Brainscape's Knowledge GenomeTM


Top Flashcards (Certified)
All Flashcards

w Molecular Biology of Disease > 1. Cystic fibrosis and gene therapy > Flashcards

1. Cystic fibrosis and gene therapy Flashcards

(60 cards)

Start Studying
1
Q

Define cystic fibrosis

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What does CF affect?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What are the common treatmetn options for CF?

A

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)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Does CF affect life expectancy?

A

Still yes - but improvement in last 50 years by introducing more treatments:
- antibiotics
- physiotherapy
- DNase
- newborn screening for CF
- channel modifiers for CF

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Explain population genetics of CF

A

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 well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

How do CFTR mutations affect normal phenotype?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Does CF have a single phenotype?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What are the two effective drugs for CF? What classes of CFTR mutations do they target?

A
  • Ivacaftor - effective for Class III - helps CFTR open/close regulation
  • Trikafta - effective for Class II - helps CFTR maturation
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Explain Class II CFTR mutation CF

A
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What kinds of base mutations are common in CF?

A
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What is lung disease in CF?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What are the problems caused by CFTR mutations?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Explain low volume hypothesis by Ric Boucher

A

The Low Volume Hypothesis by Ric Boucher - dehydration of the airway surface liquid (ASL) -> defective mucus clearance

  1. Defective CFTR doesn’t export Cl-
  2. Sodium import protein imports more Na+
  3. Ionic imbalance forces more water out of cells than normal
    => loss of water - low volume
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What is periciliary liquid?

A

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 well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

How is PCL affected in CF?

A

Periciliary liquid (PCL) is defected in CF - depleted of PCL + hyperviscous mucus because CF inhibits mucus transport

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Explain ionic imbalance in CF?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Explain the pathogenesis cascade of CF lung disease

A

Defective CFTR -> deficient CFTR protein -> abnormal Cl- permeability in cells -> decreased water in ASL -> bronchial obstruction -> bacterial infections -> inflammation -> bronchiectasis + lung insuffiency => death

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

What are the models used for studying CF?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

What are the pros and cons of using mice CF models?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

What are the pros and cons of using pig CF models?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

Which animal is the best model for CF?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

Do all CFTR mutations cause severe CF phenotypes?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

What are the two types of gene therapies?

A

Gene therapies:
- indirect (ex vivo) - cell manipulation in vitro + transplantation
- direct (in vivo) - gene injection straight into organ

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

How can gene therapy be topically delivered?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Approximately what promortion of CF is asymptomatic?
50% symptomless
26
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
27
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**
28
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
29
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)
30
What is vector pseudotyping?
Gene therapy **vector pseudotyping** - **modifying the viral surface proteins** to **enhance** its ability to specific cells or tissue **targeting**
31
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
32
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**
33
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**
34
Comparison of gene vectors in CF
35
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**
36
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**
37
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**
38
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**
39
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
40
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**
41
What are the clinical trial stages in approving CF gene therapy?
1. **Nose**: spray gene and liposome into nose - test nose for safety and correction of CF 2. **Lungs**: spray gene and liposome into lungs - test lungs for safety and CF correction 3. **Long-term clinical trials** to prove safety and clinical benefit
42
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
43
What is the process of developing a gene therapy?
44
What has been the best gene therapy vector developed so far?
read more on rSIV trial with rSIV
45
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
46
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**
47
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 b**one 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
48
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
49
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
50
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
51
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
52
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
53
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
54
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**
55
Explain how gene therapy is performed using re-implantation of stem cells
56
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)
57
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
58
Explain how gene therapy attempted to treat spinal muscular atrophy (SMA)
**Spinal muscular atrophy (SMA) ** - l**oss 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
59
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**
60
Explain Strimvelis gene therapy
**Strimvelis** - **gene therapy for ADA-SCID** - **gammaretroviral** therapy administered in **haemotopoietic stem cells**

w Molecular Biology of Disease (8 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2025 Bold Learning Solutions. Terms and Conditions