Some basics about stem cell therapy and gene therapy

1. Stem Cell Therapy
Gene Therapy
Presented by
T.Hemanth kumar
Department of Pharmacology

2. Stem cell Therapy
▪Stem-cell therapy is the use of stem cells to treat or prevent a disease or
condition.
▪ What are stem cells?
▪Stem cells are a special class of cells characterized by their ability to self-
renew (i.e. multiply to generate same kind of cells) and produce progenitor
cells which are committed to give rise to fully differentiated cells.
▪ Most cells of the body, such as heart cells or skin cells, are committed to
conduct a specific function, a stem cell is uncommitted and remains
uncommitted, until it receives a signal to develop into a specialized cell.

3. Origin of the term stem cell
▪The words stem cells were first used by Haeckel in 1868 to describe origin of
multicellular organisms from unicellular organisms.
Sources of stem cells
1) Embryonal stem cell
2) Adult stem cell

4. Embryonic stem cells
 As their name suggests they are derived from embryos (blastocyst) that
develop from eggs that have been fertilized in vitro—in an in vitro
fertilization clinic and then donated for research purposes with informed
consent of the donors.

5. Differentiation into specific cell types

6. Adult stem cells
▪ Adult or somatic stem cells exist throughout the body after embryonic
development and are found inside of different types of tissue. These stem cells
have been found in tissues such as the brain, bone marrow, blood, blood
vessels, skeletal muscles, skin, and the liver. They remain in a quiescent or
non-dividing state for years until activated by disease or tissue injury.
Types of Adult Stem Cells
 Hematopoietic stem cells
 Bone marrow stromal cells

7. ▪ Hematopoietic stem cells forms all the types of blood cells in the body.
▪ Stromal cells are a mixed cell population that generates bone, cartilage, fat
and fibrous connective tissue.

8. Potency
 Stem cells are categorized by their potential to differentiate into other types
of cells. Embryonic stem cells are the most potent since they must become
every type of cell in the body. The full classification includes:
▪ Totipotent - the ability to differentiate into all possible cell types.
Examples are the zygote formed at egg fertilization and the first few cells that
result from the division of the zygote.
▪ Pluripotent - the ability to differentiate into almost all cell types.
Examples include embryonic stem cells and cells that are derived from the
mesoderm, endoderm, and ectoderm germ layers that are formed in the
beginning stages of embryonic stem cell differentiation.

9. ▪ Multipotent - the ability to differentiate into a closely related family of cells.
Examples include hematopoietic (adult) stem cells that can become red and
white blood cells or platelets.
▪ Oligopotent - the ability to differentiate into a few cells.
Examples include (adult) lymphoid or myeloid stem cells.
▪ Unipotent - the ability to only produce cells of their own type, but have the
property of self-renewal required to be labeled a stem cell.
Examples include (adult) muscle stem cells
o Embryonic stem cells are considered pluripotent instead of totipotent
because they do not have the ability to become part of the extra-embryonic
membranes or the placenta.

10. Uses of stem cells
Leukemia : For over 30 years, bone marrow has been used to treat cancer
patients with conditions such as leukaemia and lymphoma; this is the only
form of stem-cell therapy that is widely practiced.
 During chemotherapy, most growing cells are killed by the cytotoxic agents
It is this side effect of conventional chemotherapy strategies that the stem-cell
transplant attempts to reverse; a donor’s healthy bone marrow reintroduces
functional stem cells to replace the cells lost in the host’s body during
treatment.
 Another stem-cell therapy called Prochymal, was conditionally approved in
Canada in 2012 for the management of acute graft-vs-host disease in children
who are unresponsive to steroids.

11. ▪ The FDA has approved five hematopoietic stem-cell products derived from
umbilical cord blood, for the treatment of blood and immunological diseases.
Neurodegeneration :
Research has been conducted on the effects of stem cells on animal models of
brain degeneration, such as in Parkinson’s, Amyotrophic lateral sclerosis, and
Alzheimer’s disease.
▪ Healthy adult brains contain neural stem cells which divide to maintain
general stem-cell numbers, or become progenitor cells. In healthy adult
laboratory animals, progenitor cells migrate within the brain and function
primarily to maintain neuron populations for olfaction.

12. Brain and spinal cord injury:
Stroke and traumatic brain injury lead to cell death, characterized by a loss of
neurons and oligodendrocytes within the brain. A small clinical trial was
underway in Scotland in 2013, in which stem cells were injected into the brains
of stroke patients.
Heart:
Stem-cell therapy for treatment of myocardial infarction usually makes use of
autologous bone-marrow stem cells (a specific type or all), however other
types of adult stem cells may be used, such as adipose-derived stem cells.
Possible mechanisms of recovery include:
 Generation of heart muscle cells

13. ▪ Stimulation of growth of new blood vessels to repopulate damaged heart
tissue .
▪ Secretion of growth factors .
▪ Assistance via some other mechanism .
Blood-cell formation :
Fully mature human red blood cells may be generated ex vivo by
hematopoietic stem cells (HSCs), which are precursors of red blood cells. In
this process, HSCs are grown together with stromal cells, creating an
environment that mimics the conditions of bone marrow, the natural site of red-
blood-cell growth. Erythropoietin, a growth factor, is added, coaxing the stem
cells to complete terminal differentiation into red blood cells

