Molecular therapy (NWI-BM078) is a compulsory course for the human biology track in the medical biology master. This document does not contain all lecutres and tutorials, but the table of contents at the beginning of the documents clearly shows which components are covered. Every lecture or tutoria...
Lectures molecular therapy
(research highlights not
included)
Table of contents
Week 1 Monday 06/09/21: Personalized healthcare by Alain van Gool.................................................4
Personalized medicine........................................................................................................................4
Personalized healthcare.....................................................................................................................5
Translational medicine.......................................................................................................................6
Week 1 Tuesday 07/09/21: Drug transporters and metabolism by Tom Schirris...................................8
Transporters.......................................................................................................................................8
ABC transporters................................................................................................................................9
Measuring transport activity............................................................................................................12
SLC transporters...............................................................................................................................12
Biotransformation............................................................................................................................13
Phase I reactions...............................................................................................................................13
Phase II reactions..............................................................................................................................14
Interplay between phase I, II, and III reactions.................................................................................15
Factors that influence biotransformation.........................................................................................15
Week 2 Monday 13/09/21: Drug targeting and delivery part I Roland Brock.......................................16
Limitations of classical drugs............................................................................................................16
Nanomedicine..................................................................................................................................17
Barriers in drug delivery...................................................................................................................18
Types of drug delivery vehicles.........................................................................................................21
Nanoparticles...................................................................................................................................21
Liposomes.........................................................................................................................................21
Targeting strategies..........................................................................................................................25
Passive targeting: Enhanced Permeation and Retention (EPR).........................................................25
Protein particles as nanoformulations..............................................................................................27
Polyester polymers...........................................................................................................................27
Week 2 Tuesday 14/09/21: Advanced drug delivery II.........................................................................28
Active targeting: Antibody drug conjugates (ADCs)..........................................................................28
Factors that influence tissue targeting.............................................................................................29
Targeted cytokines...........................................................................................................................30
, Cellular molecule import..................................................................................................................31
Lipid nanoparticles (LNPs)................................................................................................................32
Cellular uptake..................................................................................................................................33
Other mechanisms for endosomal release.......................................................................................34
Week 2 Tuesday 14/09/21: Drug development....................................................................................37
Current small molecule drug development pipeline........................................................................37
Classification of therapeutics............................................................................................................40
Biologics............................................................................................................................................40
Small molecules versus biologics......................................................................................................41
mAb production................................................................................................................................42
Clinical development........................................................................................................................42
Week 3 Monday 20/09/21: Genetic therapy for retinal disease. Rob Collin, department of Human
Genetics, RadboudUMC........................................................................................................................42
The eye and the retina......................................................................................................................43
Inherited retinal diseases (IRDs).......................................................................................................43
Possible therapeutic interventions...................................................................................................44
Week 3 Monday 20/09/21: Gene augmentation therapy. Rob Collin, department of Human Genetics,
RadboudUMC.......................................................................................................................................46
The RPE65 story................................................................................................................................46
Alternatives for AAV’s.......................................................................................................................48
Week 3 Monday 21/09/21: Splicing modulation therapy. Rob Collin, department of Human Genetics,
RadboudUMC.......................................................................................................................................49
CEP290-associated LCA.....................................................................................................................49
Stargardt disease..............................................................................................................................51
Degrading toxic of dominant-negative RNA.....................................................................................53
Week 3 Tuesday 22/09/21: Genome editing. Rob Collin, department of Human Genetics,
RadboudUMC.......................................................................................................................................53
Zinc finger nucleases (ZFNs).............................................................................................................53
Transcription activator-like effector nucleases (TALENs)..................................................................53
CRISPR-Cas........................................................................................................................................53
Base editing......................................................................................................................................55
Prime editing....................................................................................................................................55
RNA editing.......................................................................................................................................55
Week 4 Monday 27/09/21: Renal physiology. Jojanneke Huck, department of physiology.................56
Functions of the kidney....................................................................................................................56
Kidney diseases.................................................................................................................................56
Kidney mechanism............................................................................................................................56
, Segmental ion transport along the nephron.....................................................................................57
Glomerulus.......................................................................................................................................58
Proximal tubule................................................................................................................................59
Intermediate tubule.........................................................................................................................59
Distal tubules....................................................................................................................................59
Collecting system..............................................................................................................................59
Regulation of ion transporters..........................................................................................................59
Week 4 Monday 27/09/21: Current studies and therapy of renal tubulopathies. Jojanneke Huck,
department of physiology....................................................................................................................60
Kidney anatomy................................................................................................................................60
Mineral disturbances........................................................................................................................60
Tubular transport disorders..............................................................................................................60
Barter syndrome...............................................................................................................................62
Tubular transport disorders..............................................................................................................64
Week 4 Tuesday 28/09/21: Drug-induced renal pathologies. Jojanneke Huck, department of
physiology.............................................................................................................................................66
Acute renal failure............................................................................................................................67
Week 5 Monday 04/10/21: Tutorial RTK signalling. Helma Pluk, department of biochemistry............71
Signalling pathways..........................................................................................................................71
Dimerization of receptors.................................................................................................................72
Classic RTK signalling pathway..........................................................................................................73
Jak/STAT...........................................................................................................................................74
Phosphatidylinositol molecules........................................................................................................75
More signal transduction pathways.................................................................................................76
Signalling has to be turned off..........................................................................................................76
Week 5 Tuesday 05/10/21: from here lectures and tutorials are not included....................................77
,Week 1 Monday 06/09/21: Personalized healthcare by
Alain van Gool
Personalized medicine
The principle of personalized healthcare, is that a patient group with the same diagnosis is
subdivided into smaller groups, namely:
- Patients where the drug is toxic but beneficial.
