Gene therapy Flashcards
1
Q
Gene therapy
A
- Transfer of genetic material into cells or tissue to either prevent or cure disease
- Initially developed to cure single gene hereditary disease (CF and haemophilia)
- Now directed to treat polygenic or non-inherited disease e.g. cancer, CVD, HIV
2
Q
In vivo gene therapy
A
Introduction of the therapeutic gene into vector which is then administered directly into a patient
3
Q
Ex vivo
A
Transfer of therapeutic gene into cultured cells derived from the patient then reintroduced into the patient
4
Q
Steps in Developing gene therapy
A
- Gene defect identified
- Functional copy of gene available
- Target cells must be available and amenable to transfection resulting in long-term expression
- Route of administration by which vector will be introduced to patient
- Have an accurate animals model
5
Q
What is needed for tissue-specific expression?
A
Tissue-specific promoters
6
Q
What makes an ideal gene therapy vector?
A
- Effeciently transduce target cells
- Does not activate immune response either against itself or therapeutic gene
7
Q
Choice of vector depends on …
A
- Target tissue
- Whether disease requires short term or chronic treatment
8
Q
Viral vectors
A
- Viral genes replaced by therapeutic gene
- Viruses incapable of causing disease - pathogenic genes elimated
9
Q
4 most common viruses in gene therapy
A
- Retroviral vectors
- Lentiviral vectors
- Adenoviral vector
- Adeno-associated vector
10
Q
Retroviral and lentivirals vectors
A
- Both derived from retroviruses and RNA genome
- RV requires host cell to be in division process
- LV can induce dividing and nondividing cells
11
Q
Adenoviruses
A
- dsDNA genome
- Vector enters endosome by endocytosis
- Released from endosome and DNA enters nucleus where it persists in extrachromosomal form
12
Q
Adeno-associated viral vectors
A
- ss DNA
- Maintained in extrachromosal form
13
Q
Limitations to viral gene therapy
A
Limited packaging capacity
14
Q
Non-viral delivery methods
A
Physical
- Electroporation
- Ultrasound
Chemical
- Polyplexes
- Lipoplexes
- Polymeric micelles
- Dendrimers
15
Q
Physical methods
A
Increase the cell membrane permeability to plasmids
16
Q
Chemical methods
A
Polyplexes
- Complexes of polymers with DNA
- Cannot release DNA into cytoplasm
- Positively charged binds with negatively charged DNA
- e.g. Polyethylenimine (PEI) and Poly-L-Lysine (PLL)
17
Q
Chemical method :Lipoplexes
A
- Complexes of cationic lipids or liposomes with nucleic acids
- Transfection = Lipofection
- e.g. Lipofectin, lipofectamine
18
Q
Non-viral vectors for delivery : advantage vs disadvantage
A
Advantages
- Versatility
- Protection of DNA
- Fusion with cell membrane
- No size limit
- No immune response against vector
- Re-adminstered if necessary
Disadvantages
- Lower transfer efficiency than viral vectors
- Stability
- Labor intensive
- expensive
- Clearance by macrophage (innate immunity)
19
Q
Haemophilia gene therapy
A
AdV and AAV injection of correct copy of factor IXinto blood stream –> hepatic transduction
20
Q
Clinical phase
A
- Pre-clinical: assess safety, toxicity, pharmacodynamics, pharmacokinetics
- Phase 1: Drug given to 20-100 people
- Phase 2: Drug given to 100-500
- Phase 3: Drug given to 300-3000 people
- Phase 4: Post marketing studies delineate additional information - drug risk, benefits
21
Q
X-SCID
A
= severe combined immunodeficiency disease
- No B, T, NK cells
- Mutation in the gene encoding CD132 protein or interleukin 2 receptor subunit gamma chain
- ADA: SCID - lack of enzyme adenosine deaminase
- Treated with lentiviral vector
22
Q
Side effects of gene therapy
A
- Immune response to vector
- Immune response to new/foreign gene products
- General toxicity of viral vectors
- Random integration in genome
- Insertional mutagensis: mutagenesis of DNA by insertion of 1 or more bases
- Insertional oncogenesis
- Incsertional virogenesis: patient may be infected by other viruses at the time, and recombination could create a new virus capable of infecting other people.
23
Q
Gene therapy strategies
A
- Replacement of a missing or defective gene
- introduction of genes to influence cellular processes
- Interference with gene products
24
Q
Commonly used vectors
A
25
Human B-globin locus
* Located on short arm of chromosome 11
* Multiple interactions between regulatory regions are required to stabalise an active chromatin hub
26
Thalassemia treatment
* Blood transfusions and iron chelation therapy to remove excess iron
* Bone marrow or stem cell transplants - not readily available
* Stem cell gene therapy - common myeloid progenitor (CMP) cells
* Introduce functional b-globin under control of LCR in lentiviral vectors
27
Lentiviral vectors
* Based on lentiviruses
* 3 plasmid components: all with promoters
* Transfer vector containing transgene and flanking LTRs
* Packaging vector - Gag, pol, tat, rev
* Envelope - env
28
Data to support gene therapy for b-thalassaemia
* effecient gene transfer into target cells
* adequate level of b-globin expression
* Persistence of gene expression for life of animal
* Tissue-specific expression
* Tolerance to transgene product
* No adverse effects in mice
29
Gene Therapy Vectors
* Retro/lentiviral the most popular
* Accept relatively short DNA sequences
* Gene expression may be inappropriately regulated
* Susceptible to transcriptional silencing (heterochromatin modification after integration)
* Random integration associated with genotoxicity - site-specific integration may avoid genotoxicity
30
Integration at defined chromosome location
* AAV (non-pathogenic human parvovirus) integrates at site AAVS1 on human chromosome 19
* AAVS1 ideal target for gene therapy
* AAV capable of site specific integration or persist episomally
31
AAV site-specific integration
* ss linear DNA genome consists of short inverted terminal repeats required for replication, packaging and site-specific integration
* Rep protein - integrase
32
FISH
* DNA probe tagged with fluorescent marker
* Probe and target DNA denatured and probe is allowed to hybridize with target
* Fluorescent tag detected with fluorescent microscope
33
B-globin locus is too big for common viral vectors. What is used?
Herpes virus
34
Herpes virus
* dsDNA virus, recombinant vector
* Advantages: transduction of non-dividing cells, accomodates large fragment of foreign DNA (150-170kb), affinity to neuronal tissues
* Disadvantage: transient gene expression
35
Genome editing
Technique as a means to avoid issues with current gene therapy - random integration, genotoxicity (lentiviral)
Precise manipulation of desired gene of interest
~cut and paste at DNA level
36
Genome editing sequence-specific nucleases
Genome editing tools:
1. Recognise specific DNA sequences
2. Cut DNA then a scar is left behind
e.g. zinc finger nuclease, TAL effector nuclease, CRISPR-associated nuclease
37
CRISPR/Cas system
Prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity
* Deliver Cas9 protein and guide RNAs into cell, the organism's genome can be cut at any desired location
38
Zinc finger nucleases
* ZFNs make targeted ds breaks in genomic DNA and stimulate recombination and repair processes at specific sites
* Each ZF recognises 3 nucleotides
* ZFN linked to Fok1 nuclease
* Outcomes:
1. Double knockout - same or different chromosomes
2. Large deletion - hundred kb deletion
3. Gene modification
4. Gene integration