14. Blindness and vision impairment :
▪ Since 2003, researchers have successfully transplanted corneal stem cells into
damaged eyes to restore vision.
▪ “Sheets of retinal cells used by the team are harvested from aborted fetuses,
which some people find objectionable.”
▪When these sheets are transplanted over the damaged cornea, the stem cells
stimulate renewed repair, eventually restore vision.
Pancreatic beta cells:
▪Diabetes patients lose the function of insulin-producing beta cells within the
pancreas.
▪ In recent experiments, scientists have been able to coax embryonic stem cell
to turn into beta cells in the lab.

15. Wound healing
▪Stem cells can also be used to stimulate the growth of human tissues. In an
adult, wounded tissue is most often replaced by scar tissue
▪A possible method for tissue regeneration in adults is to place adult stem cell
“seeds” inside a tissue bed “soil” in a wound bed and allow the stem cells to
stimulate differentiation in the tissue bed cells.
Infertility
▪Human embryonic stem cells have been stimulated to form Spermatozoon-like
cells, yet still slightly damaged or malformed.
▪It could potentially treat azoospermia.

16. Gene Therapy
Genes, the functional unit of heredity, are specific sequences bases that encode
instructions to make proteins.
 When genes are altered, encoded proteins are unable to carry out their normal
functions, resulting in genetic disorders.
 Gene therapy is the insertion, alteration, or removal of genes within an
individual's cells and biological tissues to treat diseases.
 Gene therapy is a technique for correcting defective genes responsible for
disease development.

17. Researchers may use one of several approaches for correcting faulty genes:
i. A normal gene may be inserted into a nonspecific location within the
genome to replace a non functional gene.
ii. An abnormal gene could be swapped for a normal gene through
homologous recombination.
iii. The abnormal gene could be repaired through selective reverse mutation,
which returns the gene to its normal function.
( Reverse mutation: Mutation that restores a mutant phenotype or genotype to
the original or normal form)

18. Gene therapy history
• The girl was treated on 14th September, 1990, at the National Institutes of
Health's Clinical Center, Bathesda, Maryland.
• Dr. W. French Anderson and his colleagues at the health center, carried out
the proceedings.
• White blood cells were extracted from the body. After the implantation of
genes that produce ADA, the cells were transferred back to the girl's body.
• Considerable improvement in the immune system of the girl was noticed.
Meanwhile, the trials of gene therapy continued on various diseases.
• The patients with skin cancer, melanoma were treated by means of gene
therapy.

19. Principle of gene therapy
• An abnormal gene could be swapped for a normal gene through homologous
recombination.
• The abnormal gene could be repaired through selective reverse mutation,
which returns the gene to its normal function.
• The regulation (the degree to which a gene is turned on or off) of a particular
gene could be altered.

20. • Approaches of gene therapy
1. Gene modification
o Replacement therapy
o Corrective Gene therapy
2. Gene transfer
o Physical
o Chemical
o Biological
3. Gene transfer in specific cell line
o Somatic gene therapy
o Germ line gene therapy
4. Eugenic approach (gene insertion)

21. Flow chart shows gene therapy

22. Vectors in gene therapy
Some of the different types of viruses used as gene therapy vectors:
Retroviruses - A class of viruses that can create double-stranded DNA copies of
their RNA genomes.
 These copies of its genome can be integrated into the chromosomes of host
cells. Human immunodeficiency virus (HIV) is a retrovirus.
- One of the problems of gene therapy using retroviruses is that the integrase
enzyme can insert the genetic material of the virus into any arbitrary position in
the genome of the host; it randomly inserts the genetic material into a
chromosome. 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.

23. Adenoviruses- A class of viruses with double-stranded DNA genomes that cause
respiratory, intestinal, and eye infections in humans.
• The virus that causes the common cold is an adenovirus.
Adeno associated viruses- A class of small, single-stranded DNA viruses that can
insert their genetic material at a specific site on chromosome 19.
Herpes simplex viruses- A class of double-stranded DNA viruses that infect a
particular cell type, neurons.
• Herpes simplex virus type 1 is a common human pathogen that causes cold
sores

24. Non-viral methods
• Non-viral methods present certain advantages over viral methods
• Previously, low levels of transfection and expression of the gene held non-
viral methods at a disadvantage; however, recent advances in vector
technology have yielded molecules and techniques with transfection
efficiencies similar to those of viruses.
• Ormasil (organically modifided silica or silicate) used as non viral method.

25. Physical Methods to Enhance Delivery
Electroporation- Electroporation is a method that uses short pulses of high voltage
to carry DNA across the cell membrane. This shock is thought to cause
temporary formation of pores in the cell membrane, allowing DNA molecules to
pass through.
• However, a high rate of cell death following electroporation has limited its
use, including clinical applications.
Gene Gun- The use of particle bombardment, or the gene gun, is another physical
method of DNA transfection.
• In this technique, DNA is coated with gold particles and loaded into a device
which generates a force to achieve penetration of DNA/ gold into the cells.
eg:- If the DNA is integrated in the wrong place in the genome, for example in a
tumor suppressor gene, it could induce a tumor.

