L10, Gene therapy Flashcards
1
Q
Gene therapy definition:
A
- 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
2
Q
4 Key groups of targets for gene therapy:
Include examples for each
A
- Single gene recessive LOF -> Gene addition or replacement (e.g. CF, haemophilia)
- Single gene haploinsufficiency -> Gene addition (e.g. DSH)
- Single gene dominant negative -> allele silencing or replacement (e.g. HD)
- Multi-gene or acquired -> addition of therapeutic gene (e.g. cancer, heart disease, rheumatoid arthritis)
3
Q
Gene therapies of cancer:
A
- Mostly CAR-T
- Largest portion of gene therapies in use
4
Q
In vivo gene therapy:
A
- Single step
- Vector administered (injected or inhaled) directly to patient, targeted specifically to organ or tissue
- Targeting somatic cells only
5
Q
Ex vivo gene therapy:
A
- Two steps
- Cells removed from patient, vector added to cells in vitro then engineered cells returned to patient
- May be combined with (stem) cell-based therapy
- Targeting somatic cells
6
Q
Discuss the processes and relevant barriers to gene therapy:
A
- Circulating antibodies target the vector for immune destruction -> often a basis for exclusion from trials
- Uptake into cells requires expression of specific receptors
- Transport into nucleus
- Must replicate either by integrating into host DNA forming an episome for independent replication
- Transcript must be processed to produce a protein; epigenetic effects can prevent this (e.g. insertion into heterochromatic region) -> no control over where vector inserts into genome
- If the protein is unrecognised by the immune system, it will be attacked (e.g. in MD if patient totally lacks dystrophin)
7
Q
List possible vectors for gene therapy with examples where relevant:
A
- Adenovirus
- Adeno-associated virus
- Gamma-retrovirus (e.g. Moloney murine leukaemia virus-derived)
- Lentivirus (e.g. HIV-derived)
- Routine plasmids
- Mini circles
- Transposons (e.g. sleeping beauty)
8
Q
Sites for in vivo gene therapy:
A
- Accessible organs: lungs, skin, muscles
- Inaccessible: liver, retina, brain
9
Q
Vectors and key targets for treatment (in vivo):
A
- Adenovirus, AAV, some use of retroviral vectors
- Single gene disorders and acquired disease
10
Q
Advantages and disadvantages of adenoviral vectors:
A
+
- Large capacity
- Easily purified
- Infect broad range of cell types
- Efficient transduction
-
- High incidence of neutralising antibodies (common cold)
- Capsid protein is highly immunogenic
- Potentially fatal inflammatory response
- Transient expression of transgene
11
Q
Adeno-associated virus: Overview
A
- Small
- Non-pathogenic, minimal immune response
- rep and cap genes can be replaced with expression cassettes (limited capacity)
- Can be used in non dividing cells (maintained as episome)
- Different serotypes target different tissues
12
Q
Leber’s congenital amaurosis (LCA):
Clinical features and molecular basis:
A
- Amaurosis: vision loss without obvious physical signs
- Early onset blindness
- Autosomal recessive (14 genes including RPE65)
- RPE65 codes for retinal pigment epithelium-specific 65 kDa protein -> required for photoreceptor function
13
Q
Use of gene therapy for LCA:
A
- Vision restored in mouse and dog LCA models using AAV vectors containing RPE65
- Successful phase II clinical trials
- Subretinal injection -> AAV2 serotype capsids -> virus taken up by retinal epithelium
- -> RPE65 gene expressed from episomal vector -> light sensitivity restored, maintained for >3 yrs
14
Q
Ex-vivo gene therapy for SCID: Basic principle
A
- IL2RG or ADA gene
- Use f a gamma-retroviral vector
- Aiming for long-term reconstitution of lymphoid lineages
14
Q
Potential target organs for AAV vectors:
A
- Liver: Gene factory, metabolic disorders
- Muscle (gene factory; trials for haemophilia B, alpha antitrypsin deficiency, LPL deficiency
- Repair of muscle disorders (DMD)
- Brain (immunoprivileged site, BBB, trials for Parkinson’s disease, Canavan’s disease, Batten’s disease)
15
Q
SCID gene therapy trials: ADA vs X-linked
A
- gamma-retrovirus vectors
- ADA enzyme replacement therapy withdrawn ensuring transduced cells have selective advantage -> 9 out of 10 had apparently permanent curative effect
- X-linked therapy, immune function restored in all but 5 out of 20 developed leukaemia (insertion into LMO2 proto-oncogene -> activation acts synergistically with IL-2R to promote cell proliferation)
- Issue: gamma-retroviruses tend to insert near promoters (LTR: Enhancer activity for nearby promoters)
16
Q
Alternatives to gamma-retroviruses:
A
- Lentiviruses (LTRs lack strong enhancer; self-inactivating vectors delete LTRs for additional safety)
- DNA vectors (simple plasmids/minicircles; no pre-existing immunity, high capacity but delivery in vivo is very difficult)
17
Q
Targeted changes for gene therapy:
A
- Programmeable nucleases to recognise specific site (e.g. CRISPR-Cas9)
- DSBs induced-> NHEJ leads to gene disruption
- Gene editing or replacement by homology directed repair is more efficient, with staggered cuts
- Challenging to avoid apoptosis, off-target mutations, optimal vector design, need for DNA replication for HDR
18
Q
+ 3 types of programmable nucleases for genome engineering:
A
- Zinc finger nucleases (ZFNs)
- Transcription-activator-like effector nucleases (TALENs)
- Cas9 RNA-guided engineered nucleases (RGENs)
19
Q
+ Outline the basic principle of genome engineering with programmable nucleases:
A
- DSBs induced -> repaired by homology-directed repair
- Leads to gene insertion, correction and point mutagenesis/NHEJ -> gene disruptions
20
Q
+ What is an alternative to programmable nucleases which induces single breaks:
A
- Programmable nickases -> single-strand breaks
- Repair of these leads to precise genome editing
- Can be used in pairs -> much more specific than nucleases
