5. Principles and methods of gene therapy Flashcards
What is gene therapy?
Treatment and correction of disease via gene transfer
What ilnesses can gene therapy treat?
Inherited/acquired diseases Cancer (most heavily researched right now) Virus infections
What is the goal of gene therapy?
Effectively deliver a gene to the disease site with subsequent expression, of a therapeutic protein, under physiologic conditions
What is a phase 1 trial?
small ~ 15 individuals Determines toxicity
What is a phase 2 trial?
10-50, individuals Tests efficacy/delivery methods Gather data on effectiveness
What is a phase3/4 trial?
Large trial ~several hundred Determine safety and effectiveness
What are the most common genes transferred in current clinical trials?
Antigens
What is transduction?
Process whereby DNA is transferred from one bacterium to another by a virus (e.g. via vector)
What is a transposon?
DNA sequence that can change its position within the genome
What is a retrotransposon?
DNA sequence that can move and amplify itself via reverse transcription
What are long terminal repeats? (LTRs)
Identical sequences of DNA that repeat hundreds of times, found at either end of retrotransposons. They are used by viruses to insert genetic material
What are the current 3 approaches to gene therapy?
1) Gene addition 2) Gene replacement 3) Gene silencing
What are the two types of delivering therapeutic transgenes to patients?
Direct Delivery (virus injected straight to patient Cell-based (indirect) delivery - adult stem cells introduced to virus in vitro - modified cells reintroduced to patient
What are the advantages of direct delivery?
Simplicity
What are the advantages of indirect (ex vivo) delivery?
Cells manipulated more precisely Can be expanded Better tissue targeting`
What are the disadvantages of direct (in vivo) delivery?
Little control of therapeutic gene Random integration into chromosomes Poor tissue targeting specificity
What are the disadvantages of indirect delivery?
Relatively complex-cell isolation and proagation Cell biomarker knowledge Possible cell transformation
What are the considerations of effective gene therapy?
1) manufacture of gene delivery vehicle 2) Delivery to the target cell 3) Intracellular trafficking 4) Functional and sustainable gene expression for required duration 5) Safety of delivery system 6) Potential immune responses “off-target effects”
What are the characteristics of the ideal vector?
Insert size = one or more genes Targeted Stable - (not mutated) Production - (easy to produce many) Regulated - (switched on when required and produce enough protein to have an impact)
What are the two major vector categories?
- Viral 2. Non viral
What are the major barriers to gene transfer?
1) Extracellular trafficking to target cells (Immune clearence) 2) Internalization by target cells (lack of surface receptors) 3) Intracellular trafficking to nucleus (lysosomes reking) 4) Incorporation and transcription in nucelus (impermeable to nuclear membrane)
What types of viruses are associated with viral vectors?
1) Retrovirus 2) Adenovirus 3) Adeno-associated virus 4) Lentivirus (HIV vectors) 5) Herpes simplex virus
How is a viral vector manufactored?
1) Viral packing signal removed and transfected to cell line (packing cells) 2) Packaging signal fused with therapeutic gene 3) Resulting consctruct transfected into packaging cell line to produce replication-deficient virions containing therapeutic gene 4) Virions infect target cell and express therapeutic gene
What are majority of retroviral vectors based on?
Mouse leukemia virus Only invades dividing cells ssRNA 8Kb size limit
What are the limitations of the mouse leukemia virus?
Immune response Low viral titres Insertion may disrupt host genome
What is a plasmid?
Small circular DNA that has separated from chromosomal DNA
What do packaging cells do and what do contain?
packaging cells have been transfected with vector containing packaging genes and therapeutic genes. They then go on to produce virions
What cell type does PMX retrovirus vector transfect?
Plat E cell
How does the adenovirus transduce non-dividing cells?
Coxsackie and adenovirus receptor (CAR)
What is the most successful vector so far?
Adenoviral vectors 7.5Kb
What limitations are associated with adenoviral vectors?
Immune response No integration
Are adeno-associated viruses pathogenic?
No Latent until helper virus infects
What are some limitations with adeno-associated viruses?
Replication requires helper virus Low titre production
What type of DNA does the lentivirus (8kb) contain? and what are their safety concerns?
ssRNA Fear of recombinant live HIV
What type of DNA is the herpes simplex virus?
dsDNA (30Kb)
What are the benefits of non-viral carriers of DNA?
low immunogenicity Easy to manufacture
What are the three main methods of non-viral DNA carriers?
1) Naked DNA 2) Cationic Lipids or polymers 3) Particle bombardment
What is naked DNA?
Plasmid injected Works for muscle and skin can induce immune response
What are cationic lipids/liposomes?
Positively charged lipids interact with negatively charged DNA Stable complex traverses membrane Can carry lots of DNA No immune response
What are the limitations associated with cationic liposomes?
Poor targeting Transient epression Inactivated by serum
What is particle bombardment?
Plasmid coupled to surface of small beads Fired into tissue
What are the limitations of particle bombardment?
Poor targeting Efficiency tissue type dependent Surgical procedure required Transient expression
What are all limitations etc of each virus type?
