Drug Delivery Flashcards

Question

What are the extracellular antibody strategies?

Answer 1

Direct Targeting of Cancer Cells The antibody binds to a specific antigen on the surface of cancer cells and: - Blocks growth signals (e.g., Trastuzumab against HER2) - Induces cell death by signaling pathways Immune System Activation The antibody flags cancer cells for destruction by the immune system through: - Antibody-dependent cellular cytotoxicity (ADCC) - Complement-dependent cytotoxicity (CDC) Delivery of Cytotoxic Agents Some antibodies are conjugated to: - Chemotherapy drugs (antibody-drug conjugates, ADCs) - Radioactive isotopes (radioimmunotherapy) - These deliver toxic payloads directly to cancer cells, sparing normal cells

Answer 2

Goal: Target the stromal cells and extracellular matrix (ECM) that support tumor growth. The stroma includes: - Fibroblasts - Immune cells - ECM proteins (collagen, fibronectin) Stromal cells often protect tumors and facilitate growth, invasion, and therapy resistance. Ablating or modifying the stroma can: - Enhance drug delivery - Reduce tumor support systems - Increase sensitivity to other treatments

Answer 3

Goal: Destroy or disrupt the blood vessels (vasculature) that supply tumors. Tumors need blood vessels for oxygen, nutrients and waste removal Methods include: - Anti-angiogenic drugs - e.g., Bevacizumab (anti-VEGF antibody). - Vascular disrupting agents (VDAs) - drugs that damage existing tumor blood vessels, causing rapid shutdown of blood flow.

Answer 4

- Starves the tumor - Targets tumor microenvironment - Highly selective + relatively stable - Coupling of toxic agents

Answer 5

Tumor Hypoxia can make tumors more aggressive and resistant to therapy, may promote invasion and metastasis. Incomplete Ablation (tumors can develop alternative blood supply) Side Effects Complex Tumor Microenvironment

Answer 6

- High specificity - Multiple mechanisms of action - Personalised medicine (receptors) - Combine well with other therapies - Long half-life - Antibodies easy to make and validate - Direct genetic or chemical coupling to make chimeras possible | delete

Answer 7

- Very expensive, and complex manufacture - Infusion reactions/allergies - Limited tumor penetration - Developing resistance - Immune side effects - Complex multidose schedules initially considered

Answer 8

The immune system uses checkpoint proteins to prevent overactivation and avoid attacking normal cells. Cancer cells exploit these checkpoints to evade immune detection by “turning off” immune cells. PD-1 (Programmed Death-1) receptor on T cells PD-L1 (Programmed Death Ligand-1) on tumor or immune cells CTLA-4 (Cytotoxic T-Lymphocyte-Associated protein 4) on T cells

Answer 9

- Bi-specific antibodies: engineered antibodies designed to bind two different antigens at the same time. Typically, one arm binds a tumor antigen and the other binds an effector immune cell (like a T cell). One arm binds to a tumor cell antigen, the other arm binds to an effector cell receptor, often CD3 on T cells. This physically brings the T cell into close proximity to the cancer cell, it is then activated locally and releases cytotoxic molecules to kill the tumor cell. Causes - Directs immune attack precisely at cancer cells - Bypasses need for major histocompatibility complex presentation, which tumors often downregulate - Can activate T cells even if they are not tumor-specific initially - Leads to potent, targeted immune-mediated tumor cell killing. - antibody-dependent cellular phagocytosis - antibody-dependent cell-mediated cytotoxicty - triggers involve CD16A engagement by TCR/CD3 engagement of blockage of immunocheckpoints | delete

Answer 10

- High precision - Efficient and versatile - Easy to engineer + multiplex - Cost effective

Answer 11

A cytokine storm is an excessive and aggressive immune response where the body releases a large amount of cytokines very quickly. - Can last up to 3 days - Causes headaches, rigors, lumbar myalgia, fever, tachycardia and more - Can cause multi-organ failure between 12hrs and 15 days

Answer 12

- Good in theory but doesn't work - Works in blood but not solid tumor - Tumors are only about 25% cells, which send cytokines to supress function of other cells - White blood cells neutralised - T cells are 'exhausted' (LoF)

