Gene therapy Flashcards
(27 cards)
Uncommon genetic disorders that cannot be cured
Cystic fibrosis
Sickle cell anaemia
Thalassaemia
Duchenne Muscular Dystrophy
Haemophilia A
Familial hypercholesterolaemia
Phenylketonuria
Tay-Sachs disease
Targets of gene therapy
Single gene, recessive loss of function - Cystic fibrosis, haemophilia - Therapy would be gene addition/replacement
Single gene, haploinsufficiency - Dyschromatosis Symmetrica Hereditaria (DSH) - Gene addition
Single gene, dominant negative - Huntington disease - allele silencing/replacement
Multi-gene or acquired - Cancer, heart disease, rheumatoid arthritis - Addition of therapeutic gene
What rescued chronic non-ischemic heart failure in minipigs?
Cardiac bridging integrator 1 gene therapy
Gene therapy is used to treat…
- Mostly cancer
- Also genetic disease, infection etc
- Treatment been used to treat haemophilia, HIV, multiple myeloma
What types of genetic diseases are treated using gene therapy
Metabolic disease, eye disease, blood coagulation disorders, immunodeficiency, neuromuscular disease, haemoglobinpathy
Types of gene therapy
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
Barriers to gene therapy
- Neutralising antibodies bind to antigen on virus
- Uptake, transport and uncoating of virus
3.
Vectors for gene therapy
- Adenovirus
- Adeno-associated virus
- y-Retrovirus (e.g. Moloney Murine Leukaemia Virus-derived)
-Lentivirus (e.g. HIV derived)
- Routine plasmids
- Mini-circles
- Transposons
In vivo gene therapy pros/cons
- Difficulty of delivery
- Accessible organs - lungs, skin, muscles
- Less accessible - liver, retina, brain
- Vector - adenovirus, adeno-associated virus, some use of retroviral vectors
- Treatment of single gene disorders and acquired disease
What are the advantages of adenoviral vectors
- Large capacity - up to 30kb if helper virus provided
- Easily purified
- Infects broad range of cell types
- Efficient transduction
Disadvantages of adenoviral vectors
- Common cold virus - high incidence of neutralising antibodies
- Capsid proteins in highly immunogenic
- Potentially fatal inflammatory response (death of Jesse Gelsinger during OTC trail in 1999)
- Transient expression of transgene
Adeno-associated virus
- Limited capacity (4.7kb ssDNA genome)
- Non-pathogenic, minimal immune response
- rep and cap genes can be replaced with expression cassette
- Can be used in non-dividing cells - maintained as episome
- Different serotypes target different tissues
Successful AAV trails
Glybera (alipogene tiparvovec) - UniQure - EMA approved, 11-2-12 - targets LPL gene - Major indication is LPL deficiency
AAV case study – Leber’s congenital amaurosis (LCA)
Amaurosis (‘darkening’) is vision loss without obvious physical signs
Early-onset blindness
Autosomal recessive (14 genes, including 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
- Three seperate clinical trials
- AAV2 serotype capsids directly injected beneath retina
- Virus taken up by retinal epithelium
- RPE65 gene expressed from episomal vector
- Light sensitivity restored - maintained for >3 years
- Early intervention required for best results
Other AAV target organs - Liver
- Gene factory (plasma protein deficiencies)
- Metabolic disorders
- Haemophilia B (factor IX), clinical trials (unsuccessful - immune responses)
Other AAV target organs - muscle
Delivery by intramuscular injection
Gene factory: trials for haemophilia B, α1 antitrypsin deficiency, LPL deficiency (Glybera)
Repair of muscle disorders: Duchenne’s Muscular Dystrophy
Other AAV target organs - Brain
Immunoprivileged site
Blood Brain Barrier (AAV9 can cross BBB)
Trials for Parkinson’s disease, Canavan’s disease, Batten’s disease
Ex vivo gene therapy
Haematopoietic stem/precursor cells
- well established techniques for culture and transplantation
- treatment of single gene blood/immune disorders e.g. SCID, chronic granulomatous disease, thalassaemias
- engineered immune cells (e.g. to target cancer cells)
Epidermal stem cells
Cardiac stem cells
Neural stem cells
What is required for long-term transgene expression?
Chromosome integration
ADA SCID gene therapy
γ-Retrovirus vector
Ten children treated
ADA enzyme replacement therapy withdrawn (ensures transduced cells have selective advantage)
Nine patients had immune function restored – no life-threatening opportunistic infections
Cure appears permanent (up to 8 years after treatment)
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-oncogene – activation acts synergistically with IL-2R to promote cell proliferation
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 site downstream of 5’ LTR (can splice to exons of oncogenes)
Solve by using self-inactivating vectors – most of LTRs removed during integration
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-existing immunity
High capacity
Integration via transposase?
Delivery in vivo very difficult
