L17 Gene Therapy Flashcards
Gene Therapy
Transfer of genetic material into cells or tissue to either prevent or cure disease
Commonly used vectors
see onenote
- retrovirus/lentivirus
- adenovirus
- adeno-associated
- naked DNA/liposomes
Categories of inherited genetic disorders
see onenote
- recessive
- x-linked
- mitochondrial
- dominant
Hb molecule
see onenote
- alpha chain x 2
- beta chain x 2
- iron
- heme group
helical shape
beta-haemoglobin disorders
see oneonte
caused by mutations in beta-gene locus
beta-thal
see onenote
mutations resulting in low or absent beta-globin synthesis
- excess of free alpha-globin chains
- anaemia
- splenomegaly
- marrow expansion
Sickle cell disease (SCD)
see onenote slides
Glutamate => valine
- vaso-occlusive crises
- when the microcirculation is obstructed by sickled RBCs, causing ischemic injury to the organ supplied and resultant pain - haemolysis
- acute chronic organi damage
Polymerises in deoxygenated state => changes structure of cell into sickle shape, blocks the capillaries?
Thalassaemia-related costs
see onenote
blood transfusions
>$200,000 per year per patient
Treatment options
see onenote
- blood transfusions
- iron chelation
- bone marrow/stem cell transplants
- gene therapy
Successful gene therapy in mice
see onenote
Gene therapy
- Viral vectors to deliver functional beta globin gene into stem cell of mice, to establish efficacy
- Was able to demonstrate correction of beta thalassemia and sickle cell anaemia in mice
- Human beta globin gene inserted in the reverse orientation to prevent splicing
Lentiviral gene therapy vector for treatment of beta-thal
see onenote
If truncate large LCR into micro LCR and add beta globin gene and beta globin promoter to it => gene therapeutic vector
Lentiviral vectors are based on lentiviruses
see onenote
To produce lentiviral particles, need 3 plasmid components:
- transfer vector containing transgene and flanking LTRs
- packing vector or set of packaging plasmids
- envelope vector
Producing lentiviral particles
see onenote
The LV vector itself is the only genetic material transferred to the target cells
- comprises the transgene cassette flanked by cis-acting elements necessary for encapsidation, reverse transcription and integraiton
Success of gene therapy based on
- efficient gene transfer into target cells
- adequate levels of transgene expression
- persistence of gene expression
- regulation of gene expression
- tolerance of transgene product
- safety
HSC are targets cell or gene therapy
see onenote
HSC - self renewing stem cells
- Can harvest from bone marrow
- Can differentiate into other types of blood cells
Gene therapy of beta-thal
see onenote
gene transfer of beta-globin gene in HSC would reduce imbalance between alpha and beta globin chains in erythroid cells
Paul Beauchesne
see onenote
underwent gene therapy for beta-gal
long term transfusion independence 1 year after transplant
Lentiviral vector design with marked beta-globin gene
see onenote
Beta globin gene has been modified
- Contains T => Q aa modification, prevents sickling from taking place, inhibit sickling
HPLC
- Can separate individual globin chains
Concentration of Hb in blood
see onenote
Inhibition of physiological HMGA2 regulation following random LV integration
see onenote
overexpression of HMGA2 protein (which has been linked to cancer), detected in high proportion of genetically modified cells
BB305 lentiviral vector in subsequent studies
see onenote
- improvements made in vector design
BB305 lentiviral vector in clinical studies
Other lentiviral vector in clinical studies
see onenote
Rapid and sustained transfusion independence in patients with non-beta0/beta0 genotypes
see onenote
X-SCID
see onenote
severe combined immunodeficiency, “bubble boy”
Lentiviral vectors
- Also used to treat disorders other than blood disease e.g. immunodeficiency diseases
Rhys Evans
see onenote
Was diagnosed with disorder that effects boys, known as x-linked SCID
Able to cure immunodeficiency
- Lentiviral vector able to deliver functional gene, allows functional development of t-cells
Gene therapy treatment for Haemophilia
see onenote
Blood clotting
- Deficient blood clotting factors
- Used adeno associated virus, vector carrying the clotting factor can be injected into the liver - liver able to produce therapeutic protein and prevent bleeding disorder for the rest of their life
- Long term efficacy
Gene therapy to treat sight disorder
see onenote
Lack of RPE65 causes leber’s congenital amaurosis
AAV carrying normal copy of RPE65 gene is injected into retina, allowing photoreceptor cells to detect light
Gene therapy pricing
see onenote slides
Cost
- Very expensive to treat rare disorders
- Is it cost effective?
- LPLD trial withdrawn due to lack of demand and its high expense
- Needs a high demand
Gene therapy vectors
see onenote
retroviral/lentiviral the most popular vehicle
- accept relatively short DNA sequences
- gene expression may be inappropriate regulated
- susceptible to transcriptional silencing
- random integration associated with genotoxicity
Integration at defined chromosome location
see onenote
Using site specific feature of adeno-associated virus
- Integrate into AAVS1 site on human chromosome 19
- AAVS1 has characteristics that make it an ideal target for somatic gene therapy
Site-specific integration
Adeno-associated virus (AAV)
see onenote
- AAV is a non-pathogenic human parvovirus
- evolved a unique ability to integrate specifically at a defined site, AAVS1 on human chromosome 19
- the 4.7kb single-stranded linear DNA genome consists of short ITR required for replication, packaging and site-specific integration
WT and recombinant adeno-associated viruses
see onenote
Without rep
- Free roaming in the nucleus
With rep
- Able to integrate into AAVS1
Rep-based site specific integration into AAVS1 site
see onenote
If we can harness genetic info from ITR and transplant into gene therapy vector system
- Deliver rep protein together with ITR
- Rep protein can recognise ITR, facilitates gene vector interaction with AASV1 site - promote site specific integration
Fluorescence in situ hybridisation (FISH) used to follow integration
see onenote slides
Able to identify site specific integration of 20kb vector
Targeted genome editing?
see onenote slides
Ultimate goal: change mutated DNA sequence back to normal sequence and treat disease
Evolution of genome editing tools
see onenote
Sequence specific nucleases
- ZFN, TALEN, CRISP-Cas system
- recognises specific DNA sequences
- cut DNA (designer nucleases) then a scar is left behind
- this scar is what allows permanent modification of the genome
Genome modification using repair of DS breaks
see onenote
NHEJ - indels
Homologous template - induce recombination
Bacterial acquired immunity against phage infection
see onenote
CRISPR/Cas9
see onenote slides
Cas9 = reprogrammable RNA dependent restriction enzymes
- Cas9 protein
- crispr RNA, homologous to the target site
- trans-activating crisprRNA, recruits the nuclease complex
gRNA
- 20 bases + PAM (NGG)
Genome editing - GFP to BFP
see onenote
