BMS3031 Exam Workshop Notes PDF

BMS3031 EXAM WORKSHOP NOTES PRECISION MEDICINE (WS2): GENE THERAPY GENE EDITING Does not alter the patients genome/genetic Changes the patient’s DNA material...

BMS3031 EXAM WORKSHOP NOTES PRECISION MEDICINE (WS2): GENE THERAPY GENE EDITING Does not alter the patients genome/genetic Changes the patient’s DNA material Requires a nuclease to cut the genome (Zinc No nuclease required (DNA not cut) Fingers, TALENS, CRISPR-CAS9) Inserts DNA with HDR Can’t use NHEJ – no insertion Uses an episome which is not passed to daughter DNA changes are passed onto daughter cells cells ZINC FINGERS VS TALENS VS CRISPR CAS ETC (TABLE FROM NOTES HERE) SITE DIRECTED RECOGNITION MECHANISM SPECIFICITY NUCLEASE 1. Zinc finger recognises a specific triplet Protein is a Can arrange ZF’s with nucleases of DNA chimera while different specificities to Fok1 nuclease is design a protein that ZF need to be G rich bound to its c recognises a specific because ZF cannot terminus sequence. having 2 ZF recognise every single Actively cleaves sequences you increase the nucleotide. DNA when a specificity. homodimer ZF downstream of the target site (on one strand) and upstream of the target site (on the other strand) initiates a double stranded cut 2. Transcription Recognises a individual brings the Fok1 nuclease to Can make specific activator-effector nucleotide the recognition site sequences. nucleases Module for each individual nucleotide means that any sequence can be targeted. 3. RNA-guided uses a complementary Tracer RNA forms a Part of the guide sequence engineered strand of RNA (tracer RNA) complex with cas9 and is complementary to the nucleases holds guide sequence in tracer and part is (CRISPR CAS9) place. complementary to the tracer??? CRISPR CAS9 finds dsDNA complementary to whatever is wanted can be the guide strand put in the guide RNA dsDNA opens and the RNA causes a conformational change Activating the nuclease in cas9 and causing a double stranded break o Limitation = target DNA needs a pam protospacer – sequence of N-G-G to hold it in place in the cas9 enzyme (N can refer to any nucleotide) Advantages of CRISPR over ZFN/TALEN Disadvantages of CRISPR over ZFN/TALENs o Classic base pairing rules for DNA o Overall recognition sequence is small (20 base pairs binding, doesn’t require proteins. approximately), this means there can be recognition at a o Making custom sgRNA used by site that wasn’t intended to be cut CRISPR is much easier, quicker and cheaper PATHOPHYSIOLOGY OF HUNTER SYNDROME Genetically associated lysosomal storage disorder due to a deficiency of the enzyme I2S (iduronate 2- sulfatase) Chronic progressive o IDS = produces the enzyme I2S which breaks down glycosaminoglycans (GAGs) in lysosomes o Spectrum of symptoms includes enlarged/dysfunctional organs, thickened connective tissue and intellectual decline. Thickened tissue is due to the accumulation of partially degraded GAGs STRUCTURAL CAUSE OF HUNTER SYNDROME  - I2S protein active site binds to the GAG to remove a sulfate group – facilitating breakdown in the lysosome - Multiple mutation types in I2S structure: o Buried steric clash – misfolding o Catalytic inactivation o Local charge inversion o Disulfide bridge disruption o Hydrophobic core destabilisation o Backbone geometry distortion o Surface charge alteration o Loss of N-linked glycosylation § Incorrect glycosylation of the I2S protein may impair binding to the mannose 6-phosphate receptor Interferes with lysosomal targeting ELAPRASE (IDURSULFRASE) PRODUCTION FOR ENZYME REPLACEMENT THERAPY Therapy has the IDS gene inserted into the cells of the body ELAPRASE MCHANISM OF ACTION - Administered via in IV infusion once weekly CLINICAL SUCCESS OF ELAPRASE Improves walking capacity in patients 5 years and older. No data available for 16 month – 5 years of age for improvement in disease symptoms/long term outcomes. Reduced spleen volume. JAPANESE study found long term treatment was well tolerated/effective in improving clinical features and slowing disease progression. DISADVANTAGES OF ELAPRASE Safety/efficacy not established in patients under 16 months Does not cross the blood brain barrier o Poses challenges