ To insert new genes directly into cells, scientists use a vehicle called a
“vector.”
´ Vectors are genetically engineered to deliver the necessary genes for treating
the disease.
´ When a normal gene is inserted into the nucleus of a mutant cell, the gene
most likely will integrate into a chromosomal site different from the defective
allele; although that may repair the mutation, a new mutation may result if
the normal gene integrates into another functional gene.
´ If the normal gene replaces the mutant allele, there is a chance that the
transformed cells will proliferate and produce enough normal gene product
for the entire body to be restored to the undiseased phenotype.
´ Both inherited genetic diseases (e.g., hemophilia and sickle cell disease) and
acquired disorders (e.g., leukemia) have been treated with gene therapy.

10. ´ One of the most often used techniques consists of recombinant
DNA technology, in which the gene of interest or healthy gene is
inserted into a vector, which can be a plasmidial, nano-strutured, or
viral;
´ the latter is the most often used due to its efficiency in invading cells
and introducing its genetic material.

11. ´ Although several protocols have been successful, the gene therapy process
remains complex, and many techniques need new developments.
´ The specific body cells that need treatment should be identified and accessible.
´ A way to effectively distribute the gene copies to the cells must be available, and
the diseases and their strict genetic bonds need to be completely understood.

12. Difference between cell and gene therapy
´ the most important difference between cell and gene therapy: in
cell therapy, the cells are not genetically modified but instead are
subjected to a certain manipulation involving cell culture and
exposure to specific types of media whereas gene therapy is
mediated by the addition of any nucleic acid.

13. History of Gene Therapy

14. ´ The idea that a gene can be delivered into specific cell types and its
expression can lead to therapeutic efficacy, dramatically improving the
patients' quality of life, was originally introduced by Theodore
Friedmann 45 years ago and was later strongly encouraged and realized
by George Stamatoyannopoulos, one of the founding members of the
American Society of Gene and Cell Therapy (ASGCT).
´ In this setting, the drug, which in the case of gene therapy is a gene, is
packaged within a vector used to facilitate its entrance into the patients'
cells.
´ Of course, the notion of gene therapy has evolved, and in general, we
refer to gene therapy when a therapeutic process involves genetic
manipulation of the patients' cells with the use of a nucleic acid.
History of Gene Therapy

16. Classes of gene therapy based on target cells
´ There is also the important issue of the target cell type of gene
therapy that currently is subdivided into two large groups:
´ Gene therapy of somatic cells.
´ Gene therapy of the germline cells
´ Gene therapy of stem cells

17. Somatic cell gene therapy:
´ Somatic cell gene therapy is when therapeutic genes are transferred to a
patient’s somatic cells.
´ This type of gene therapy is used when modifying nuclear DNA
´ Somatic cells cured by gene therapy may reverse the symptoms of
disease in the treated individual.
´ Any modification and any effects of somatic gene therapy are restricted
only to that patient and are not inherited by future generations.
´ Somatic cells gene therapy could treat diseases such as cystic fibrosis,
adenosine deaminase deficiency, familial hypercholesterolemia, cancer,
and severe combined immunodeficiency (SCID) syndrome.
´ This technique is considered as the best and safest method of gene therapy.

18. Germline gene therapy:
´ The aim is to genetically modify the DNA of a gamete, zygote, or early embryo
during in vitro fertilization.
´ Modified cells will undergo meiosis and provide a normal gametic contribution
to the next generation.
´ The modifications are hereditary and pass on to subsequent generations.
´ Germ-line gene therapy aimed at modifying nuclear DNA has been widely
banned in humans for ethical reasons.
´ However, germ-line gene therapy aimed at modifying mitochondrial DNA to
prevent transmission of severe mtDNA disorders is a rather different matter and
has already been legalized in the UK.
´ Essentially all current human gene therapy trials and protocols involve
modifying the genome of somatic cells.

19. Gene therapy of stem cells
´ Scientists have also explored the possibility of combining gene
therapy with stem cell therapy.
´ In a preliminary test of that approach, scientists collected skin cells
from a patient with alpha-1 antitrypsin deficiency (an inherited
disorder associated with certain types of lung and liver disease),
reprogrammed the cells into stem cells, corrected the causative
gene mutation, and then stimulated the cells to mature into liver
cells.
´ The reprogrammed, genetically corrected cells functioned
normally.

20. Types of Gene Therapy based on
experimental approach
´ There are basically three types of gene therapy:
´ ex vivo/in-vitro
´ in vivo, and
´ in situ.

