L10, Gene therapy

Question 1

Gene therapy definition:

Answer 1

• 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

Question 2

4 Key groups of targets for gene therapy:

Include examples for each

Answer 2

• 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)

Question 3

Gene therapies of cancer:

Answer 3

• Mostly CAR-T
• Largest portion of gene therapies in use

Question 4

In vivo gene therapy:

Answer 4

• Single step
• Vector administered (injected or inhaled) directly to patient, targeted specifically to organ or tissue
• Targeting somatic cells only

Question 5

Ex vivo gene therapy:

Answer 5

• 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

Question 6

Discuss the processes and relevant barriers to gene therapy:

Answer 6

• 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)

Question 7

List possible vectors for gene therapy with examples where relevant:

Answer 7

• 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)

Question 8

Sites for in vivo gene therapy:

Answer 8

• Accessible organs: lungs, skin, muscles
• Inaccessible: liver, retina, brain

Question 9

Vectors and key targets for treatment (in vivo):

Answer 9

• Adenovirus, AAV, some use of retroviral vectors
• Single gene disorders and acquired disease

Question 10

Advantages and disadvantages of adenoviral vectors:

Answer 10

+

• 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

Question 11

Adeno-associated virus: Overview

Answer 11

• 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

Question 12

Leber’s congenital amaurosis (LCA):

Clinical features and molecular basis:

Answer 12

• 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

Question 13

Use of gene therapy for LCA:

Answer 13

• 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

Question 14

Ex-vivo gene therapy for SCID: Basic principle

Answer 14

• IL2RG or ADA gene
• Use f a gamma-retroviral vector
• Aiming for long-term reconstitution of lymphoid lineages

Question 14

Potential target organs for AAV vectors:

Answer 14

• 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)

Question 15

SCID gene therapy trials: ADA vs X-linked

Answer 15

• 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)

Question 16

Alternatives to gamma-retroviruses:

Answer 16

• 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)

Question 17

Targeted changes for gene therapy:

Answer 17

• 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

Question 18

+ 3 types of programmable nucleases for genome engineering:

Answer 18

• Zinc finger nucleases (ZFNs)
• Transcription-activator-like effector nucleases (TALENs)
• Cas9 RNA-guided engineered nucleases (RGENs)

Question 19

+ Outline the basic principle of genome engineering with programmable nucleases:

Answer 19

• DSBs induced -> repaired by homology-directed repair
• Leads to gene insertion, correction and point mutagenesis/NHEJ -> gene disruptions

Question 20

+ What is an alternative to programmable nucleases which induces single breaks:

Answer 20

• Programmable nickases -> single-strand breaks
• Repair of these leads to precise genome editing
• Can be used in pairs -> much more specific than nucleases