5.9 - Emerging Treatments Flashcards
1
Q
What are inborn errors of metabolism?
A
- largest group of genetic diseases
- affects various pathways including carbohydrate, fatty acid and protein metabolism
- generally caused by lack of enzyme in that pathway - means no product is formed which are vital for health
- also causes increase in substrate concentration which can cause disease symptoms
- also the substrate could be pushed to make an alternate product which can result in disease phenotype
2
Q
What are some examples of inborn errors of metabolism?
A
- PKU - phenylketonuria
- MCAD deficiency
- Maple Syrup Urine Disease
- homocystinuria
3
Q
What is PKU?
A
- phenylketonuria
- lack of phenylalanine hydroxylase which converts phenylalanine to tyrosine = less tyrosine, more phenylalanine
- this pushes phenylalanine down an alternate path to produce phenylketones which are toxic and cause disease symptoms
- untreated PKU symptoms include major cognitive impairment, behavioural difficulties, fairer skin/hair/eyes than siblings (due to lack of melanin) and recurrent vomiting
4
Q
How is PKU treated?
A
- with a low protein diet and tyrosine supplements
- 1934 - PKU identified but thought to be untreatable
- 1953 - lack of PAH identified as cause & role of dietary phenylalanine in disease found
- 1954 - low protein diet therapy introduced, to be effective needs to start before symptoms (as some are irreversible)
- 1960s - PKU screening introduced
- 1984 - PAH mutation identified
- before treatment, need to identify the cause (mutation in PAH) but not necessarily the gene involved
5
Q
What is haemophilia?
A
- blood clotting disorder, known since ancient times
- uncontrolled bleeding
- bleeding into joints - causes excruciating pain
- bleeding into brain
- internal bleeding
- fatal if untreated
6
Q
How is haemophilia treated?
A
- 1930s - diluted snake venom (contains clotting factors) used topically
- 1940s - whole blood transfusions (very large volumes required = painful)
- 1952 - factor VIII discovered
- 1955 - first infusions of factor VIII in plasma form (large volumes required)
- 1964 - cryoprecipitate discovered
- 1968 - first FVIII concentrate available
- 1970s - freeze-dried plasma-derived factor concentrates available
7
Q
What was the haemophilia treatment blood scandal?
A
- in 70s&80s, between 5k and 30k were given clotting factors contaminated with HIV and hepatitis, and so far 2500 have died from this
- there is evidence this contamination was known about at the time
- how was this solved?
- 80s - methods of heat treating to kill virus, FVIII gene cloned
- 90s - recombinant FVIII treatment, gets around the issue of blood donated FVIII
8
Q
What other diseases are treated by replacement?
A
- growth hormone deficiency - injection of growth hormone which is now recombinant (originally was cadaver-derived and caused patients to develop CJD)
- lysosomal storage disease which affects lysosomal breakdown of products in cell e.g:
- Fabry disease - injection of recombinant alpha galactosidase A
- Pompe disease - injection of alpha galactosidase
- these treatments are not mutation specific so can be given to any patient with the condition
9
Q
What are the stages of drug development?
A
- first stage - discovery/preclinical - testing in cells, longest part is lab-based
- then testing in animals
- moves on to clinical phase - 3 phases:
- phase I - check safety in healthy volunteers (<100)
- phase II - check therapeutic effect in few patients (100-300)
- phase III - large scale therapeutic trials (200-3000)
- approval by EMA (Europe) / FDA (America) needed
10
Q
How is drug approval for NHS done?
A
- by NICE in England & Wales
- by Healthcare Improvement Scotland in Scotland
- they also give guidelines for treating conditions
11
Q
What do pharmacological therapies targeting the protein do?
A
- treat the condition not the symptoms
- these are treatments not cures
- try to normalise function of mutant protein
12
Q
What are pharmacological chaperones?
A
- protein folding in cells is complex and sometimes fails
- chaperones aid in folding process
- system in ER degrades misfolded proteins
- misfolded proteins are subject to degradation by various pathways
- some mutations prevent proteins folding properly
- if correctly folded, protein is active and moved down secretory pathway –> cytoplasm / out of cell
- quality control system, ensures only correctly folded proteins end up in target
13
Q
What is Fabry disease?
