Gene Therapy Flashcards
What is gene therapy?
Treatment of a disease by genetically modifying the cells of a patient.
-usually involves the insertion of a normal gene (DNA) directly into a cell
Why gene therapy?
Done to correct or prevent a pathological process
- Acquired diseases
- Genetic diseases
How is gene therapy done?
- Normal (therapeutic) DNA isolated
- Gene therapy
- (a) therapeutic DNA is packaged into a vector
- (b) vector unloads its DNA into target cell and
- (c) faulty or damaged cell restored to normal
What is somatic gene therapy?
- Modification introduced into somatic (non-reproductive ) cell implicated in the disease
- Confined to a patient
- Changes are not heritable
- may have to be repeated in future generations
What is germline gene therapy?
- Modification in gamete or fertilized egg (zygote or early embryo)
- Changes are heritable
- prevents the diseas/trait from being passed on to future generations
Ethical implications:
-technology is not perf3ct
- no right to decide
- therefore not currently performed in humans
What are strategies for transecting human cells?
In vivo gene therapy
Ex vivo gene therapy
What is gene augmentation therapy?
Insert a normal gene into a nonspecific location within the genome to replace a nonfunctional gene. A most common approach
- to treat inherited disorders caused by loss of function of a gene
- effect of disorder must be reversible
What is gene replacement therapy?
Swap an abnormal gene for a normal gene through homologous recombination
What is gene inhibition therapy?
Silencing a gene: exogenous gene inhibits gain of function caused by mutation, or a pathogen
Antisense RNA or DNA hybridized to normal mRNA. Protein synthesis is blocked
What is gene repair therapy used for?
Repair an abnormal gene
What is gene alteration?
Alater the regulation (the degree to which a gene is turned on or off) of a particular gene
What is viral mediated gene therapy?
Also called transduction
- Therapeutic DNA is delivered to the cell using a “vector” (virus)
- Viruses can recognize and insert their DNA into certain cells
- Most protocols have used mammalian viral vectors because of their high efficacy
- The viral vectors are modified:
- So they are unable to cause disease
- to increase their ability to deliver specific genes to a patient’s cells
Why choose a particular viral vector for viral-mediated gene therapy?
- ability to recognize and infect certain cells
- targeted gene therapy e.g. a strain of Herpes virus (HSV-1) primarily infects cells of central nervous system
-ability to transfer the genes to cells
- Ability to alter DNA:
- permanently
- temporarily
What are common viral vectors used for gene therapy ?
Integrating viral vectors:
Retroviruses- single stranded RNA viruses e.g. Maloney murine leukemia virus (Mo-MLV)
Lentiviruses- specialized retroviruses that can infect both dividing and non-dividing cells (e.g. human immunodeficiency virus)
What are the non-integrating viral vectors of common viral vectors used for gene therapy?
Adeno-associated viruses: nonpathogenic single stranded DNA viruses, capable of infecting both dividing & non dividing cells, e.g. human parvovirus
-Adenovirus- non-enveloped viruses containing a linear double stranded DNA genome (e.g. the viruses causing common cold)
Poxviruses
Herpes viruses- have a linear double stranded DNA molecule in its genome (e.g. Herpes simplex virus type 1) a human neurotropic virus
Describe Non-viral mediated gene therapy
Safety concerns of recombinant viruses have prompted increasing interest into non-viral vector systems including:
Transfection (chemical, liposome mediated, lipofection, electroporation, and receptor mediated)
Direct transfer (microinjection t& particle bombardment)
Bacterial gene transfer involves live invasive bacteria (salmonella and shigella) or those who transfer DNA through their pilus eg Agrobacteria tumefaciens
What is In vivo liposome gene delivery ?
Used of artificial lipid here with an aqueous core
Liposome carries therapeutic DNA and is capable of passing the DNA through the target cell’s membrane when injected or sprayed into the tissue
What is receptor mediated endocytosis ?
Viral coat protein binds a specific receptor on the plasma membrane of cells, followed by endocytosis
Explain the introduction of a 47th (artificial human chromosome into target cells)
47th chromosome would be a large vector
- capable of carrying substantial amounts of genetic code
- would exist autonomously alongside the standard 46
- wouldn’t cause any mutation
Hopefully, the body’s immune systems wouldn’t attack it because of its construction and autonomy
-Difficulty to deliver such a large molecule into the nucleus of a target cell
Give the example of the first human gene therapy
1990- Ashanti DaSilva
Ex vivo gene therapy strategy used in a 4 year old severe combined immunodeficiency (SCID) patient with a deficiency in adenosine deaminase (ADA) gene
What are the challenges facing human gene therapy?
- human immune system may fight off viral vector
- appropriate vectors
- gene therapy is still in its infancy
- current therapies are primarily experimental
- in research & human clinical trials stages
- Gene therapy is short lived
- Problem with adequately targeting and sustaining therapeutic effects
- problems with viral vectors
- gene therapy used to treat children with X-SCID (“bubble boy” disease) is stopped (2002) in France when the treatment caused leukemia in one of the patients
- multi gene or multifactorial disorders
- e.g. heart disease, Ahlzeimer’s disease, arthritis and diabetes -would be especiall difficult to treat effectively using gene therapy
-need for increased public funding for biomedical research
What are the recent advances?
- Gene therapy rids men of cancer
- sickle cell disease corrected in human models using stem cell based gene therapy
- gene therapy prevents the onset of diabetic symptoms in mice
- gene therapy for HIV
Give the history of the human immune system may fight off viral vector
E.g. gene therapy that led to death of Jesse Gelsinger in 1999
- an 18 year old patient who suffered from orthinine transcarbamoylase deficiency,
- an inherited disorder: commonly causes death in affected new born males from inability to properly process nitrogen in food proteins
Gelsinger’s died from immune reaction to engineered adenovirus that was infused into his liver
How can gene therapy be used to rid men of cancer?
- Blood taken from patient
- T cells infected with virus to carry key genes into them
- DNA from genes helps cells develop receptors
- Modified cells injected back into patient
- Receptors target cancerous cells to be killed
