1. Cystic fibrosis and gene therapy Flashcards
Define cystic fibrosis
Cystic fibrosis - a hereditary exocrine gland disease characterized by secretion of sticky, thick mucus which leads to chronic lung infection and inability to absorb nutrients from food
What does CF affect?
CF is a multiorgan disease - compromises function of many organs by covering them with thick mucus
Effects:
- lung susceptibility to infection
- abnormal sweat - Na+, Cl- ions -> saltier sweat
- osteoporosis
- male infertility
- chronic sinusitis
- intestinal blockage
What are the common treatmetn options for CF?
CF commonly treated by:
- antibiotics
- physiotherapy to physically remove mucus from the lungs
- DNase - mucus viscosity created by DNAs - digest them
- small molecule modulators (for a few genotypes of CF)
- lung transplant (last resort) - but mucus will still be produced in other organs (???) - read
–> gene therapy (still experimental)
Does CF affect life expectancy?
Still yes - but improvement in last 50 years by introducing more treatments:
- antibiotics
- physiotherapy
- DNase
- newborn screening for CF
- channel modifiers for CF
Explain population genetics of CF
Mostly affected by CF
- Caucasian descent
- autosomal recessive - need two carriers to create a homoz. recessive affected child
- all mutations linked to chr7
- Cystic fibrosis transmembrane conductance regulator (CFTR) gene affected by mutations
How do CFTR mutations affect normal phenotype?
CFTR produces a channel protein in cells for transporting ions - mainly Cl- - has 2 transmembrane domains, 2 nucleotide ATP binding domains - regulates how much the pore opens for ions transport into/out of cell
Does CF have a single phenotype?
No, one CFTR gene effected but several different mutations of CFTR - affect CFTR structure and function - DeltaF508 most common mutation
5 main CFTR mutation phenotypic classes:
I: affects CFTR synthesis (degraded)
II: affects CFTR maturation (post-translational modification)
III: affects CFTR regulation open/close
IV: affects CFTR conductance (structural changes in the pore)
V: affects CFTR quantity in cells
What are the two effective drugs for CF? What classes of CFTR mutations do they target?
- Ivacaftor - effective for Class III - helps CFTR open/close regulation
- Trikafta - effective for Class II - helps CFTR maturation
Explain Class II CFTR mutation CF
What kinds of base mutations are common in CF?
What is lung disease in CF?
Lung disease - chronic lung infection caused by CF - defective CFTR protein causes reduced Cl- secretion and increased Na+ absorption ->
- thick, sticky mucus in the airways
- impaired mucociliary clearance (the ability to clear mucus and trapped particles from the airways) -> clogs the airways -> environment conducive to chronic bacterial infections => lung related problems -> death
Opportunistic lung infection
Most common - Pseudomonas auruginosa
Can be infected by fungi, viruses, bacteria
What are the problems caused by CFTR mutations?
CFTR mutations cause:
- fluid imbalance:
low volume hypothesis by Ric Bouche, reduced water
- ionic imbalance:
pH & salt-sensitive anti-bacterial activities - defensins, lysozyme
- defective bicarbonate secretion
- defective mucus production
- pro-inflammatory state in CF:
chronic neutrophil infiltration, imbalance of cytokines
Explain low volume hypothesis by Ric Boucher
The Low Volume Hypothesis by Ric Boucher - dehydration of the airway surface liquid (ASL) -> defective mucus clearance
- Defective CFTR doesn’t export Cl-
- Sodium import protein imports more Na+
- Ionic imbalance forces more water out of cells than normal
=> loss of water - low volume
What is periciliary liquid?
The periciliary liquid (PCL) is a thin, watery layer of fluid that surrounds the cilia - part of the airway surface liquid (ASL), which plays a crucial role in the mucociliary clearance system of the respiratory tract
How is PCL affected in CF?
Periciliary liquid (PCL) is defected in CF - depleted of PCL + hyperviscous mucus because CF inhibits mucus transport
Explain ionic imbalance in CF?
Ionic imbalance -> reduced airway surface pH (more acidic) impairs bacterial killing
Experiment: placed bacteria in normal / CF pigs -> CF struggled in clearing + samples found CF trachea to be more acidic
Explain the pathogenesis cascade of CF lung disease
Defective CFTR -> deficient CFTR protein -> abnormal Cl- permeability in cells -> decreased water in ASL -> bronchial obstruction -> bacterial infections -> inflammation -> bronchiectasis + lung insuffiency => death
What are the models used for studying CF?
CF animals:
- CF mice - but don’t resemble human well
- CF pigs - resemble humans quite well
- CF ferret
- CF rat
- CF rabbit
- CF sheep - resemble humans really well but not widely accessible
What are the pros and cons of using mice CF models?
CF mice
Pros:
- genetics: variety of CFTR nulls / homologous of human alleles
- effects: many molecular level effects similar to humans
- gut phenotype also observed as in humans
Cons:
- CF more severe - causes death more easily
- don’t form spontaneous lung disease
=> limits usefulness of CF mice as models - rodents overall not the best model for CF
What are the pros and cons of using pig CF models?
CF pigs
Pros:
- spontaneously develop lung disease
- severe gut phenotype
- underdeveloped at birth
Cons:
- large for big studies - high cost
- high neonatal mortality
- some species differences still present
Which animal is the best model for CF?