A
- deficiency of alpha-galactosidase A and subsequent build up of globotriaosylceramide (glycosphingolipid)
- some mutations that cause Fabry are ones that cause protein misfolds
- treatment is with a small molecule chaperone Migalastat - similar to beta-D-galactose which is the substrate for alpha-galactosidase A
- stabilises enzyme in correct shape
- Migalastat binds to the misfolded protein and rescues the folding process = not degraded = protein can travel through ER and increases activity of enzymes in lysosome
- NICE approved in Feb 2017
- mutation specific - only treats those where cause of disease is a mutation causing misfolding
14
Q
What are pharmacological modulators?
A
- commonly used drugs
- can be receptor agonists/antagonists
- can be ion channel activators/blockers
- can be enzyme activators/inhibitors
- can design one that has these effects on mutant receptor/channel e.g. Bcl-abl Kinase inhibitors for treatment of cancers caused by Philadelphia chromosome
15
Q
How have pharmacological modulators been used to treat cystic fibrosis?
A
- CF - defective chloride channel where mutations (33) cause the channel not to open
- can design a drug which causes activation - Ivacaftor
- mutation specific - only effective where a mutation prevents channel from opening
16
Q
How has combination therapy been used to treat cystic fibrosis?
A
- some cases of CF caused by mutation (f508del) that causes both misfolding and an inactive channel
- can be treated with combination of a chaperone and activator e.g. Orkambi (Ivacaftor/lumacaftor) - NICE approved in Oct 2019
- is not a cure but improves lung function - reverses effect of mutation instead of just treating symptoms
17
Q
What are drugs that read through premature stop codons?
A
- some diseases are caused by a non-sense mutation that introduces a stop codon prematurely in the protein sequence
- this prevents protein production and ends up with a shorter protein because release factors bind = truncated protein released
- can use drugs that prevent this e.g. aminoglycoside antibiotics bind to bacterial ribosomes and cause mistranslation
- drugs based on these read through non-sense mutations and blocks the effect of release factors = allows full length protein with only a minor change to the sequence (sometimes has a point mutation as tRNA may not always be correct) so still mostly active
18
Q
How do drugs that read through premature stop codons treat DMD?
A
- Duchenne muscular dystrophy caused by premature stop codon = truncated dystrophin
- Becker muscular dystrophy = missing section
- if we read through the premature stop codon we get a phenotype more like BMD (milder)
- Ataluren approved in EU not US, and NICE approved 2016
- non-sense mutation specific so MD caused by other mutation types cannot be treated by Ataluren
- not a cure but reduces symptoms and increases life expectancy
19
Q
What is gene therapy?
A
- for a recessive disease, we replace defective gene
- for a dominant disease, we delete defective gene
- very difficult to achieve in practice - achieving specificity, getting therapy to right place, maintaining expression
- much easier in vitro (in glass - test tube) + ex vivo (out of the living) than in vivo (in living)
20
Q
What is mitochondrially inherited disease therapy?
A
- only effective therapy is gene therapy
- requires IVF
- maternal spindle transfer - take chromosomes from unfertilised egg with mutated mitochondrial DNA and transfer to unfertilised donor egg with removed nucleus which is then fertilised in vitro and develops into an embryo
- pronuclear transfer - egg with mutated mitochondrial DNA is fertilised in vitro, resulting pronucleus is removed and transferred to a fertilised donor egg with a removed pronucleus, and fused egg develops normally
- approved for use in UK but controversy - known as ‘three-parent babies’ but inaccurate as genome has 3bil bp vs 16k bp in mitochondrial DNA, so minute in comparison
21
Q
What is gene silencing?
A
- used for dominant diseases - gain of function, switches off mutant copy of the gene
- can be used for recessive diseases - downstream effects
- can be used for non-genetic diseases
- several potential methods: antisense oligonucleotides, RNAi
22
Q
How are anti-sense oligonucleotides used as treatment?
A
- can be included in pharmacological agents but targeted against the genes that cause disease rather than proteins
- short modified nucleic acid complementary to mRNA of target gene
- they are modified to prevent degradation and allow entry to cell
- they bind to target and block translation of target mRNA causing it to be degraded, or can alter splicing pattern of mRNA
- relatively cheap to make
- ASO = in-vivo therapy gene silencing
23
Q
What are anti-sense oligonucleotides useful to treat?
A
- useful for diseases caused by gain of function and a number of them are now approved
- e.g. Inotersen - treats transthyretin-related hereditary amyloidosis (TRHA), caused by mutation in transthyretin (TTH) that prevents it from forming tetramers and causes it to form aggregates instead which damages tissue - progressive and fatal disease
- Inotersen can bind to both wild type and mutant form of TTH meaning they are degraded and can no longer produce protein and therefore aggregates - slows down progression
- mutation specific
24
Q
What is RNAi (siRNA)?