26. Sonoporation- Sonoporation uses ultrasonic frequencies to deliver DNA into
cells. The process of acoustic cavitation is thought to disrupt the cell membrane
and allow DNA to move into cells.
Magnetofection- In a method termed magnetofection, DNA is complexed to a
magnetic particles, and a magnet is placed underneath the tissue culture dish
to bring DNA complexes into contact with a cell monolayer.
Chemical Methods to Enhance Delivery
Oligonucleotides - The use of synthetic oligonucleotides in gene therapy is to
inactivate the genes involved in the disease process. There are several methods
by which this is achieved. One strategy uses antisense specific to the target
gene to disrupt the transcription of the faulty gene. Another uses 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, and therefore expression of the gene.

27. Lipoplexes and polyplexes - To improve the delivery of the new DNA into the cell,
the DNA must be protected from damage and (positively charged).
Initially, anionic and neutral lipids were used for the construction of lipoplexes
for synthetic vectors.
Dendrimers- A dendrimer is a highly branched macromolecule with a spherical
shape. The surface of the particle may be functionalized in many ways and
many of the properties of the resulting construct are determined by its surface.

28. Advantages and disadvantages of gene therapy Advantages of gene therapy
 In case of ‘silence’ a gene. In the case of someone with HIV, which had not
yet developed into AIDS, scientists could save them the pain and suffering of
the disease by using gene therapy to ‘silence’ the disease before its onset.
 Gene therapy has the potential to eliminate and prevent hereditary diseases
such as cystic fibrosis and is a possible cure for heart disease, AIDS and
cancer.
Disadvantages of Gene Therapy
Short-lived nature of gene therapy.
Immune response - Genes injected with a virus may trigger an immune
response against the virus.Problems with viral vectors (once inside the
patient, the viral vector could recover its ability to cause disease).
 Multigene disorders - The genetic material might not get into the right cell,
or the right place in the cell’s DNA.

29. Ethical issues surrounding gene therapy are
• Who decides which traits are normal and which constitute a disability or
disorder?
• Will the high costs of gene therapy make it available only to the wealthy?
• Could the widespread use of gene therapy make society less accepting of
people who are different?
• Should people be allowed to use gene therapy to enhance basic human traits
such as height, intelligence, or athletic ability?
Applications of gene therapy
In case of Parkinson’s disease
o Gene therapy has been proven to work for Parkinson’s disease.
o A new procedure is implemented which is intended to boost levels of a brain
chemical called GABA, which is lacking in people with Parkinson’s.

30. In case of Alzheimer’s disease
• Scientists have successfully switched off a gene thought to cause Alzheimer’s
by using a new way to deliver drugs directly to the brain.
In case of Diabetic Neuropathy
• Gene therapy shows promise in treating diabetic polyneuropathy, a disorder
that commonly affects diabetics who've had the disease for many years, a new
study finds.
• Researchers in Boston found that intramuscular injections of vascular
endothelial growth factor (VEGF) gene may help patients with diabetic
polyneuropathy.
Cancer treatment: Gene therapy is used to treat many types of cancers by altering the
genes responsible for mutations.

31. Gene therapy in Cancer

32. Future aspects of gene therapy
• Nanotechnology + gene therapy yields treatment to torpedo cancer.
• Results of world's first gene therapy for inherited blindness show sight
improvement.
• Researchers at the National Cancer Institute (NCI), part of the National
Institutes of Health, successfully reengineer immune cells, called
lymphocytes, to target and attack cancer cells in patients with advanced
metastatic melanoma
• Gene therapy is effectively used to treat two adult patients for a disease
affecting non lymphocytic white blood cells called myeloid cells.

33. References
www.researchgate.net
 http://creationwiki.org/index.php/Stem_cell
 http://www.medicalnewstoday.com/info/stem_cell
 stem Cell Information The official National Institutes of Health resource for stem cell research
 Stem cells: A new paradigm Sachin Kumar, N. P. Singh Maulana Azad Medical College and Associated Lok
Nayak and G. B. Pant Hospitals, New Delhi - 10002, India
 Stem Cells: Introduction and Prospects in Medicine
 Stem cell therapy: a novel & futuristic treatment modality for disaster injuries G.U. Gurudutta, Neeraj
Kumar Satija, Vimal Kishor Singh, Yogesh Kumar Verma, Pallavi Gupta & R.P. Tripathi
 Stem cell therapy: a novel & futuristic treatment modality for disaster injuries G.U. Gurudutta, Neeraj
Kumar Satija, Vimal Kishor Singh, Yogesh Kumar Verma, Pallavi Gupta & R.P. Tripathi
 http://www.thefreedictionary.com/reverse+mutation
Human Gene Therapy : A Brief Overview of the Genetic Revolution Sanjukta Misra
 http://sgugenetics.pbworks.com/f/1301871442/how%20it%20works-gene%20therapy.gif