to treating neurological aspects of the disease § Possible solution = intracerebroventricular injection NORMAL AAV GENOME - All viral genes are removed from AAVs when used for gene therapy/editing so there is no viral insertion - Insert genome info from lecture notes PRODUCTION OF AAV WITH THE IDS GENE Construct 3 different AAV: 1. IDS gene 2. ZF1 3. ZF2 When collecting cells with AAV, need to select for those with all 3 plasmids AAV DELIVERY TO TARGET - Via IV administration - Travel to the hepatocytes and ZFN plasmids enter the nucleus to transcribe respective mRNA transcripts MECHANISM OF IDS GENE INSERTION 1. A FokI is attached to each ZFN and brought together by complementarity to the flanking gene sequence 2. FokI homodimerization induces a ds break with overhanging ends 3. IDS gene inserted Cutting the genome occurs via: Insertion of IDS into the albumin gene occurs via: homology directed repair IMPACT OF IDS GENE THERAPY VIA AAV - IDS inserted into albumin locus and hepatocytes express the I2S protein Theory = I2S protein produced in the liver from transgene facilitates breakdown of GAGs in hepatocyte cells – lessens the burden of hunter syndrome RESULTS OF THE AAV GENE THERAPY - Urine GAG levels rose 9% on a low dose - Middle dose declined by 51% after 4 months o Blood tests did not detect enzyme - Highest dose (10x starting) caused more plasma IDS Overall = study did not meet clinical expectations (potency/delivery needs improvement)  DRUGS AND THERAPY (WS3): VOLUME OF DISTRIBUTION = Total dose given/concentration of plasma, to determine how much of the drug is left o Units = volume/kg body weight Determined via: o Dose a human intravenously so that the level of drug is known o Immediately take a blood sample (within seconds) o Can compare the dose to the plasma concentration to gain an estimate Numbers: o If volume of distribution = total body water, then it is distributed everywhere o Consider that all values are for a 70kg person. FACTORS INFLUENCING VD: 1. Plasma binding 2. Lipophilicity 3. Molecular weight/size RENAL CLEARANCE OF DRUGS o Blood passes the glomerulus and gets filtered into urine. o Can be reabsorbed back into plasma circulation. If lipophilic and wants to get back into the peritubular capillaries Can them re-enter systemic circulation. § Anion/cation transporters shuttle the drug back into the bowman capsule to increase excretion. o Excretion is dependent on the level of filtration and the glomerular filtration rate, remove reabsorption from this equation, add tubular secretion from the peritubular capillaries. Reabsorption is dependent on filtration and urine flow rate. Total elimination via the kidneys = amount filtered + amount secreted PH BALANCE KIDNEYS – amount reabsorbed - Reabsorb bicarbonate back FACTORS EFFECTING RENAL CLEARANCE: into the blood - Secrete hydrogen ENZYMATIC DRUG METABOLISM ions into the urine  o Makes lipid soluble drugs more water soluble so that they can be excreted from the kidneys rather than being continually reabsorbed. o Depends on enzymes (most are located intra-cellularly) o Metabolism under 2 reaction type: 1. PHASE 1 o Catabolic o Exposes functional group or introduces it via oxidation, reduction etc o Purpose – increase solubility and conjugate other molecules onto the drug if active sites are exposed. o Phase 1 can lead to phase 2 2. PHASE 2 o Anabolic o Conjugation style o Attach endogenous molecule that is very water soluble to the drug to supercharge water soluble. Always inactivates the drug. Example of phase ½ - paracetamol PHASE 2 DRUG METABOLISM o Metabolite it usually a large molecule attached to a small substrate o Needs a functional group in image to create a conjugate o In overdose the enzyme system needed to break the drug down becomes saturated For the paracetomol this is due to a build up of the toxic intermediate which can cause hepatotoxicity PHASE 1 DRUG METABOLISM o Occurs in liver but can be in other tissues o Some metabolites are more active/toxic Eg: paracetamol (metabolite – hepatocellular death) Prodrugs (codeine -morphine) o Prodrugs are drugs that don’t have any pharmacological activity but can be converted into something that does o When