Answer 13

The cancer stem cell hypothesis proposes that within a tumor, there exists a small subpopulation of cells called cancer stem cells (CSCs) or tumor-initiating cells that: Have stem cell-like properties: self-renewal and differentiation. Are responsible for tumor initiation, growth, metastasis, and recurrence. Can give rise to the heterogeneous cell types found in the tumor. - Widely accepted that stem cells cause recurrance of cancer (no proof) - Theory 1 - stem cells differentiate into cancer cells - Theory 2 - cancer cells differentiate into stem cells

Answer 14

- Low specificity and off-target effects - Limited in vivo delivery - Immune responses

Answer 15

Tumor-Associated Antigens (TAAs) - Antigens that are overexpressed or aberrantly expressed in tumor cells but may also be found at low levels in normal cells. - Pros: More widely applicable across patients. - Cons: Risk of autoimmunity since antigens are not truly tumor-specific. Tumor-Specific Antigens (TSAs) / Neoantigens Unique antigens arising from tumor-specific mutations not found in normal cells. - Highly specific targets for immune attack. - Personalized vaccines can be designed based on individual tumor mutations. Viral Antigens Some cancers are caused by viruses (e.g., HPV in cervical cancer, EBV in certain lymphomas). - Vaccines target viral proteins expressed by tumor cells. - Example: HPV vaccines prevent cervical cancer.

Answer 16

- Only works if viral antigen - Other targets more complicated as they are 'self', very few truly cancer specific antigens - Concerns around immune system overactivation - Even if we could vaccinate, too many types

Answer 17

- Tumor ablation - Chemotherapy - Radiotherapy - Small molecules - Oncolytic viruses

Answer 18

DC vaccines involve: - Harvesting a patient’s dendritic cells through leukapheresis - Making a TNF PGE2 cocktail - Reinfusing them into the patient to stimulate a targeted anti-tumor T cell response - Very difficult to do but very effective

Answer 19

- Cancer always trying to survive, develop escape mechanisms to get out of treatment Techniques - Antigen loss - MHC downregulation - Checkpoint expression - By attacking all cells at once, more likely to reduce escape - Huge effort comes with risk of off-targets

Answer 20

- Removes cancer cell from patient modifies and replaces - Very specific - Makes cell look more problem than self - Doesn't always work

Answer 21

- Vaccination + checkpoint inhibition, more agressive treatment - Leading tumor destruction + regression - Checkpoint inhibitors induce TNF

Answer 22

- Analysis can identify specific changes to genome which resulted in cancer - Can indicate whether certain drugs are suitable or not - May identify mutation which may result in cancer

Answer 23

- Substances whose production is increased in cancer cells or healthy cells in response to cancer - May be secreted + found in blood or other samples - Can be used to diagnose, monitor and manage treatment as conc often correlated to prognosis

Answer 24

- Most genetic analysis techniques rely on hybridisation, joining two completmentary strands of nulceic acids - Complementary probe can bind to DNA/RNA to detect presence of sequence - Complementary primer can bind to DNA/RNA to amplify specific sequence - Occurs below mekting temp of nucleic acid - DNA + RNA can hybridise together (heating breaks to ssDNA, then can bind to hybridise

Answer 25

- Method for looking at gross changes to genes + chromosomes - Fluorescently labelled complementary probes hybridised to target - Must be sufficient size but not too large Diagnostic tool - gene amplifications - chromosomal translocations - deletions or duplication

Answer 26

High sensitivity and specificity. Works on interphase (non-dividing) cells. Can be used on paraffin-embedded tissue.

Answer 27

Only detects sequences you specifically probe for. Labor-intensive and requires microscopy expertise. Resolution is lower than some sequencing methods.

Answer 28

- a genetic condition where a cell has either too few or too many chromosomes Aneuploidy can lead to: - Overexpression or loss of key genes (e.g., oncogenes or tumor suppressors) - Imbalances in cellular machinery and metabolism - It can drive tumor evolution and adaptation - up to 93% of breast cancers have changes involving the whole of Chr 17 - chromosomal changes linked to decreased apoptosis, increased proliferation, cell mobility + angiogenesis - these and other significant genomic changes

Answer 29

- HER2 overexpression in 25-30% of BCs - 90-95% due to gene amplification - Results in shorter disease-free survival - Therapy influenced by HER2 status

Answer 30

- Chromosomal translocation - a translocation is when a segment of one chromosome is moved to another chromosome - In some leukaemias, chromosomal translocation results in fusion of genes ABL-BCR - Results in philadelphia chromosome leading yo BCR-ABL fusion protein, an 'always on' tyrosine kinase

