Gene Therapy Flashcards
Describe cell therapy.
Replacement of a diseased cell population via transplantation with normal cells. Includes embryonic stem cell transplantation as well.
Broadly define gene therapy.
Transfer of genetic material into a patient via transfection.
What are the classes of gene therapy?
Somatic cell- Modifies the somatic cell genes of an individual. Are not transferred to progeny, and can be done in vivo or ex vivo.
Germline- Modifications of genes in germ cells. Can be done preimplantationally to affect all cells of the individual and future progeny.
Describe ex vivo gene therapy. What are advantages and disadvantages?
- Cells are removed from patient, such as hematopoietic stem cells.
- Vector carrying a therapeutic gene is transduced into the cell.
- Cells are returned into the patient.
Advantages- Can control which cells are altered, high transduction efficiency.
Disadvantages- Difficulty removing an adequate amount of cells and difficulty regrafting them correctly.
Describe in vivo gene therapy. What are advantages and disadvantages?
Gene is transferred directly into cells of a patient without their removal.
Advantages- Can access any site of the patient.
Disadvantages- Dosage is difficult to control, the therapeutic vector may act toxically to other tissues or to the resident tissue if too much is given, and there is a risk of movement throughout the body and contamination of the germline.
What are the major obstructions to efficient gene therapy?
- Transfection of DNA into patient cells is inefficient, and not enough enters so ineffectual amount of gene product is produced.
- Vectors may induce heavy inflammatory responses.
- Effect is only transient if DNA remains epichromosomal, and is lost after cell division.
- Transfection with large DNA adducts is difficult.
- Integration near a protooncogene can lead to cancer development.
What are non-viral vector transfection modes? What are advantages and disadvantages?
Naked DNA, DNA coated gold particles, liposomes.
Advantages- Do not require receptors on target cells, immune response is minimal, can deliver large DNA sequences.
Disadvantages- Low transfection efficiency, transient expression.
Describe retro viral vectors, as well as their advantages and disadvantages.
Virus enters cell and its DNA with normal gene is reverse transcribed into a random position of dividing cell genome.
Advantages- Transfections efficiency is high, expression is long term through daughter cells.
Disadvantages- Gene size must be small, random insertion can cause mutagenesis.
Describe adenovirus vectors, as well as their advantages and disadvantages.
Virus binds receptor on PM and is internalized in an endosome. Extrachromosomal is the method of viral expression.
Advantage- Very large DNA sequences can be added in an efficient manner, dividing and no dividing cells are infected, risk of mutagenesis is low.
Disadvantages- Transient expression, high doses can be toxic and cause an immune response.
What is the structure of a transgene within a vector, and what are types of promoter used?
Are a promoter region followed by exon cDNA of the gene of interest.
The promoter may be tissue specific, ubiquitous with high activity, or drug inducible.
What are the three overarching mechanisms of correcting genetic issues, as in what do the introduced genes now cause?
Gene augmentation- Insertion of genes to replace a missing product (loss of function).
Gene inhibition- Decreases expression of a pathogenic gene, or interferes with the activity of its product (gain of function, novel mutation) via antisense oligonucleotides, ribozymes, or siRNAs.
Genetic editing or deletion by CRIPR/CAS.
What are the three mechanisms prevalent in cancer therapies?
Corrective gene therapy- Restore normal patterns and function of tumor suppressor genes, and suppress tumor promoting oncogenes via antisense oligos and ribozymes.
Cytoreductive therapy- Genetic induction of apoptosis, activation of a prodrug within the tumor to express cytotoxic drugs here, antiangiogenic gene expression to limit tumor blood supply, or accumulation of radioactive iodine in the tumor cells.
Immunoregulatory gene therapy- Generates an effective local immune response via causing foreign antigens, immunostimulatory molecules, or costimulatory molecules to be expressed on tumor cells.
