Stem Cell Therapy

Gene Therapy

Presented byT.Hemanth kumar

Department of Pharmacology

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 progenitorcells which are committed to give rise to fully differentiated cells.

▪ Most cells of the body, such as heart cells or skin cells, are committed toconduct a specific function, a stem cell is uncommitted and remainsuncommitted, until it receives a signal to develop into a specialized cell.

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 cells1) Embryonal stem cell

2) Adult stem cell

Embryonic stem cells

As their name suggests they are derived from embryos (blastocyst) thatdevelop from eggs that have been fertilized in vitro—in an in vitrofertilization clinic and then donated for research purposes with informed

consent of the donors.

Differentiation into specific cell types

Adult stem cells

▪ Adult or somatic stem cells exist throughout the body after embryonicdevelopment and are found inside of different types of tissue. These stem cellshave been found in tissues such as the brain, bone marrow, blood, bloodvessels, skeletal muscles, skin, and the liver. They remain in a quiescent ornon-dividing state for years until activated by disease or tissue injury.

Types of Adult Stem Cells

Hematopoietic stem cells

Bone marrow stromal cells

▪ 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.

Potency

Stem cells are categorized by their potential to differentiate into other typesof cells. Embryonic stem cells are the most potent since they must becomeevery 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 thatresult 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 themesoderm, endoderm, and ectoderm germ layers that are formed in thebeginning stages of embryonic stem cell differentiation.

▪ 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.

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 onlyform of stem-cell therapy that is widely practiced.

During chemotherapy, most growing cells are killed by the cytotoxic agentsIt is this side effect of conventional chemotherapy strategies that the stem-celltransplant attempts to reverse; a donor’s healthy bone marrow reintroducesfunctional stem cells to replace the cells lost in the host’s body duringtreatment.

Another stem-cell therapy called Prochymal, was conditionally approved inCanada in 2012 for the management of acute graft-vs-host disease in childrenwho are unresponsive to steroids.

▪ 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 ofbrain degeneration, such as in Parkinson’s, Amyotrophic lateral sclerosis, andAlzheimer’s disease.

▪ Healthy adult brains contain neural stem cells which divide to maintaingeneral stem-cell numbers, or become progenitor cells. In healthy adultlaboratory animals, progenitor cells migrate within the brain and functionprimarily to maintain neuron populations for olfaction.

Brain and spinal cord injury:

Stroke and traumatic brain injury lead to cell death, characterized by a loss ofneurons and oligodendrocytes within the brain. A small clinical trial wasunderway in Scotland in 2013, in which stem cells were injected into the brainsof stroke patients.

Heart:

Stem-cell therapy for treatment of myocardial infarction usually makes use ofautologous bone-marrow stem cells (a specific type or all), however othertypes of adult stem cells may be used, such as adipose-derived stem cells.

Possible mechanisms of recovery include:

Generation of heart muscle cells

▪ Stimulation of growth of new blood vessels to repopulate damaged hearttissue .

▪ Secretion of growth factors .

▪ Assistance via some other mechanism .

Blood-cell formation :

Fully mature human red blood cells may be generated ex vivo byhematopoietic stem cells (HSCs), which are precursors of red blood cells. Inthis process, HSCs are grown together with stromal cells, creating anenvironment that mimics the conditions of bone marrow, the natural site of red-blood-cell growth. Erythropoietin, a growth factor, is added, coaxing the stemcells to complete terminal differentiation into red blood cells

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 cellsstimulate renewed repair, eventually restore vision.

Pancreatic beta cells:

▪Diabetes patients lose the function of insulin-producing beta cells within thepancreas.

▪ In recent experiments, scientists have been able to coax embryonic stem cellto turn into beta cells in the lab.

Wound healing

▪Stem cells can also be used to stimulate the growth of human tissues. In anadult, 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 tostimulate differentiation in the tissue bed cells.

Infertility

▪Human embryonic stem cells have been stimulated to form Spermatozoon-likecells, yet still slightly damaged or malformed.

▪ It could potentially treat azoospermia.

