PHRM3041 - Biotech II Flashcards
gene therapy
technique to treat disorders by inserting genes into patient’ cells instead of using drugs or surgery
3 approaches to gene therapy
- Replacing a mutated gene that causes disease with a healthy copy of the gene
- Inactivating or ‘knocking out’ a mutated gene that is functioning improperly
- Introducing a new gene into the body to help fight a disease
cystic fibrosis
- Autosomal recessive disease
- Mutations in cystic fibrosis transmembrane conductance regulator (CFTR) gene
- Chloride channel in apical membrane of exocrine epithelial cells
- Current approaches include: antibiotics, anti-inflammatory mucolytics, nebulized hypertonic saline etc
- Could create a therapy to correct the channel defect
2 ways to deliver genes
non-viral transfection
virus-mediated transfection (transduction)
non-viral transfection
2 classifications
•The process of introducing nucleic acids into cells by non-viral methods is defined as “transfection”
•Can be broadly classified as either
o Chemical reagents
o Physical method
non-viral, chemical reagent
- Positively charged chemicals make complexes with negatively charge nucleic acids
- Attracted to negatively charged cell membrane
- Pass through cell membrane
non-viral, chemical reagent
example: liposomes
oA lipid with overall net positive charge at physiological pH is used to create an artificial liposome
oThe cationic portion of the lipid molecules associates with the negatively charge nucleic acids
oA liposome/nucleic acid complex is formed
oThe complex is taken up by the cells
oThe complex is transported to the nucleus
non-viral, physical methods
3 types
- direct microinjection
- electroporation
- biolistic particle delivery
non-viral, physical methods
direct mircoinjection
directly inject nucleic acid into cytoplasm of nucleus
non-viral, physical methods
electroporation
expose the cells to an electrical impulse to perturb the cell membrane and allow pores to form
-allows nucleic acids to enter the cell
non-viral, physical methods
biolistic particle delivery (gene gun)
this method relies upon high velocity delivery of nucleic acids on micro projectiles to recipient cells
non-viral drawbacks
inefficient
variable
laborious
viral (infection)
what
common types
•Delivery of DNA by viruses •Can be almost 100% efficient in some cases •Most common vectors used un clinical trials include o Adenovirus o Retrovirus o Adeno-associated virus o Lentivirus o Herpes simplex virus
viral delivery drawbacks
- potential immunogenicity
- insertional mutation (random insertion could disrupt tumour suppressor gene, activate oncogene)
drawbacks and limitations of gene therapy
4 and defintions
•Gene therapy is short lived
oTherapeutic DNA must remain functional during cell division and be resistant to degradation by the cell
oMultiple rounds of gene therapy may be necessary
•Immune response
oEffectiveness may be reduced due to an immune response
oThis could be a problem if gene therapy needs to be given repeatedly
•Problems with viral vectors
oViruses can cause toxicity, immune and inflammatory responses
oProblems with gene control and targeting
oViral vectors could regain virulence, i.e. cause disease
•Multigene disorders
oConditions that arise from a single gene are best candidates for gene therapy
oMany conditions (e.g. heart disease, diabetes) are multi-gene disease