Answer 31

- Some changes in the ALK gene implicated in variety of cancer - ALK is a receptor tyrosine kinase - its activity can be altered by mutation of gene, amplification or chromosomal rearrangement a well known fusion/inversion of ALK is with EML4 gene implicated in 2-5% of NSCLC - EML4-ALK fusion screened for using FISH 'breakapart' assay, postive test leads to treatment with ALK inhibitors

Answer 32

- Combination of IHC and FISH - Can dual stain a sample using probes with different antigen + secondary antibodies with dfferent enzyme (horeradish peroxidase common) - Enzyme reaction used to cause certain ions to precipitate (gold-faciliated) and become coloured - Can be seen under standard microscope but less sensitive the FISH

Answer 33

- Detect the presence of a gene or mutation. - Measure gene expression (when used with reverse transcription for RNA → cDNA). - Prepare DNA for sequencing, cloning, or analysis

Answer 34

- template (DNA of any origin or cDNA) - primers (two synthetic oligonucleotides, usually 18-22 nucletides, complementary to 3' end of each - DNA polymerase (replicate template from 3' end)

Answer 35

1 - denaturation (94C for 10 mins) 2 - annealling (30-65C in presence of complimentary primers for ~30 sec) 3 - DNA synthesis (65-75C for 2-5 mins repeat until complete)

Answer 36

RT-PCR is a technique used to convert RNA into complementary DNA (cDNA) using the enzyme reverse transcriptase, and then amplify that cDNA with PCR. It’s essential for studying gene expression because RNA levels tell us which genes are active.

Answer 37

- Traditionally, ethidium bromide is used to stain nucleic acid PCR products in agarose gels followed by visualisation under UV - Alternatively, primers with fluorescent+label or conjugated enzyme can be used - Mulitple products can be detected in different sites

Answer 38

- Regular PCR doesn't give any info regarding amount of DNA interest in intial sample - Enables quantification of product in real time - Forward+reverse primers, DNA pol + probe added to DNA sample - Following denaturing and annealing of primers, DNA pol synthesises new - On forward strand, DNA pol encounters probe - 5' nuclease activity degrades probe, releasing fluorscent reporter - SYBR green binds to dsDNA, binding increases green fluorescence 1000x - can stain gels following electrophoresis - Fluorescent signals generated during each cycle are measured - This fluorescence correlates with the amount of DNA produced, letting you quantify the initial amount of target DNA or RNA (if combined with reverse transcription).

Answer 39

- detected with PCR

Answer 40

- Examples of siRNA, shRNA etc (optimised by design, typically act to supress all cell activity) - Examples of DNA, RNA (optimised by nature, typically act to induce gain of function) - All require intracellular delivery to reach machinery involved in cellular processes

Answer 41

Fusogenic lipids - specific types of lipids that are added to liposomes to enhance their ability to fuse with cell membranes, allowing for direct delivery of encapsulated cargo into the cytoplasm of target cells - Work well in vitro, not in vivo - In vitro cytotoxicity correlates with efficiency - Often poor outcomes to deliver intended targets - Histology of liver, lung and spleen, clinical chemistry parameters and haematology look fine but DNA strand breaks in lung/spleen, inductions of antioxidant response and oxidised nucleobases - inflammatory cytokines were elevated in lung, spleen or liver

Answer 42

- Pores only in certain places - Vasculature growing fast, many defects possible - Once found tumor, must find cancer cells, only 5-25% of tumor - Some therapies are okay killing bystander cells, some aren't - Positively charged materials interact with negatively charged surface before reaching lipid-bilayer - May need very high doses to combat this

Answer 43

- Hard to do and low volume but has cell capable of efficient antigen presenting

Answer 44

- Different drug exposure (quick onset for breakthrough, metabolites, opioid naivety) - Avoid 1st pass metabolism - Efficient non-invasive delivery (transmembrane, reactal, nasal, buccal) - Patient factors (convenience, age, dexterity)

Answer 45

- Immediate release for acute or breakthrough pain (2-6 hours) - Sustained/controlled release for chronic pain (more stable pk, more prone to abuse)

Answer 46

- Better to schedule mediciations rather than waiting - Short acting - IR - every 4h - steady-state ~ 1 day - Long acting - SR - every 12h - steady-state ~ 2-4 days

