Genetic treatments Flashcards
What are inborn errors of metabolism?
Rare inherited disorders whereby the body is unable to effectively convert substrate into energy or for the synthesis of ATP (Glycolysis, and beta oxidation)
Disorders attributed to defects in specific enzymes responsible for the hydrolytic degradation (metabolism) of food
What inheritance pattern is demonstrated by phenylketonuria (PKU)?
Autosomal recessive
Which metabolism is affected by PKU?
Phenylalanine metabolism
Why is phenylalanine metabolism affected by PKU?
There is a deficiency in the enzyme phenylalanine hydroxylase (PAH)
The enzyme catalyzes the amino conversion of phenylalanine into tyrosine
Leads to an accumulation of phenylalanine and its metabolites in tissues and bodily fluids
What is the PAH gene mutation in terms of chromosomes?
12q23.2
Explain the pathophysiology of PKU in terms of phenylalanine accumulation?
Elevated levels of Phe, leads to increased production of phenyl ketones, these compounds express degrees of neurotoxicity.
Phe affects the synthesis of inhibitory monoamine neurotransmitters (Responsible for physiological and homeostatic processes)
What are the symptoms associated with untreated PKU?
Major cognitive impairments; behavioural difficulties; fairer skin tone, hair and eyes; lack of melanin and recurrent vomiting.
How is PKU treated?
Patients are given a low protein diet (protein diets have high Phe content) - in a combination of Tyrosine supplements
Screening program introduced to measure levels of phenylalanine upon initial recognition of disease; treatment succeeds in scientific understanding.
What is neutral amino-acid therapy?
Supplement partially inhibits the absorption of phenylalanine
What is the inheritance pattern of hemophilia?
Sex-linked inherited genetic condition
What is hemophilia?
Sex-linked genetic conditions which impair the body’s ability to release clotting factors in aim to produce blood clots.
Symptomatically, this increases the clotting time after injury, contributes to easy bruising and increased riskof internal bleeding (brain and joints)
The condition typically caused by hereditary lack of coagulation factor VIII,
Heat treatment of product kills HIV & HBV
What treatments are available for hemophilia (5)?
Cryoprecipitate Desmopressin Clotting preserving medications Fibrin sealants Replacement therapy
How can cryoprecipitate be used to treat hemophilia?
Prepared from plasma & purified, contains factor VIII, and platelet concentrates can be intravenously administered as a replacement therapy
Recombinant clotting factor VIII gene cloned to synthesize an increased level of factor can be used.
How can desmopressin be used to treat hemophilia?
Hormone can stimulate the release of more clotting factors, administered intravenously or as a nasal spray, only for milder variants of hemophilia.
How do clotting preserving medications work?
Medications inhibit and reduce fibrinolysis - reducing clot degradation
How do fibrin sealants work?
Medications can be applied directly to the wound sites to promote clotting & bleeding
Which recombinant protein is injected in patients with Fabry & Pompe diseases?
Alpha galactosidase A
What is the first phase of drug development?
Drug development relies on multiple phases to ensure efficacy
First stage discovery is based on preclinical understanding (lab-based experimental procedures to provide evidence to back hypothesis)
Animal testing to evaluate toxicity, and clinical testing to identify dosages
How many main phases are there for clinical drug testing?
Phase 1-3
3 phases
What is phase 1 for clinical testing?
Safety in healthy volunteers (<100)
What is phase II during clinical testing?
Check therapeutic effect (100-300)
What is phase III of clinical testing?
Large scale therapeutic trials (200-3000) Approval: EMA & FDA
How are NHS drugs approved?
Prescription of NHS drugs require approval by the National Institute for Health & Care Excellence (NICE) for England & Wales.
Healthcare improvement in Scotland
How are NHS drugs approved?
Depending on cost-effectiveness and efficacy
Provides guidelines for treating conditions
What are pharmacological chaperones?
Protein folding and processing occurs in the endoplasmic reticulum (ER), where the protein tertiary structure is maintained and constructed to confer specificity.
Chaperone proteins regulate the folding process. Implemented system in ER degrades misfolded proteins (subject degradation pathway)
How do molecular chaperones work?
Molecular chaperones in the endoplasmic reticulum are designated to support nascent proteins entering the ER lumen and assist towards attaining the correct protein conformation and prevent non-specific aggregation
Mutations can alter the polypeptide sequence, transforming the trinucleotide sequence of a codon, encoding for a different amino acid (impacting the stereochemical and polar properties of the chain) - attributing to altered bonds.
Which enzyme is misfolded in Fabry disease?
alpha-Galactosidase A
Which substrate accumulates within blood vessels, tissue, and organs as a result of Fabry disease?
Globotriosylceramide
Which molecular chaperone is used in Fabry disease?
Migalastat
How does Migalastat stabilize and treat Fabry disease
Chaperone stabilizes enzyme by binding to active site to mutant forms of alpha-galactosidase A; conforming for correct structural configuration within the endoplasmic reticulum, and facilitating normal trafficking to lysosomes. Migalablast antagonist of substrate - mutation-specific
When was migalastat approved by NICE?