few enzymes for MANY drugs are present drug-drug interactions can occur PHASE 1 DRUG METABOLISING ENZYMES o Broad substrate specificity 1 enzyme may catalyse the metabolism of many different drugs 1 drug may be metabolised by multiple enzymes/isoenzymes o Liver drug metabolising enzymes Usually embedded in ER o Important family = cytochrome P450 super family  o Catalyse oxidative metabolism of xenobiotics BILIARY CLEARANCE OF DRUGS (ENTEROHEPATIC RECYCLING) o Phase 2 elimination causing eliminated via bile o Conjugated drugs that have undergone phase 2 pool in the bile ducts and enter the GIT o - if conjugated to glucose, this conjugate is found in the GIT and will be cleaved off of the drug Recycles the drug back into it’s active form § Enteroheptic recycling What is this? What happens to a drug once it is secreted into the bile ducts? How might this affect the time a drug stays within the body. CYTOCHROME P450’s o Differ in amino acid sequences so will bind to different drugs Can increase/decrease the metabolic rate of the drug by adding an inhibitor/inducer of P450s substrate specificity o enzyme reaction requires: molecular oxygen, NADPH; NADPH–P450 reductase (a flavoprotein) forms a hydroxylated product o ~74 CYP450 gene families three major families involved in drug metabolism in liver (CYP1, CYP2, CYP3) FACTORS INFLUENCING CYP450 ACTIVITY 1. Multi-allelic polymorphisms (mainly SNPs changing AA sequences) Changes substrate binding Intermediate, extensive and ultrarapid metabolisers. o Variations in drug response during phase 1 metabolism in the liver Important for the efficacy/safety of drugs. INTERMEDIATE EXTENSIVE ULTRARAPID - Very slow metabolism - Normal level of - Extremely fast Dosage = should be lower than metabolism metabolism normal Dosage = should be normal Dosage = should be higher than normal 2. Environmental factors such as smoking, diet and co-administration of medications Smoking = binding of tobacco to an aryl hydrocarbon receptor causes increased expression of CYP450 enzymes – causing faster metabolism of drugs Diet = metabolism of citric fruits can cause intermediates which inhibit CYP450 activity – slow metabolism. Vitamin A enhances CYP450 activity – increased metabolism  Co-administration of drugs = CYP450 can be inhibited or induced by drugs. Inhibitors can block the metabolic activity of one/more CYP450 and decrease metabolism of another drug. Inducers can increase metabolic activity of CYP450 and increase metabolism of another drug. PLASMA HALF LIFE AND STEADY STATE - Dosage interval doesn’t affect mean steady state concentration achieved or rate at which it is achieved o Need to be taken dosages at roughly the half-life so that the dose in = dose out 1ST ORDER ELIMINATION KINETICS - Plasma half-life is the same over time. - Half-life doesn’t vary based on plasma concentration (always the same) o It determines time to reach steady state and removal time - Rate of elimination is driven by Cp - Constant/repeated dosing achieves a steady state 0 ORDER ELIMINATION KINETICS - Alcohol, aspirin - Rate of elimination is independent of Cp - Elimination is dependent on turnover of enzyme metabolising that drug o If alcohol consumption is modest, alcohol dehydrogenase can become saturated so only a certain amount of elimination can occur at a time PLSAMA HALF LIFE – 0 ORDER KINETICS - Half-life varies based on plasma concentration - Small changes in dose can lead to disproportionate increases in plasma concentration - Patients require monitoring o Can quickly reach toxic levels DOSING OF DRUGS Rate of elimination = CL x Cp o At steady state dose in= dose out (RE)  Loading dose = if therapeutic efficacy is needed quicker then the drug can naturally meet steady state Maintenance dose rate = RE, or DR = CL x target Cp ENZYMES INVOLVED IN PLASMA HALF LIFE Metabolising enzymes such as CYP450 FACTORS INFLUENCING ENZYME ACTIVITY – PLASMA HL - Drug-drug interactions - Diet - Environmental impacts - Polymorphisms CASE STUDIES Volume of A new drug is being developed by a pharmaceutical company for the treatment 1 distribution; of an anxiety disorder. In the early phase clinical trials, the drug is calculated to have a Vd of