Emerging Treatments Flashcards
What is the largest group of genetic diseases?
Inborn errors of metabolism
What causes inborn errors of metabolism?
Defects of single genes that code for enzymes that facilitate conversion of substrates into products (lack that enzyme)
What is the main problems with the inborn errors of metabolism?
There is an accumulation of substances that are not converted into products that may be toxic or interfere with normal function
What are four examples of diseases caused by inform errors of metabolism?
Maple syrup urine disease
MCAD Deficiency
Phenylketonuria (PKU)
Homocystinuria
What pathways do inborn errors of metabolism effect?
carbohydrates
fatty acids
proteins
What happens in phenylketonuria?
There is no phenylalanine hydroxylase meaning phenylalanine is no longer converted into tyrosine and is converted into phenylketones instead
What can untreated PKU lead to?
Major cognitive impairment
Behavioural difficulties
Lack of melanin so fairer skin, hair and eyes
Recurrent vomiting
What is used to treat PKU?
Tyrosine supplements in diet and low protein diet
Why are patients with PKU advised to keep a low protein diet?
Phenylalanine is an amino acid found in protein, so given PKU patients are advised to have a low protein diet to avoid unnecessary buildup as they struggle to degrade it
What are some symptoms of Haemophillia (blood clotting disorder)?
Uncontrolled bleeding
Bleeding into joints and brain (excruciating pain)
Internal bleeding
If untreated it is fatal
What did some patients get after being treated for haemophilia?
HIV and Hep C
How is haemophillia treated?
By replacing the missing clotting factors isolated from human blood serum, 8 and/or 9
What methods did they used to use to treat haemophilia?
diluted snake venom then whole blood transfusion
What is important to know before trying to treat through diet and replacement?
The biochemistry behind the condition as the treatment is not mutation specific
What are four other conditions which can be treated by replacement?
Growth hormone deficiency (injection of growth hormone now recombinant)
Fabry disease (Injection recombinant alpha galactosidase A)
Pompe disease (injection of alpha glucosidase)
Lysosomal storage disease (effects lysosomal breakdown)
What are the five general stages of drug development?
- Discovery/preclinical
- Lab based testing
- Testing in animals
- Clinical testing - in 3 phases
- Approval by the regulatory bodies (EMA, FDA)
What happens in phase 1 of clinical testing?
Tested on healthy volunteers, with a sample size of <100
What happens in phase 2 of clinical testing?
The drug is tested to check the therapeutic effect on patients with the condition, 100-300 people
What happens in phase 3 of clinical testing?
Large scale therapeutic trials with 200-3000 patients
Who tests and approves drugs in England?
The National Institute for Health and Care Excellence (NICE)
What is the purpose of protein-targeting therapies?
They try to normalise the function of mutant proteins
These are treatments not cures
Treat the condition not symptoms
What is a pharmacological chaperone?
A drug which serves as molecular scaffolding in order to stabilize mutant proteins and cause them to fold correctly
System in ER degrades misfolded proteins
What condition is migalastat used to treat and what is it?
Fabry disease
Migalastat is a small molecule chaperone
It stabilises enzyme in correct shape
Mutation specific
What are pharmacological modulators?
They are receptor agonists / antagonists which can act as ion channel activators and blockers
They can be designed to have an effect on mutant receptor or channel
What condition is Ivacaftor used to treat and how does it work?
Cystic fibrosis
Mutation (33) causes channel not to open
Ivacaftor causes activation of channel
What is combination therapy?
When a protein chaperone and modulator is used as the problem could be fixed with both
What is an example of a case where combination therapy would be useful?
When you have a defective chloride ion channel in CF resulting from one mutations that lead to misfolding
What is a non-sense mutation?
When a stop codon is inserted into the middle of the gene, preventing the complete read-through of the instructions for a complete protein to form
premature stop codon
How does stop codon read through therapy work?
When the therapy can recognise a mutated stop signal which differs from the normal one, and encourages the cell to ignore the stop codon
Aminoglycoside antibiotics bind to ribosome cause mistranslation
Drug based on these-> read through non-sense mutations
What condition is Ataluren used to treat and what kinda of treatment is it?
Stop codon read through therapy for Duchenne Muscular Dystrophy
What are the four methods of small molecule treatment?
- Stop codon read through therapy
- Pharmacological Modulators
- Pharmacological Chaperones
- Combination therapy
What does in vitro mean?
In Glass
What is meant by gene therapy?
The therapeutic delivery of nucleic acid into a patients cells as a drug to treat disease
What does in vivo and ex vivo mean?
In the living and out the living
What are three reasons that gene therapy is very difficult to achieve in practise?
Achieving specificity
Getting therapy to right place
Maintaining expression once it is in the right place
What is done in gene therapy when you have a recessive disease?
You replace the defective gene
What is done in gene therapy when you have a dominant disease?
Delete defective gene
What is meant by spindle transfer/ mitochondrial transfer?
Where the nuclear DNA is transferred to another healthy egg cell leaving the defective mitochondrial DNA behind
requires IVF
Does the genome have more genetic information or does mitochondrial DNA?
Genome, 16569 bp in mitochondria
3088269832 bp in genome
mitochondrial transfer is popularly known as 3-parent baby even though it’s just a minor transfer of DNA
Is mitochondrially inherited disease therapy in vivo or in vitro?
In vitro
What does virus gene therapy involve?
The use of an engineered virus to carry therapeutic genes
Give examples of conditions that virus gene therapy has been used to treat?