21. Ex vivo/in-vitro gene therapy
´ In ex vivo gene therapy, the target cells are removed from the patient's body,
engineered either by the addition of the therapeutic gene or by other genetic
manipulations that allow correction of the phenotype of the disease.
´ The “corrected” cells are subsequently re-infused to the patient.
´ This type of intervention is also termed in vitro gene therapy and is
particularly applicable to blood diseases.
´ In the case of blood cancer, the target cell may be T cells and, most recently,
NK cells, and the therapeutic gene is the chimeric antigen receptor (CAR).
´ In the case of monogenic diseases, the target cell is the hemopoietic stem cell
(HSC) and the transgene varies analogous to the disease.
´ The viral vectors utilized in both cases are mostly retroviral vectors,
belonging either in the lentiviral or the oncoretroviral families of
Retroviridae.

23. in vivo gene therapy
´ However, depending on the affected tissue, ex vivo gene therapy is
not always the intended type of corrective approach.
´ For example, if the target organ is the brain, the spinal canal, or
the liver, another type of therapy is employed, termed in vivo gene
therapy.
´ In this setting, the therapeutic vector is administered systemically
in the blood circulation or the cerebrospinal fluid of the patient,
and depending on the disease, different types of viral vectors are
utilized, such as adenoviral vectors (AVs) or adeno-associated
viral vectors (AAVs).

26. in situ
´ Finally, there is a last scheme of gene therapy, in which the viral vector is
administered in situ, i.e., to a specific organ or area in the body of the patient
either through direct injection, e.g., into the tumor (in the case of melanoma) or
into suitable brain areas (in the case of neuropathies) or by an insertion of a
catheter in the case that the organ to be treated is the heart.
´ The selection of the procedure depends entirely on the type of indication, the
affected tissue, and the cell type that requires correction.
´ In contrast to HSCs (Hematopoietic stem cells), namely, CD34+ cells, that can be
easily isolated from the patients, nerve stem cells are difficult to obtain for ex
vivo manipulation.
´ In addition, stem cells are only partially characterized in the liver.
´ Hence, gene therapy for specific organs or indications is dependent on systemic
or in situ administration of the therapeutic vector.

28. Approaches of Gene Therapy
´ Gene therapy is used to correct defective genes in order to cure a disease or
help your body better fight disease.
´ Researchers are investigating several ways to do this, including:
´ Replacing mutated gene: Some cells become diseased because certain
genes work incorrectly or no longer work at all.
´ Replacing the defective genes may help treat certain diseases.
´ For instance, a gene called p53 normally prevents tumor growth.
´ Several types of cancer have been linked to problems with the p53 gene.
´ If doctors could replace the defective p53 gene, that might trigger the
cancer cells to die.

29. ´ Fixing mutated genes: Mutated genes that cause disease could be
turned off so that they no longer promote disease, or healthy
genes that help prevent disease could be turned on so that they
could inhibit the disease.
´ Making diseased cells more evident to the immune system: In
some cases, your immune system doesn't attack diseased cells
because it doesn't recognize them as intruders. Doctors could use
gene therapy to train your immune system to recognize the cells
that are a threat.

30. ´ For obvious reasons, the idea of gene addition was particularly
applicable in monogenic diseases based on the simplified notion of
“adding the missing gene or the normal allele to compensate for
the expression of the mutated allele.”
´ However, under the view of the latest advancements, gene therapy
does not correspond to an addition of a gene, otherwise missing
in the patient's cells, but with a gene that could offer therapeutic
benefit to the affected individual.

32. Gene therapy process:
1. In spite of various methods or types of gene therapy, the therapy starts
with the identification of mutant gene which is responsible for the
cause of the disease.
2. The next step is cloning the identical healthy gene. This is called
therapeutic gene or transgene. The therapeutic gene is tailored to the
need i.e. to augment or suppress or repair.
3. Once the therapeutic gene is produced it is loaded in a vehicle called
vector. The function of the vector is to deliver the therapeutic gene to
the patient target cell.
4. After the vector reaches the target cell, it delivers the genetic material
to the nucleus. In the nucleus the genetic material gets integrated into
DNA and corrects the defective or mutated gene.

34. Gene therapy products:
´ Plasmid DNA: Circular DNA molecules can be genetically engineered to carry
therapeutic genes into human cells.
´ Viral vectors: Viruses have a natural ability to deliver genetic material into cells, and
therefore some gene therapy products are derived from viruses. Once viruses have been
modified to remove their ability to cause infectious disease, these modified viruses can
be used as vectors (vehicles) to carry therapeutic genes into human cells.
´ Bacterial vectors: Bacteria can be modified to prevent them from causing infectious
disease and then used as vectors (vehicles) to carry therapeutic genes into human tissues.
´ Human gene editing technology: The goals of gene editing are to disrupt harmful genes
or to repair mutated genes.
´ Patient-derived cellular gene therapy products: Cells are removed from the patient,
genetically modified (often using a viral vector) and then returned to the patient.