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
ethical aspect
how can good and bad be distinguished
- who decides
- will cost only make it available to the weathly
mircoarray
- Micro = small
- Array = orderly arrangement
- DNA microarray = orderly arrangement of DNA fragments representing the genes of an organism
- Outdated technology
- A microarray is usually a slide onto which thousands of DNA fragments have been arranged (spotted) in a grid pattern
- Each DNA spot corresponds to a gene fragment
pros and cons of microarray
•Pros
o Relatively cheap
o Fast
•Cons
o Requirement for a prior knowledge of the sequences being interrogated
o Problematic cross-hybridization artifacts with highly similar sequences – requires validation
o Sensitivity
RNA-seq
what is it?
4 basic steps
High throughput technology for comprehensive transcriptome analysis
•Measure expression of thousands of genes simultaneously
•Does not depend on hybridization between gene probes and target genes
oNot limited to detecting known transcripts
•Improved resolution and sensitivity of technology
-single nucleotide resolution e.g. SNPs, alternative splicing etc
•Basic steps:
1.RNA to cDNA fragments
2.Sequenced in a high-throughput manner
3.Results aligned to reference transcripts
4.Or assembled de novo to produce a transcription map (structure and/or level of expression)
drug discovery and clinical use
- disease classification
- predict outocme
- clues for gene function that an help identify appropriate targets for therapeutic intervention