AAV
Adenovirus
Lentivirus - HIV
Vaccinia Virus
What does virus choice depend on?
Target tissue
What depends on virus choice?
Amount of DNA limited
What is SCID?
Severe combined immunodeficiency
Bubble baby disease
Lack both B-cell and T-cell mediated response
There are several forms
- X-linked (X-SCID 70%)
- Adenosine deaminase deficiency (ADA-SCID 15%)
Why can virus gene therapy be useful for patient with SCID?
SCID can sometimes be treated using a bone marrow transplant, however it is not possible for all children (80% ADA-SCID no match) and it has its own risks
What is the in vitro gene therapy for ADA-SCID?
Strimvelis
Autologous transplant
Isolate patients Hematopoietic stem cells
isolate and expand CD34+
Transfected with ADA- lentivirus
Grow transformed cells
Treat patient with busulfan (kills HSC)
Reinfused transformed cells into patient
What are CAR-T cells?
Chimeric Antigen Receptor T cells
How does CAR-T cell therapy work?
T cells are genetically engineered to express chimeric antigen receptors bound to MHC, which are speicifially directive towards antigens on patients tumour cells - this means the cancer cells are killed and attacked
What thpe of antibodies recognises cancer cells using CAR-T cells?
scFv = single chain fragment variable
What are some of the side effects of CAR-T cell therapy?
Cytokine release syndrom and neurological damage
How does in vivo supplement therapy work?
When patients lack a functional copy of the gene, they can use a virus to carry a working copy into the cells
What is gene silencing used for?
Used for dominant diseases- gain of function
Can be used for recessive diseases- downstream effects
Can be used for non-genetic diseases
Several potential methods
Antisense-nucleotides
RNAi
What are anti-sense oligonucleotides?
Small sections of mRNA which bind to target mRNA and cause it to be degraded - preventing the protein being transcribed properly
Short modified nucleic acid complementary to target
Modification prevents degradation allow entry to cell
Binds to target
Blocks translation
Can also alter splicing
relatively cheap to make
What is RNAi?
RNA interference
biological process in which RNA molecules are involved in sequence-specific suppression of gene expression by double-stranded RNA, through translational or transcriptional repression.
How does RNAi work?
dsRNA (double stranded RNA) is complementary to target
Modification prevents degradation allow entry to cell
Activated via Dicer/ Risc pathway
Targeted RNA cleaved
What is an example of in vivo therapy gene silencing using RNAi?
Lumasiran-
Primary hyperoxaluria type 1
Autosomal recessive mutation in AGT
Causes excess glyoxylate build up
Broken down to oxalate
Produces calcium oxalate kidney damage
What is in vivo therapy gene silencing used for?
Useful for diseases caused by gain of function
It uses ASO
What is an example of in vivo therapy gene silencing?
Inotersen (an antisense oligonucleotide inhibitor)
Transthyretin-related hereditary amyloidosis
Mutation in Transthyretin (TTH)
TTH cannot form tetramers; forms aggregates
Mutation specific
What type of diseases is in vivo known down therapy useful for?
In diseases caused by a gain of function
What is exon skipping therapy?
Involves the use of oligonucleotides to skip disease causing exon, with the useful RNA being put back into the reading frame so active protein is still made
Generally only large proteins
What can happen in exon skipping therapy when given to patients with duchenne muscular dystropy?
Removal of the mutant exon can cause DMD to become BMD
Eteplirsen- oligonucleotide cause skipping exon 51
Will result in production partially active dystrophin
What is a future possible gene therapies?
CRISPR-Cas 9
What are the disadvantages of using CRISPR-Cas9?
Cannot target large changes like large deletions of triplet expansions
One form of SCID (severe combined immunodeficiency) is caused by a lack of Adenine deaminase (ADA) in haematopoietic stem cells. This condition is often treated by bone marrow ransplant. In some patients this is not possible what genetic approach could be used to treat this condition?
Ex vivo gene therapy
Phenylketonuria (PKU) is an autosomal recessive disease caused by lack of phenylalanine hydroxylase (PAH). Some of the mutations in PAH prevent it from folding properly. Other mutations reduce the activity of the enzyme. Which approaches might be successful in treating the disease caused by these two types of mutation.
Pharmacological chaperones
Familial Mediterranean fever is an autosomal dominant disease caused by a point mutation in FMF gene. Which approaches might be successful in treating this disease?
Knowndown approach
Marsili Syndrome is an autosomal dominant disease caused by a mis-sense mutation, in this disease the mutation is the (a point mutation) in the ZFHX2 gene. What approach might be successful in treating this disease ?
Knockdown (antisense) approach - disease is dominant and caused by a protein with a gain of function so thr activity of this protein needs to be blocked
McArdle Disease is an autosomal recessive condition caused by mutations in glycogen phosphorylase, muscle associated (PYGM). PYGM is small enzyme . One of the most common mutations is a nonsense mutation (a premature stop codon) caused by the change of a single nucleotide. What approaches might be useful for treating this disease ?
Stop codon read through therapy as it is caused by a premature stop codon
Where can you inject locally?
eye, epidurally spine and brain for in vivo therapy supplementation
What can in vivo therapy supplementation be used for example?
Leber congenital amaurosis type 2
Recessive disease caused by mutation RPE65
Progressive blindness- loss of retinal cells
Lucturna rAAV2 expressing RPE65
Not cure- greatly improves vision though
What has supplementation recently been licenced for and what is ongoing?
Haemophilia A
on going for Haemophilia B
