1. Cystic fibrosis and gene therapy Flashcards
Define cystic fibrosis
Cystic fibrosis - a herediatry 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 - affects the function of many organs by covering them with thick mucus
- 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)
–> 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
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 potential drugs for CF? What classes of CFTR mutations do they target?
- Ivacaftor - effective for Class II - 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) -> hindered 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:
- variety of CFTR nulls / homologous of human alleles
- 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 cheep models also help but can’t replace CF models
Do all CFTR mutations cause severe CF phenotypes?
No, not all CFTR cause severe CF, can also be asymptomatic
Genotype / phenotype correlations seen - CFTR mutatiosn 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)
- 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 speudotyping - modifying the viral surface proteins to enhance its ability to target specific cells or tissues
How can a gene therapy cetor be improved to target specific cells and tissues?
Can change vector surface proteins - pseudotyping
Ex. pseutyping 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 mcuus layer and survive defence / clearance mechanisms
- reach target cells
- cross extra-cell membrane and exit endosome
- traverse cytoplasm and trnaslocate 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 rpomoters
- 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 us 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 …
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 consdiering in regulatory issues depending on the country - differetn regulations in different countries - UK / EU / US
Explain how SCID gene therapy works
SCID-X1 gene therapy:
SCIF-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 imporve 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