Sheep - CFTR 90% similar to humans - similar in lung development, physiology and pathology
Normal sheep models also help but can’t replace CF models
-> but not widely availbale yet for research
Do all CFTR mutations cause severe CF phenotypes?
No, not all CFTR cause severe CF, can also be asymptomatic
Genotype / phenotype correlations seen - CFTR mutations and splice variants recognised to be strongly asscoiated with distinct clinical diagnoses - some CFTR forms not strong enough to cause lung disease but can affect health - infertility, sinusitis
Other genetic background also comes into play
What are the two types of gene therapies?
Gene therapies:
- indirect (ex vivo) - cell manipulation in vitro + transplantation
- direct (in vivo) - gene injection straight into organ
How can gene therapy be topically delivered?
Topical delivery - deliver the drug to the skin surface or mucous membranes
In CF gene therapy can be topically delivered by aerosoll to lungs - breath in
What are the possible vectors for gene transfer?
Vectors for gene transfer in gene therapy:
- adeno-associated virus (AAV)
- adenovirus (Ad)
- Lentivirus (LV) -> Sendai-pseudotyped SIV
- Lipoplex
What is vector pseudotyping?
Gene therapy vector pseudotyping - modifying the viral surface proteins to enhance its ability to specific cells or tissue targeting
How can a gene therapy vector be improved to target specific cells and tissues?
Can change vector surface proteins - pseudotyping
Ex. pseudotyping lentivirus for CFTR gene transfer - mixing surface proteins of letivirus and sendai virus - Sendai-Pseudotyped SIV -> long-lived transgene expression + efficient airway cell tropism
Explain what are lipoplexes
Lipoplexes - DNA + lipids - used in gene therapy as a vector
Created by mixing cationic lipid and therapeutic DNA -> spontaneously form lipoplexes
Comparison of gene vectors in CF
What are the biological barriers to CF gene therapy?
In order to deliver gene therapy agents must:
- penetrate mucus layer and survive defence / clearance mechanisms
- reach target cells
- cross extra-cell membrane and** exit endosome**
- transverse cytoplasm and translocate to the nucleus
If in gene therapy delivery vectors come across a barrier such as - that receptors are usually on basolateral surface - how to overcome such barrier
A barrier that target cell receptors are on the basolateral surface of the cell can be overcome by:
1. delivering vector from basolateral surface
2. transiently disrupting tigh junctions
3. engineering a new receptor on apical membrane
4. developing new formulations for the vector
5. engineeing new ligands into the vector
If in gene therapy delivery vectors come across a barrier such as - that the persistence of introduced CFTR expression is low - how to overcome such barrier
A barrier that introduced CFTR expression persistence is low - promoter activity decreases, transgene is lost, immune response may destroy transduced cells - can be overcome by:
- develop stable promoters
- stabilise episomal DNA
- eliminate or circumvent immune response (leaky viral transcription, vector DNA, input vector protein)
- develop integrating vectors
- CRISPR/Cas9
Why is gene therapy bothering with gene vectors instead of using CRISPR/Cas9 directly?
CRISPR/Cas9 is good for embryo/zygotic editing but not efficient in vivo - homologous recombination is inefficient in differentiated cells - better for it:
=> base editing
=> prime editing
Explain base and prime editing
Base editing -
Prime editing -
They are better in gene therapy differentiated cell editing that CRISPR/Cas9 because …
Also questions in CRISPR lecture
What is the process of developing a gene therapy?
What has been the best gene therapy vector developed so far?
read more on rSIV trial with rSIV
Explain somatic gene therapy
Somatic gene therapy - medical intervention designed to treat / prevent disease by introducing, removing, or altering genetic material within the somatic cells of a patient - affects only non-reproductive cells
What is the “Berlin patient” and how did the trials cure HIV?
Somatic gene therapy:
permanently cleared HIV in the “Berlin patient”: CCR5-/CCR5- genotype bone marrow donor - transplanted into the patient - CCR5 is a co-receptor used by HIV to infect immune cells - new immune cells of the Berlin patient didn’t have CCR5 - HIV couldn’t infect -> cured HIV
Berlin case - left approach - transplantation
What aspects of regulatory issues are worth considering before developing somatic gene therapy?
Worth considering in regulatory issues depending on the country - different regulations in different countries - UK / EU / US
Explain how SCID gene therapy works
SCID-X1 gene therapy:
SCID-X1 boys (X-linked) produce no fucntional gamma-c chains in immune cells -> have no immunity -> gene therapy aims to replace the gene in bone marrow cells
Explain how gene therapy is performed using re-implantation of stem cells
Explain what an adverse effect has resulted from Fischer gene therapy for SCID-XI trial
SCID-X1 gene therapy activated an oncogene in WBCs -> leukemia developed
=> major efforts to improve safety of rertroviral vectors -> self-inactivating (SIN) vectors developed - have no promoter in terminal repeats (LTRs) - would not turn on oncogene expression -> in addition using lentiviral vectors - intrinsicly safer than gammaretroviral (LTR driven)
Explain how LCA/RPE65 gene therapy helps treat blindness
Mutation of RPE65 gene in retina -
gene therapy by AAV expressing functional RPE65 - injected into retina - helps alleviate blindness but doesn’t fix retinal degeneration
Explain Strimvelis gene therapy
Strimvelis - gene therapy for ADA-SCID - gammaretroviral therapy administered in haemotopoietic stem cells
