Gene therapy Flashcards
Gene therapy
- 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
In vivo gene therapy
Introduction of the therapeutic gene into vector which is then administered directly into a patient
Ex vivo
Transfer of therapeutic gene into cultured cells derived from the patient then reintroduced into the patient
Steps in Developing gene therapy
- 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
What is needed for tissue-specific expression?
Tissue-specific promoters
What makes an ideal gene therapy vector?
- Effeciently transduce target cells
- Does not activate immune response either against itself or therapeutic gene
Choice of vector depends on …
- Target tissue
- Whether disease requires short term or chronic treatment
Viral vectors
- Viral genes replaced by therapeutic gene
- Viruses incapable of causing disease - pathogenic genes elimated
4 most common viruses in gene therapy
- Retroviral vectors
- Lentiviral vectors
- Adenoviral vector
- Adeno-associated vector
Retroviral and lentivirals vectors
- Both derived from retroviruses and RNA genome
- RV requires host cell to be in division process
- LV can induce dividing and nondividing cells
Adenoviruses
- dsDNA genome
- Vector enters endosome by endocytosis
- Released from endosome and DNA enters nucleus where it persists in extrachromosomal form
Adeno-associated viral vectors
- ss DNA
- Maintained in extrachromosal form
Limitations to viral gene therapy
Limited packaging capacity
Non-viral delivery methods
Physical
- Electroporation
- Ultrasound
Chemical
- Polyplexes
- Lipoplexes
- Polymeric micelles
- Dendrimers
Physical methods
Increase the cell membrane permeability to plasmids
Chemical methods
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)
Chemical method :Lipoplexes
- Complexes of cationic lipids or liposomes with nucleic acids
- Transfection = Lipofection
- e.g. Lipofectin, lipofectamine
Non-viral vectors for delivery : advantage vs disadvantage
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)
Haemophilia gene therapy
AdV and AAV injection of correct copy of factor IXinto blood stream –> hepatic transduction
Clinical phase
- 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
X-SCID
= 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
Side effects of gene therapy
- 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.
Gene therapy strategies
- Replacement of a missing or defective gene
- introduction of genes to influence cellular processes
- Interference with gene products
Commonly used vectors
Human B-globin locus
- Located on short arm of chromosome 11
- Multiple interactions between regulatory regions are required to stabalise an active chromatin hub
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
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
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
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
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
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
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
B-globin locus is too big for common viral vectors. What is used?
Herpes virus
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
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
Genome editing sequence-specific nucleases
Genome editing tools:
- Recognise specific DNA sequences
- Cut DNA then a scar is left behind
e.g. zinc finger nuclease, TAL effector nuclease, CRISPR-associated nuclease
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
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:
- Double knockout - same or different chromosomes
- Large deletion - hundred kb deletion
- Gene modification
- Gene integration
