Gene Editing Flashcards
What is Gene therapy?
= the introduction (using a vector) of nucleic acids into cells with the intention of altering gene expression to prevent, halt or reverse a pathological process
= biggest barrier = COST
(extremely expensive, not always used very often)
What are some target disorders of gene therapy?
Single gene, recessive loss of functions
(e.g. cystic fibrosis, haemophilia)
= gene addition / replacement
Single gene, hapolinsufficiency
(e.g. DSH - dyschromatosis symmetrica hereditaria)
= gene addition
Single gene, dominant negative
(e.g. Huntington disease)
= allele silencing / replacement
Multi-gene or acquired
(e.g. cancer, heart disease, rheumatoid arthritis)
= addition of therapeutic gene
(acquired diseases are more attractive to drug companies)
What are the different gene therapy approaches?
In vivo
= single step process
= vector administered directly to patient
= targeted to specific organ/tissue
(route of administration or specificity of vector)
Ex vivo
= two step process
= cells removed from patient
= vector added to cells in vitro
= engineered cells returned to patient
= may be combined with (stem) cell-based therapy
= both approaches target somatic cells only
= no attempt to engineer the germ line to achieve a permanent heritable cure
(illegal , apart from mitochondrial DNA in 3 parent babies)
What are some barriers to gene therapy?
EXTRA READING
= Delivery
(use viral / non-viral vectors)
= Immune response
(need to reduce this)
= Off-target effects
(leads to new health problems)
= Cost
(v. expensive)
What are some of the vectors used for gene therapy?
= Adenovirus
= Adeno-associated virus
= γ-retrovirus
(e.g. Moloney Murine Leukaemia Virus-derived)
= Lentivirus
(e.g. HIV-derived)
= Routine plasmids
= Mini-circles
(similar to plasmids but without extra DNA)
= Transposons
(e.g. Sleeping beauty)
= need to overcome immunogenicity and genotoxicity
How is in vivo gene therapy used?
= difficult to deliver
= accessible organs: lungs (inhalation), skins (topical) , muscles (injection)
= less accessible organs: liver, retina, brain
= vectors used: adenovirus, adeno-associated virus, some use retroviral vectors
= treatment of single gene disorders and acquired diseases
What are some advantages vs disadvantages in Adenoviral vectors?
ADVANTAGES:
= large capacity: up to 30kb if helper virus supplied
= easily purified
= infect broad range of cell types
= efficient transduction
= potential vector for cancer treatment
(expression of anti-cancer proteins)
DISADVANTAGES:
= common cold virus - high incidence of neutralising antibodies
= capsid protein is highly immunogenic
= potentially fatal inflammatory response
(death of Jesse Gelsinger during OTC trial in 1999)
= transient expression of transgene
What is the Adeno-associated Virus (AAV)?
= small (4.7 kb ssDNA genome)
= non-pathoenic, minimal immune response
= rep + cap genes can be replaced with expression cassette
(limited capacity)
= can be used in non-dividing cells
(maintained as episome)
= different serotypes target different tissues
What is an example of an AAV case study?
= LCA (Leber’s congenital amaurosis)
= Amaurosis (darkening) = vision loss without obvious physical signs
= early-onset blindness
= autosomal recessive (14 genes inc. RPE65)
= RPE65 codes for retinal pigment epithelium-specific 65kDa protein
(required for photoreceptor function)
= photoreceptors persist in affected individuals
= vision restored in mouse and dog LCA models using AAV vectors containing RPE65
= successful phase II clinical trials
LCA gene therapy:
= AAV2 serotype capsids directly injected beneath the retina
= virus taken up by retinal epithelium
= RPE65 gene expressed from episomal vector
= light sensitivity restored + maintained for > 3yrs
= early intervention required for best results
What are some other AAV target organs?
Liver
= gene factory (plasma protein deficiencies)
= metabolic disorders
= haemophilia B (factor IX) clinical trials
(unsuccessful - due to immune response)
Muscle
= deliver by intramuscular injection
= gene factory: trials for
- haemophilia B
- α1 antitrypsin deficiency
- LPL deficiency (glybera)
= repair of muscle disorders - e.g. DMD
Brain
= immunoprivileged site
= BBB (AAV9 can cross BBB)
= trials for Parkinson’s disease, Canavan’s disease and Batten’s disease
What is ex vivo gene therapy?
Haematopoietic stem/precursor cells
= well established techniques for culture + transplantation
= treatment of single gene blood / immune disorders
(e.g. SCID, chronic granulomatous disease, thalassaemias)
Epidermal stem cells
Cardiac stem cells
Neural stem cells
= chromosome integration required for long-term transgene expression
What is ADA SCID gene therapy?
= γ-retrovirus vector
= 10 children treated
= ADA enzyme replacement therapy withdrawn
(ensures transduced cells have selective advantage)
= 9 patients had immune function restored
(no life-threatening opportunistic infections)
= cure appears permanent
(up to 8 years after treatment)
What is X-linked SCID gene therapy?
= γ-retrovirus vector
= 20 patients treated
= immune function restored in all
(BUT 5 patients developed leukaemia)
= insertion into LMO2 proto-oncogne activation acts synergistically with IL-2R
(to promote cell proliferation)
What are some problems with γ-retroviral vectors?
= preference for insertion near promoters of active genes
= strong enhancer and promoter in LTRs
(can activate nearby oncogenes)
= splice donor sites downstream of 5’ LTR
(can splice to exons of oncogenes)
= solve by using self-inactivating vectors = most of LTRs removed during integration
What are some alternatives to γ-retroviruses?
Lentiviruses
= LTRs lack strong enhancer
= self-inactivating vectors delete LTRs for additional safety
(clinical trials underway)
DNA vectors
(simple plasmids / minicircles)
= no pre-exisiting immunity
= high capacity
= ? integration via transposase
= delivery in vivo very difficult
= random integration still a problem
How are site-directed nucleases used in gene therapy?
= guide RNA designed to match sequence in faulty or abnormal gene
= Cas9 nuclease cleaves DNA
= normal gene
= corrected faulty gene
What are some barriers to gene therapy (within the cell)?
EXTRA READING
- Delivery to target cell
= several barriers to reach target cell
(extracellular matrix, cell membranes and endosomes)
= achieved using viral or non-viral vectors - Immune response
= immune system will recognise and eliminate viral vectors
= lead to reduced gene expression and potential adverse effects
= can cause inflammation + tissue damage - Gene expression
= therapeutic gene must be expressed at appropriate levels for therapeutic benefit
= expression affected by factors such as promoter used to drive gene expression, chromatin structure of target cell, stability of mRNA - Off-target effects
= gene therapy can cause off-target effects
= where therapeutic gene expressed in unintended cells = leads to adverse effects
= can happen due to non-specific delivery of gene - Safety concerns
= potential to cause unintended mutations or genetic abnormalities
= can lead to long-term safety concerns