- Patients where the drug is non-toxic but non-beneficial.
- Patients where the drug is toxic and non-beneficial.
- Patient where the drug is non-toxic and beneficial.
By doing this, the right therapy can be administered to the right patient at the right intensity at the
right time. Molecular biomarkers are the key drivers of this patient selection.
It’s called personalized medicine, but it refers to a group of people it’s group healthcare rather
than personalized healthcare.
Personalized medicine in melanoma
Melanoma is a multistage tumour development. In this case, BRAF mutation is the first event in
tumour formation. BRAF is mutated which causes a benign lesion to grow into several cancerous
stages and finally into a metastatic melanoma. After the initial BRAF mutations, other mutations
occur, e.g. loss of CDKN2a, loss of PTEN, loss of E-cadherin, increase of CD1, etc. In case a metastatic
has arose, life expectancy decreases to ~5 months.
BRAF is part of the ERK-pathway, which is a growth pathway:
- A growth factor stimulates receptors on outside of a cell signalling to the inside RAS
RAF MEK ERK. ERK is a transcription factor that moves to the nucleus and stimulates
cell growth.
These proteins are intrinsically activated via kinases. In the BRAF-protein are two different
phosphorylation sites present in the kinase domain where ATP binds. Both threonine and serine need
to be phosphorylated for RAF to be activated. MEK has the same mechanism: if both phosphorylation
sites are phosphorylated, ERK is activated. Dephosphorylation happens through phosphatases.
BRAF is a spontaneous mutation. However, 60% of melanoma patients have a BRAF mutation.
When the ERK-pathway is inhibited, cell growth is inhibited. In a melanoma patient:
- BRAF mutation present? treat with RAF-inhibitor.
- No BRAF mutation present? use the standard therapy, usually chemotherapy.
Medication that ends in -ib? kinase inhibitor.
Vemurafenib is a BRAF-inhibitor which can be used to treat BRAF-melanomas. Before treatment
there is much activation of ERK, meaning the pathway is active. But after treatment, the
phosphorylation of ERK decreases, leading to decrease in cell growth.
Treatment is life-extending but not treating after 6-10 months most solid tumours become drug
resistant making it difficult to remove every last tumour cell. Therefore, nowadays therapies are
combined: vemurafenib + chemotherapy, vemurafenib + another targeted drug, etc.
Biomarkers
,A biomarker is a characteristic that is objectively measured and evaluated as an indicator of normal
biological processes, pathogenic processes, or pharmacologic responses to a therapeutic
intervention. This could either be a gene, SNP, protein, image, etc., as long as it can be objectively
measured.
An efficacy biomarker shows a drug is working in the way it should be working.
- For example: If the kinase inhibitor should inhibit a kinase and therefore cell growth, that’s
an efficacy biomarker.
If such a marker is known at the beginning of phase II, there is a success rate of over 80%. Without an
efficacy biomarker, there is only 30% success rate. Therefore, sometimes studies are not allowed to
move into clinical trials without having an efficacy biomarker.