A
- dsRNA complementary to target
- modification prevents degradation and allows entry to cell
- activated via Dicer/Risc pathway
- targeted RNA is cleaved –> gene silenced
- RNAi = in vivo therapy gene silencing
- e.g. Lumarisan - treats primary hyperoxaluria type 1 caused by a mutation in AGT = excess glyoxylate builds up = broken down to oxalate –> calcium oxalate = kidney damage
- not approved but on MHRA-EAMS
25
Q
How can anti-sense oligonucleotides be involved in exon skipping?
A
- during pre-RNA processing, oligonucleotides cause exons to be skipped by binding to exon acceptor sites so they can be used to skip disease-causing exons
- they can be used to put reading frame back in sync by blocking exon that causes truncated protein to form
- useful in limited circumstances e.g. exon being skipped must not be vital for protein function
- generally only used for large protein as in small proteins, skipping several exons is more likely to produce a non-functional protein
26
Q
How is exon skipping used to treat DMD?
A
- DMD caused by small deletion that shifts reading frame –> truncated protein - premature stop codon
- BMD caused by large deletion but reading frame intact
- exon skipping to convert DMD –> BMD (milder) - prevent incorporation of mutant exon that codes for premature stop codon
- Eteplirsen - oligonucleotide causes skipping of exon 51 –> production of partially active dystrophin
- approved for use in USA but controversial
- not approved in Europe - more data needed
27
Q
How does virus gene therapy work?
A
- can engineer a virus’ DNA to carry a therapeutic gene
- wide variety of viruses used e.g. AAV, adenovirus, lentivirus (e.g. HIV), vaccinia
- virus choice depends on target tissue - viral tropism (each virus infects particular kind of cells)
- amount of DNA limited depends on virus - each virus has different amount of DNA
- making use of ability to take over host cell machinery
28
Q
What is SCID?
A
- severe combined immuno-deficiency
- bubble baby disease
- lack both B-cell and T-cell mediated response
- several forms: X-linked (X-SCID 70%), adenosine deaminase deficiency (ADA-SCID 15%)
- can be treated with bone marrow transplant - not possible for all children (80% ADA-SCID = no match), and has its own risks
29
Q
What is in-vitro gene therapy for ADA-SCID?
A
- Strimvelis - similar to autologous transplant - transplanting back patient’s own bone marrow cells
- take bone marrow from patient, isolate haemopoietic stem cells, isolate and expand CD34+ cells
- transfect them with a lentivirus that encodes ADA and grow transformed cells
- treat patient with busulfan to kill their own HSCs, then reinfuse transformed cells into patient to replace faulty HSCs with healthy ones
30
Q
How does supplementation (in vivo therapy) work?
A
- where you lack a copy of a functional gene (recessive diseases) and can use a virus to carry in a working copy
- can inject systemically but most successful in vivo therapies have been local injections in eye, spine and brain
- many in vivo treatments in development - only one approved
31
Q
How is supplementation used to treat Leber congenital amaurosis type 2?
A
- recessive disease caused by mutation RPE65 (retinal pigment epithelium-specific kilodalton 65 which is involved in generation of visual pigment)
- causes progressive blindness through loss of retinal cells
- treated through Luxturna rAAV2 expressing RPE65 - recombinant AAV virus contains RPE65
- injected directly into eye behind retina - vector taken up by cells and produces RPE65 proteins = some restoration of visual function
- not a cure - greatly improves vision but patients need sufficiently remaining cells
- EMA approved Nov 2018, NICE approved Sep 2019
32
Q
How is supplementation used to treat haemophilia?
A
- recently licensed gene therapy for haemophilia A in both USA and Europe, not approved for NHS used
- AAV based therapy - virus injected into liver and factor VIII produced
- compared to injections: fewer side effects, no weekly injections, better QOL
- currently on trial in haemophilia B - AAV based
33
Q
How can gene therapies be used in the future?
A
- number of clinical trials of gene cell therapies in UK increasing annually
- large number of potential treatments being investigated
- cures more distant but may be possible
- use of gene therapy approaches to treat non-genetic disease e.g. COVID vaccine, inclirisan (siRNA to treat high cholesterol - switches off enzyme involved in cholesterol synthesis)
- patient expectations high - plays role
