Zygotic stem cells which have ability to differentiate into any cell type to form whole organisms including extra-embryonic membranes and so are also pluripotent and multipotent
Embryonic stem cells which have ability to differentiate into almost any cell type to form any organ BUT not those of extra embryonic membranes or type of cell and so are not totipotent but multipotent
Blood stem cells which have ability to differentiate into a limited range of cell type and so are not totipotent or pluripotent
i.e adult stem cells or somatic stem cells
heimatopoietic stem cells
Stem cells have 3 characteristics
capable of self renewal via mitotic cell division while maintaining undifferentiated state
able to differentiate into specialised cell types under presence of appropriate chemical signals
is a technique for correcting defective gene responsible for disease development
Introducing a copy of a normal functional gene into target cells with non-functional genes
A vector is required - can be viral/non-viral.
Newly introduced gene is transcribed and translated for synthesis of normal functional protein product thus restoring correct function of target cells, altering phenotype
Types of gene therapy vectors
2 types of gene delivery methods
Ex Vivo (outside body)
In Vivo (inside body)
cells are removed from the body and cultured in vitro
gene of interest is inserted
these transformed cells are then allowed to multiply to a sufficient number and then return to the body by infusion or transplant
patient's own cell used, no rejection
gene of interest is introduced directly into cells with the body
gene could be introduced either systematically or topically
not as controlled or specific compared to ex vivo
Severe combined immunodeficiency (SCID)
inheritable congenital disease
poor or no immune response
mutated interleukin-2 receptor gamma (IL2RG) gene
sex-linked recessive - IL2RG gene is found on X chromosome - hence more affected males XY (X dominant over Y)
controls production of the common gamma chain subunit of the protein receptor on the surface of immature blood stem cells in the bone marrow.
The receptor directs the development of T, B and natural killer cells of the immune system from blood stem cells.
Mutations to the IL2RG gene
results in production of a non functional version of the common gamma chain or no protein at all.
Without this common gamma chain, important chemical signals are not relayed to the nucleus. - near complete failure of immune system due to immature T B and natural killer cells.
adenosine deaminase deficiency
Autosomal recessive disorder of the ADA gene at chromosome 20
ADA (adenosine deaminase) is an enzyme
involved in the breakdown of dATP (deoxy-adenosine triphosphate)
Lack of ADA results in dATP accumulation.
Immature T and B cells are affected by the toxic effects of dATP accumulation and hence fail to reach maturity.
Gene therapy of SCID using retroviral gene delivery
Normal gene is cloned and inserted into an inactivated, non pathogenic retrovirus
Target cells are then isolated from patient and cultured in the lab
Target cells are then infected with the retroviral vector and further cultured before they are re-introduced into the patient
Advantages of using stem cells in gene therapy
capable of differentiating into different cell types
capable of continual mitotic divison -> ensures that inserted gene is not lost
Injections with retroviral T cells had to be repeated as T cells live for only 6-12 months in the blood.
Risk of insertional mutagenesis (inapporopriate insertion of retroviral vector near a proto-oncogene promoter) -> Uncontrolled proliferation of T cells
Risk of stimulating an immune response
Risk of insertional mutagenesis
Cystic Fibrosis (CF)
autosomal recessive trait affecting chromosome number 7
caused by inheriting two mutated copies of gene coding for a transmembrane protein (cystic fibrosis transmembrane conductance regulator) (CFTR)
NORMAL CFTR protein
is a transmembrane chloride channel that allows the efflux of chloride ions out of epithelial cells
Mutation in CF
involves the deletion of 3 base pairs (equals to 1 codon) from the CFTR gene.
As such, mutated CFTR protein is missing the A.A phenylalanine at position 508.
3D configuration of mutated CFTR protein changed
Preventing normal efflux of Cl- ions out of epithelial cells.
This build up of Cl- ions in the cells, attract water into the epithelial cells and away from the external mucus layer.
Thick and sticky mucus forms and clogs up the airways of the lungs, pancreatic duct, bile duct, sweat gland ducts and reproductive ducts.
Bacteria thrive in the thick mucus causing infections. i.e pneumonia, diabetes (blocked insulin release)
are phospholipid vesicles that encapsulate the gene of interest, and fuses with the plasma membrane of the target cell.
Gene therapy of CF using non-viral gene delivery
Normal CFTR gene is cloned into a plasmid, which is placed into cationic liposomes and sprayed into the nose and mouth of CF patients as an aerosal spray.
Cationic to minimise repulsion btw liposome and cell membrane as both are -vely charged.
Cationic liposomes fuse with the cell membrane of tracheal cells, releasing the normal CF gene into the cytoplasm of cells.
Gene therapy of CF using non-viral gene delivery
This introduced CF gene then enters the nucleus, transcribed into mRNA and translated into the normal CFTR protein.
Normal CFTR protein embeds into cell membrane and begins to transport Cl- ions out of cells, thinning the mucus and water moves out of cell to dilute thick sticky mucus and alleivate CF symptoms.
Disadvantage of CF gene therapy
multiple reapplications are required, since the DNA delivered via liposomes does not integrate into the chromosomes & epithelial cells constantly being shed, thus delivered gene is lostLow efficiency of transfer due to non-specificity, gene does not incorporate into target cells genome
Advantage of CF gene therapy
Liposomes can be safely administered to patients without invoking any immune response
Factors that prevent gene therapy from becoming an effective treatment for genetic diseases
Difficult to get DNA to integrate into target cell genome
Short-lived nature of gene therapy
Difficulty in controlling gene expression
Risk of stimulating an immue response Virus vector may develop virulence
Incorrect gene insertion (insertional mutagenesis) may cause cancer
Size constraint (difficult to find suitable vectors for large genes)
Difficulty in treating multi-gene disorders (impossible to introduce many genes into target cell at the same time)
Difficulty correcting a disease which is due to presence of a dominant allele (as dominant allele has to be removed first, before introducing copies of the recessive normal allele)
Not applicable to non dividing cells
Low efficiency of liposomes
Social Considerations for Gene therapy
Directed to treatment of diseases with an enomous social impact
Difficulty in determining which conditions are normal and which are disorders
Gene therapy very expensive -> accessible only to wealthy, possible genetic enhancements creating an advantage for those who can afford treatment
Concerns about widespread use of gene therapy making society less accepting of people who have genetic diseases or less abled
Germ-line gene therapy (correct genetic defect in future generations, forever changes genetic makeup of an individual's descendants)
Safety of gene therapy to humans(unknown risks)
Protection of privacy and confidentiality of medical information
Concerns that gene therapy maybe abused for genetic enhancement -> eugenics