Pharma strategy based on personalized medicine
Use the 5R’s assessment to choose a personalized medicine:
1. Right target.
2. Right tissue.
3. Right safety.
4. Right patients.
5. Right commercial potential.
In the past, drugs failed in clinical development because the pharmacokinetics were unknown. When
this was solved, drugs showed much toxicity. This has been reduced because of toxic screening and
predication models. Nowadays, most drugs that fail in phase II clinical trials, fail because lack of
efficacy.
Phases in drug development:
1. Research phase: identify a key drug target, e.g. BRAF.
2. Lead finding: identify a lead, which is a compound or antibody or some other therapy that is
able to inhibit/influence the key drug target.
3. Milestone: Is the drug good enough to go to clinical development?
4. Pre-clinical toxicity and bioavailability: dose the drugs into a rodent and non-rodent. This is
obligatory before moving onto humans.
5. Drug is available and safe? move into phase I: healthy volunteers take the drug in low
doses to test for side effects. Increase the dose until side effects are observed. Ideally,
effective level should be much lower than the toxic level.
6. Select a dose and move into phase IIa. This dose is administered to ~50 patients.
7. Phase III: Drug is administered ~200-3000 patients.
8. Market.
Personalized healthcare
Case: personalized healthcare in rare metabolic diseases
Healthy parents got a son in 2002. First son had a low birth weight, lactic acidosis, hypoglycaemia,
intellectual disability, movement disorder, and epilepsy. The first son died from respiratory failure at
the age of 3.5. Cause of the disease was unknown. In 2009 a second son was born, which had the
same clinical phenotype but worse. The second son died at the age of 1.5 from an epileptic seizure.
The clinical phenotype was a suspicion of mitochondrial dysfunction. Mitochondria create energy by
converting glucose ATP via five complexes: the OXPHOS enzyme complex I-V. These complexes are
encoded of many different proteins, of which most are encoded by mDNA and some are encoded by
nDNA. It was shown that ATP production and creatine phosphate production were decreased in the
children. However, the activity of the complexes was normal. Candidate genes were screened, but
, without results. In 2010 WES was introduced, which allows screening of all genes at once. With the
help of WES, a mutation in the WARS2 gene was found. Interestingly, WARS2 was not associated with
mitochondrial dysfunction. WARS2 is mtDNA-coded tryptophanyl-tRNA synthases. Normally, the RNA
is brought to the ribosomes via tRNA. In this case, the tRNA is loaded with tryptophane. This
mutation caused instability of the WARS2 protein, as shown in a Western Blot. In these patients the
WARS2 protein is practically absent, and the tRNA is loaded less often. Now that the mutation was
identified, prenatal testing could be used on the foetus.
Technology innovation is driving impact in personalized healthcare. Personalized healthcare is a
combination of diagnosis, treatment, and successful therapy. The diagnosis is crucial: without
diagnosis no treatment. It is important to combine different molecular views: X-omics. Also, fast
translational of the biomarker research to clinical implementation is needed.
The mutation was X-linked when the parents received a daughter, she was healthy.
Role of molecular (omics) biomarker data in personalized healthcare
Molecular biomarker data are becoming more and more important in personalized healthcare. A
personalized therapy can either be a certain drug (e.g. BRAF targeted drug) or an advice (e.g.
prenatal test, liver transplantation, etc.). Once a therapy is chosen, omics can be used to monitor the
therapy.
To investigate proteins, metabolites, and glycans Mass
spectrometry can be used.
Omics helps to increase the diagnostic field and can
contextualise change. For example, a single biomarker tests
for protein X. The figure on the right shows protein X is
increased. If protein X does not show a difference, protein Y
has to be tested. If protein Y does not show a difference,
protein Z has to be tested, et cetera. When using the omics
approach all genes/cytokines/lipids/etc. are tested at once, creating the table on the bottom of the
figure. This omics figure shows that protein X is a factor 2 increased whilst the rest is silent. This helps
to create a complete indication.
- High diagnostic yield many biomarkers are tested.
- Change can be put into context can compare the biomarker with other molecules.
Clinical omics data give a bigger picture merge the omics outcome with imaging and biochemical
data.
Translational medicine
There are currently many apps on the market which test certain
markers and to predict/diagnose disease from home. However,
there is a big innovation gap; more markers, data, and models are
produced, but the ones that are already known are not
necessarily improved. There are 3 translational innovation gaps:
1. Research to research.
2. Research to diagnostics.
3. Research to society.
Errors in research often lead to irreproducibility replication crisis.
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