Lecture 10 Flashcards
What is gene therapy
- Introduction of nucleic acids into a cell
- Uses vectors
- Alters gene expression to prevent, halt or reverse pathological process
4 targets of gene therapy
- Gene addition/replacement - Used in single genes which lost function e.g. cystic fibrosis or haemophilia
- Gene addition for single gene haploinsufficiency e.g. DSH
- Allele silencing/replacement for dominant negative single gene e.g. HD
- Addition of therapeutic gene for multi-gene or acquired disorders e.g. cancer, heart disease, rheumatoid arthritis
Which diseases are gene therapy clinical trials mostly focused on?
Cancer e.g. leukaemia, lymphoma, multiple myeloma
Genetic disease e.g. DMD, Spinal muscular dystrophy
Infections e.g. HIV, COVID-19
genetic diseases types which gene therapy trials were focused on
Metabolic disease
Eye disease
Coagulation disease
Immunodeficiency
Neuromuscular disease
Haemoglobinopathy
Gene therapy approach comparsion
in vivo vs ex vivo
in vivo:
- Single step
- Vector administered to patient
- Targeted to specific organ
Ex vivo:
- Two steps
- Cells removed from patient
- Vector added in vitro
- Engineered cells returned to patient
- May be combined with stem cell based therapy
gene delivery
- Viral vector binds cell receptor
- Up taken by endocytosis
- Viral genome released from vector -> nucleus
- DNA remains episomal or integrates to host genome
- Transgene trasncrived to RNA and translated in cytoplasm
- Protein processed in golgi apparatus
- vector antigens presented on MHC molecules - recognised by T-cell receptors on cytotoxic T lymphocytes -> immune response
Give examples of vectors for gene therapy
- Adenovirus
- Adeno-associated virus
- y-Retrovirus
- Lentivirus
- Routine plasmids
- Mini circles
- Transposons
Problems with in vivo gene therapy
- Difficulty of delivery
- Accessible organs - lungs, skin, muscles
- less accessible: Liver, retina, brain
- uses adenovirus or adeno-associated virus
- Treats only single gene disorders or acquired disease
Advantages vs disadvantages of adenoviruses
Advantages:
- Large capacity: up to 30kb
- Easily purified
- Infects different cells
- Efficient transduction
- Vector for cancer treatment
Disadvantages:
- High incidence of neutralising antibodies
- Capsid protein immunogenic
- Fatal inflammatory response
- Transient expression of transgene
Adeno-associated virus
- Small (4.7kb ssDNA genome)
- Non-pathogenic
- rep and cap can be replaced with expression cassette
- used for non-dividing cells
- Different serotypes target different tissues
AAV case study
- Amaurosis - vision loss without signs
- Early-onset blindness
- Autosomal recessive (14 genes, including RPE65)
- Encodes for retinal pigment epithelium-specific protein (65kDa)
- Photoreceptors persist in affected individuals
- Vision restored in mouse/dog LCA models
- Successful phase II clinical trials
LCA gene therapy
- Three separate clinical trials
- AAV2 serotype capsids injected beneath retina
- Virus taken up to retinal epithelium
- RPE65 gene expressed from episomal vector
- Light sensitivity restored
- Early intervention required for best results
Other AAV target organs
Liver: gene factory, metabolic disorders, Haemophilia B clinical trial
Muscle: IV injection, trials for haemophilia B, α1 antitrypsin deficiency, LPL deficiency (Glybera), repair DMD muscles
Brain: Immunoprivilged site, AAV9 crosses BBB, Trails for Parkinson’s , Canavan’s, Batten’s
Stem cells for Ex vivo gene therapy
- Haemopoietic stem/precursor cells
- Epidermal stem cells
- Cardiac stem cells
- Neural stem cells
Ex vivo gene thrapy for SCID
Hematopoietic stem/progenitor cells (HSPCs) are isolated from the patient’s bone marrow.
These cells are genetically modified ex vivo using a γ-retroviral vector to correct defective genes.
The modified HSPCs are infused back into the patient after conditioning.
Before therapy, the patient lacks proper immune cell development, leading to immune deficiency.
After therapy, modified HSPCs restore normal blood and immune cell production, reconstituting immune function
ADA SCID gene therapy
- Gamma-retrovirus
- 10 kids
- ADA enzyme replacement therapy withdrawn
- 9 patients had immune function restored
- Cure permanent
X-linked SCID gene therapy
Gamma-retrovirus
20 patients
Immune function restored in all, but 5 developed leukaemia
- LMO2 proto-oncogene activation acts synergistically with IL-2R to promote cell proliferation