Feb 2017
What are pharmacological modulators?
Are receptor agonists/antagonists behaving through ion channel activation/inhibition
What is the function of the BCL-ABL kinase inhibitor?
Product of Philadelphia chromsome, drug only binds to mutant kinase form
How does the pharmacological modulator, ivacaftor treat cystic fibrosis?
CFTR protein defection causes a dysfunctional chloride ion channel. Mutations (33) attributed to channel closure
ANSWER: Facilitates increased chloride transport by potentiating the channel open probability if the G55ID-CFTR protein. Drug is mutation-specific
How can combination therapy be used to treat cystic fibrosis?
Orkambi involves a combination of chaperone and activator molecules
Lumacaftor and ivacaftor respectively.
Refolded protein channel does not exhibit complete activity
Which cystic fibrosis mutation causes chloride channel dysfunction?
f508del
How does lumacaftor work in regards to cystic fibrosis treatment?
A pharmacological chaperone that enhances conformational stability of f508del-CFTR, increases processing and trafficking of mature CFTR protein to the cell surface
Combination of both drugs leads to elevated transmembrane chloride transport
How are missense mutations caused?
Caused by premature stop codon within the sequence of mRNA. Stop codon inhibits and ceases ribosomal translation of protein, therefore prematurely truncating the polypeptide chain. Protein is degraded (sensed as incorrect combination).
Which antibiotic causes mistranslation of proteins by impacting RNA proofreading (binding to 30s ribosomal subunit)?
Aminoglycosides
How does nonsense suppressor binding works?
Enables cognate amino-acyl tRNAs to compete with release factors.
How does DMD &BMD in terms of deletions and stop codons occur?
DMD Premature stop codon in DMA
BMD -> Deletion causes omitted section, dystrophin exhibits partial functionality
Reading through premature stop codon can cause the deleterious condition to be transformed into BMD
How can DMD be treated using Ataluren?
Stop codon read through
Inhibits premature stop codon expression
Which drug can cause a stop codon readthrough in DMD?
Ataluren
In terms of gene therapy what happens in dominant conditions to the gene?
Delete defective gene
In terms of gene therapy what happens in recessive conditions?
Replace defective gene
How can mitochondrial diseases be therapeutically treated in terms of genetics?
Requires IVF, DNA derived from fertilised ovum is extracted and transferred to donor egg normal mitochondria Spindle transfer (unfertilised egg). Spindle complex is removed and transferred to the donor egg, the fused egg is fertilised in vitro
PRONUCLEAR TRANSFER
How do viral vectors work?
Viral genes conferring for proteins that result in stimulating adverse effects are removed or inactivated, and the desired gene is inserted. Thereby, this integrates the desired gene into the viral genome
Lysogenic viruses are used to transfer the desired gene into the cell to be transformed
The recombinant DNA can thus be integrated into the host’s genome
Which viruses are commonly used as viral vectors in the treatment of genetic diseases?
AAV, adenovirus, lentivirus (HIV) and vaccinia
What are gene guns?
Gene guns bombard cells with minute particles of tungsten/gold, these particulates are coated with copies of desired DNA, the particles that have entered the cell and be integrated into the host genome
Who do liposomes work?
The desired gene is enclosed in a spherical phospholipid bilayer, the liposomes can fuse with the cell surface membrane of the host cell delivering the DNA into the cytoplasm
Which enzyme is dysfunctional in Severe Combined Immunodeficiency Disease (SCID)?
Adenosine deaminase
How can SCID be treated?
Involves the removal of T-lymphocytes and insertion of the correct allele into them using a vector. The successful cells are cloned - replenish numbers
What is the inheritance pattern of cystic fibrosis?
Autosomal recessive
Which transporter protein is affected in patients with cystic fibrosis?
CFTR Transmembrane conductive receptor
What is the purpose of the CFTR protein?
Present on epithelial cells within the airways; pumps chloride ions through channels in the plasma membrane, and hence a low water potential outside the cells.
Water moves out of the cells by osmosis down the water potential gradient, diluting the mucous and reducing its viscosity.
Which chromosome is the CFTR gene located on?
Chromosome 9
How can liposomes be used to treat CF?
Liposomes containing the dominant allele can be introduced into the cells lining the respiratory tract. Adenovirus is the vector used for treating this disorder, although the effects of cystic fibrosis are suppressed for a small period of time
Which is the most common viral vector for CF treatment/
Adenovirus
What is germline gene therapy?
Involves changing the genes in cells that would go on to become gametes
What does CAR-T cell gene therapy stand for?
Chimeric antigen receptor -T cell (CAR-T)
Which diseases are commonly treated by CAR-T cell therapy?
Cancer, mainly forms of lymphoma
What is the T cell receptor antigen associated within CAR-T cell gene therapy?
Major histocompatibility complex, low affinity
How can the TCR-MHC affinity increase in CAR-T cell gene therapy?