Gene TherapyGenes, 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 normalfunctions, resulting in genetic disorders.

Gene therapy is the insertion, alteration, or removal of genes within anindividual's cells and biological tissues to treat diseases.

Gene therapy is a technique for correcting defective genes responsible fordisease development.

Researchers may use one of several approaches for correcting faulty genes:

i. A normal gene may be inserted into a nonspecific location within thegenome to replace a non functional gene.

ii. An abnormal gene could be swapped for a normal gene throughhomologous 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 tothe original or normal form)

Gene therapy history

• The girl was treated on 14th September, 1990, at the National Institutes ofHealth's Clinical Center, Bathesda, Maryland.

• Dr. W. French Anderson and his colleagues at the health center, carried outthe proceedings.

• White blood cells were extracted from the body. After the implantation ofgenes 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 genetherapy.

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.

• 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)

Flow chart shows gene therapy

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 hostcells. Human immunodeficiency virus (HIV) is a retrovirus.

- One of the problems of gene therapy using retroviruses is that the integraseenzyme can insert the genetic material of the virus into any arbitrary position inthe genome of the host; it randomly inserts the genetic material into achromosome. If genetic material happens to be inserted in the middle of one ofthe original genes of the host cell, this gene will be disrupted (insertionalmutagenesis). If the gene happens to be one regulating cell division,uncontrolled cell division (i.e., cancer) can occur.

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 coldsores

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 vectortechnology have yielded molecules and techniques with transfectionefficiencies similar to those of viruses.

• Ormasil (organically modifided silica or silicate) used as non viral method.

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 causetemporary formation of pores in the cell membrane, allowing DNA molecules topass through.

• However, a high rate of cell death following electroporation has limited itsuse, 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 devicewhich 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 atumor suppressor gene, it could induce a tumor.

Sonoporation- Sonoporation uses ultrasonic frequencies to deliver DNA into

cells. The process of acoustic cavitation is thought to disrupt the cell membraneand 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 dishto 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 methodsby which this is achieved. One strategy uses antisense specific to the targetgene to disrupt the transcription of the faulty gene. Another uses smallmolecules of RNA called siRNA to signal the cell to cleave specific uniquesequences in the mRNA transcript of the faulty gene, disrupting translation ofthe faulty mRNA, and therefore expression of the gene.

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 lipoplexesfor 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 andmany of the properties of the resulting construct are determined by its surface.

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 notyet developed into AIDS, scientists could save them the pain and suffering ofthe disease by using gene therapy to ‘silence’ the disease before its onset.

Gene therapy has the potential to eliminate and prevent hereditary diseasessuch as cystic fibrosis and is a possible cure for heart disease, AIDS andcancer.

Disadvantages of Gene Therapy

Short-lived nature of gene therapy.

Immune response - Genes injected with a virus may trigger an immuneresponse against the virus.Problems with viral vectors (once inside thepatient, 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.

Ethical issues surrounding gene therapy are

• Who decides which traits are normal and which constitute a disability ordisorder?

• 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 ofpeople who are different?

• Should people be allowed to use gene therapy to enhance basic human traitssuch 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 brainchemical called GABA, which is lacking in people with Parkinson’s.

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 disorderthat commonly affects diabetics who've had the disease for many years, a newstudy finds.

• Researchers in Boston found that intramuscular injections of vascularendothelial growth factor (VEGF) gene may help patients with diabeticpolyneuropathy.

Cancer treatment: Gene therapy is used to treat many types of cancers by altering the

genes responsible for mutations.

Gene therapy in Cancer

Future aspects of gene therapy

• Nanotechnology + gene therapy yields treatment to torpedo cancer.

• Results of world's first gene therapy for inherited blindness show sightimprovement.

• Researchers at the National Cancer Institute (NCI), part of the NationalInstitutes of Health, successfully reengineer immune cells, calledlymphocytes, to target and attack cancer cells in patients with advancedmetastatic melanoma

• Gene therapy is effectively used to treat two adult patients for a diseaseaffecting non lymphocytic white blood cells called myeloid cells.

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