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
Approximately what promortion of CF is asymptomatic?
50% symptomless
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 are the pros and cons of Adenovirus (Ad) in delivering gene therapy, ex. in CF?
Adenovirus in gene therapy as a vector:
- efficiently infects differentiated cells
- large DNA carrying capacity
- rapid onset of gene expression
But:
- short lived expression
- immune response
- limited re-administration - no expression after second dose - humans gains immunity
What are the pros and cons of Adeno-asscoiated virus (AVV) in delivering gene therapy, ex. in CF?
AVV in gene therapy as a vector:
- serotybe flexibility
- efficiently infects differentiated cells
- stable, long lasting gene expression
But:
- small cloning capacity - CFTR cDNA can only fit using special tricks
- high cost of production
- exposure to WT AVVs - immunity against
What are the pros and cons of Lentivirus (LV) in delivering gene therapy, ex. in CF?
LV as a vector in gene therapy delivery:
- repeated delivery possible
- pseudotype confirms tropism for airway epithelium
But:
- stable integration raises genotoxicity concerns
- public fear of vector based on a deadly virus (ex. HIV)
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
What are the pros and cons of DNA/liposomes in delivering gene therapy, ex. in CF?
DNA/liposomes - lipoplexes - can be used in delivering gene therapy
Pros:
- liposomes molecularly and chemically defined - known what is being introduced
- repeated gene delivery possible
Cons:
- gene delivery inefficient
- short-lived gene expression
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 parameters are measured in deciding if CFTR gene therapy has been successful?
To determine if CFTR gene therapy has been successful - check:
- **CFTR protein **- immuno, ion channel activity
- mucus rheology, mucociliary clearance
- inflammation and infection
- anti-bacterial activity
- lung function
What are the clinical trial stages in approving CF gene therapy?
- Nose: spray gene and liposome into nose - test nose for safety and correction of CF
- Lungs: spray gene and liposome into lungs - test lungs for safety and CF correction
- Long-term clinical trials to prove safety and clinical benefit
What is the panel which works on improving CF gene therapy?
UK CF Gene Therapy Consortium - formed after first CF gene therapy clinical trial in 2000 (no clinical benefits were found) - consortium share core facilities, techniques, data and strategy for better therapy development
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
Is gene therapy the best approach for CF?
Gene therapy is feasible but high efficiency is challenging - small molecule drugs:
- are effective
- easy to take
But need new one for each subtype of CF
Gene therapy remains a major contender:
- would work in all CF types
But delivery is tricky and would only work for lungs (?? why)
=> combination approach might be best
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
Which gene therapy vectors integrate into the human genome upon gene therapy?
Lentiviruses
Retroviruses
During this process, the vectors integrate into the genome of the target cell, unlike AAVs and adenoviral vectors. The ability to integrate into the cell genome makes lentiviral and retroviral vectors best suited for dividing cells, which are targets of an ex vivo treatment approach
What aspects in the nature of disease are worth considering before developing somatic gene therapy?
Consider nature of disease before creating somatic gene therapy for a genetic disease:
- inherited (monogenic) / acquired (multifactorial)
- genotype of the disease - dominant / recessive
- cell types affected
- onset of diagnosis
- severity
- alternative treatments
- genetic model systems for R&D
What is worth considering before developing somatic gene therapy?
Consider before creating somatic gene therapy for a genetic disease:
- nature of the disease
- vector development
- regulatory issues in therapy development
What aspects in vector development are worth considering before developing somatic gene therapy?
In vector development it is important to consider:
- choice of vector: viral / non-viral / naked DNA
- model of delivery: in vivo / ex vivo / oral, aerosol, direction injection, implantation
- therapeutic dose: single / multiple dose, on off, life-long (provide effect for the duration of life), life-time (given once in lifetime, but it may not last for the entire life)
- preclinical evaluation: in vitro / in vivo / genetic model systems, scale-up to clinical quantities of reagents
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
What are the monogenic diseases already targeted by gene therapy?
Diseases already targeted by gene therapy:
- X-linked severe combined immune defficiency (SCID): ex vivo, retrovirus, haemotopoietic stem cells
- Haemophilia A and B: ex vivo, retrovirus, haemotopoietic stem cells
- Leber’s congenital amaurosis: in vivo, AAV, retina
- Duchenne muscular dystrophy (DMD): in vivo, adenovirus, adenoassociated virus, liposomes, direct DNA injection, dekeltal and cardia muscle
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 what are SIN vectors
Self-inavctivating (SIN) vectors used in gene therapy - prevents unwanted gene (ex. oncogene) activation - safer
Read more about SIN vectors
Explain how gene therapy attempted to treat spinal muscular atrophy (SMA)
Spinal muscular atrophy (SMA) ** - loss of motor neurons** - progressive muscle wastage - SMN1 mutation
SMN2 is exon-skipped malfunctioning form of SN1 -> if SMN2 exon skipping would be inhibited - could partially compensate for SMN1 loss -> alleviate the disease progression
Spinraza gene therapy developed to change exon skipping in SMN2 - even if SMN1 missing - at least some function would be performed by expressed SMN2
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