Attachment of antibody to YCR enhances affinity, thereby can recognize antigen directly without the assistance of proteins.
Which antibody region recognizes cancer cells during CAR-T cell gene therapy?
Variable region monoclonal antibody scFV receptor recognizes cancer cells.
T-cell receptor and the co-receptor signaling domain
Where is the antigen recognition domain exposed on the extracellular surface on CAR-T cells?
Ectodomain portion of the receptor
What is the purpose of the ectodomain portion of the receptor in CAR-T cells?
Interacts with potential target molecules and is responsible for targeting the CAR-T cell to any cell expressing a matching molecule
What is the antigen-recognition domain derived from in CAR-T cells?
Derived from single-chain variable fragments (scFv)- Chimeric protein consists of light and heavy chains of immunoglobulins, connected to shorter linked peptides
Light and heavy chains are used for binding affinity to the target antigen.
Which virus is used to insert the chimeric antigen gene into T-cell DNA during CAR-T cell therapy?
Lentivirus (Retrovirus undergoes reverse transcription and inserts specific scFv gene into the genome)
CAR-T cell proliferate and reinfused into the patient
How is in vitro CAR-T cell therapy conducted?
Patient T-cells are isolated, these are expanded, transfected with chimeric antigen receptor using lentivirus
Which drugs are prescribed to patients alongside with CAR-T cell gene therapy?
Immunosuppressants
Why are immunosuppressants provided to patients undergoing CAR-T cell gene therapy?
Reduces host T-cell production
What are the symptoms and side effects associated with CAR-T cell therapy?
Cytokine release syndrome attributed to neurological damage, expensive treatment
Which congenital disease is usually treated with in vivo therapy supplement?
Leber congenital amaurosis type 2
What is the inheritance pattern of Leber congenital amaurosis type 2?
Autosomal recessive
Which mutated allele causes Leber congenital amaurosis type 2?
RPE65
What are the symptoms associated with the mutated RPE65 allele?
Results in progressive blindness attributed to a reduction in retinal cells
Which virus is used to insert the corrected RPE65 gene for patients with Leber congenital amaurosis type 2?
Luxturna rAAV2
How can in vivo therapy supplement be used to treat retinal blindness?
Viral vector (rAAV2) expressing the correct RPE65 gene is injected into the retina, replaces defective recessive allele); minimizes retinal damage and inhibiting further deterioration Patients require sufficient remaining cells
What are anti-sense oligonucleotides?
Antisense oligonucleotides are short modified single-stranded nucleic acids that interact with mRNA to prevent translation of a targeted gene
How do anti-sense oligonucleotides work?
Non-coding RNA nucleotide antagonists prevents the action of RNA polymerases from catalyzing the formation of phosphodiester bonds between adjacent nucleotides - thereby inhibiting translation of DNA sequence is complementary to specific mRNA target binding leads to degradation of the DNA sequences - failure of protein production
Alter splicing during post-translational modification of pre-mRNA transcript –> transcription is inhibited
What is in vivo therapy knockdown?
RNA interference results in knocking and downregulating gene expression of the target gene responsible for the pathogenesis of the genetic condition. Useful for diseases caused by gain of function - currently no successful therapies
How can in vivo therapy be used to treat Hungtington’s?
Oligonucleotides injected into CSF of the spinal cord (intrafoetally) - Blocks the translation of mutant huntingtin protein (RG602 Huntington’s disease) PHASE III trial
What is exon-skipping?
Pre-RNA processing oligonucleotides cause exons to be skipped, thereby this skips the disease-causing exon, and enables the RNA to be reintegrated into the reading frame with the removed mutant exon, resulting in partially active protein.
Removed exon cannot be vital to protein function; effective for large proteins (minor impact on polypeptide chain)
Why is exon skipping more effective in large proteins?
Removed exon cannot be vital to protein function; effective for large proteins (minor impact on polypeptide chain)
Which genetic disorder is exon skipping used for?
Duchenne muscular dystrophy; transformed into Becker phenotype
Which drug can be used to exon skip for DMD?
Eteplisern
What is the mechanism of action for Eteplisern?
Oligonucleotide causes skipping of exon 51, therefore truncated active dystrophin is produced
What does CRISPR stand for?
Clustered regularly interspaced short palindromic repeats
Which enzymes are associated with the CAS-9 protein?
Endonucleases, these nucleases are molecular enzymes responsible for catalyzing and cleavage of phosphodiester bonds between adjacent mononucleotides
CAS-9 cleves the double-stranded DNA at specific restriction sites along with the genome, these sections can be added.remvoed
What is the purpose of guide-RNA in CRISPR?
Consists of pre-designated RNA sequence located within a longer RNA scaffold
The scaffold section binds to DNA, pre-designated sequence guides CAS-9 to the correct genomic location. Cas-9 cleaves a specific target sequence of DNA as designated by gRNA.
What are the limitations of gene editing?
Gene editing corrects relatively small errors, cannot correct large changes (deletions and triple expansions)
Off-target effects and ineffective intracellular mechanisms.