Answer 47

- potential for abuse predicted on pk profile (high Cmax and short Tmax - correlated with euphoria) - IR forms offer easiest form to recover opioid

Answer 48

Physical barrier - gel formation - tablet formulation with highly viscous structure, becomes gel like with addition of water/alcohol Aversion - utilises noxious component added to powder formulations to discourage abuse Agonist/antagonist - pellets of morphine sulfate with core of sequestered naloxone hydrochloride in ratio 100:4 - if caps are chewed or tampered with, orally active naloxone released Pro-drug - biologically inactive substances metabolised in vivo

Answer 49

Analgesic with narrow therapeutic index - Post-op pain, cancer pain - 1-2mg/ml = effective Cp - Dose titration essential Multiple systems on market - Duragesic = 1st product, a reservoir system, ethanol/hydroy-ethylcellulose gel - 10, 20, 30, 40 cm2 - Up to 72 hrs

Answer 50

- Opioid with partial agonist and antagonist actions - Indicated for moderate-severe pain, peri-operative analgesia and opioid dependence - Short half-life, zero oral bioavailability - Delivered at 5-75ug/hr

Answer 51

- Gel removed + boiled to extract opioid (liquid injected or drunk) - Patches are chewed for mucosal delivery - Multiple patches at once

Answer 52

Causes higher levels of - background pain - peak pain - depression and anxiety - functional impairment Significantly impacts daily life Requires additional dose

Answer 53

- Taken as normal - patient advises not to wait - Most commonly morphine, oxy, fenty, hydromorphone

Answer 54

- Potent u-opioid analgesic with rapid onset of analgesia - Most lipophilic of IR opioids (variety of forms, quickly crosses cell barrier, broad tissue distribution) - Oral mucosal fentanyl citrate (first rapid onset poioid to be approved for BTcp)

Answer 55

- Convenient and easy-to-use - Takes advantage of oral mucosa characteristics facilitating rapid absorption (large SA, high permeability, high vascularity) - High bioavailability (no 1st pass)

Answer 56

- Mutations in the genome can increase probability of developing a certain cancer, or may affect choice of treatment - PCR can be used to detect a number of different mutations (point mutations/SNPs, deletions, insertions) - PCR methods rely on reaction products being different sizes in absence/presence of mutations or the reaction not working

Answer 57

- EGFR is a cell surface receptor that binds EGF and triggers signaling pathways promoting cell division and survival, normally, tightly regulated. - NSCLC counts for 80% of all LC - Drugs including gefitinib and elrotinib inhibit EGFR tyrosine kinase + used to treat NSCLC - Mutations found in exons 18, 19 + 21 of EGFR gene encoding the intracellulat TK domain - These mutations increase sensitivity to 1st, 2nd + 3rd gen EGFR TKIs and can inform treatment options - Sequencing is costly and can be time-consuming so PCR can be used to detect changes by designing primers to give different sized products in absence + presence of mutation

Answer 58

- ASPCR can detect point mutations without reliance on changes in restriction site - Primers are designed so that their 3’ end matches exactly the nucleotide of the allele of interest. - Because DNA polymerase requires a perfect match at the 3’ end to efficiently extend the primer, only the specific allele (wild-type or mutant) gets amplified. - If there’s a mismatch at the 3’ end, no or very little amplification occurs.

Answer 59

- Real-time PCR 1. DNA-binding dyes (e.g., SYBR Green) These dyes bind all double-stranded DNA. - As PCR products accumulate, fluorescence increases. - Simple and cost-effective but less specific (may bind non-specific products). 2. Sequence-specific probes (TaqMan probes) - Probes carry a fluorescent reporter and quencher. - When the target DNA is amplified, the probe is cleaved, releasing fluorescence. - Highly specific to the target sequence. - One specific for wild-type, one for mutant

Answer 60

- A microarray is a microchip or flow cell onto which thousands of known RNA/DNA probes are fixed in a grid-like pattern. - Each probe corresponds to a specific gene or DNA sequence, (large number) - Used to assess expression of large numbers ends determine genotype or sequence nucleic acids - Applied in basic research, human diagnostics, personalised medicine - Advantages: high-throughput, complex patterns - Disadvantages: requires known sequences for probes, less sensitive

Answer 61

- BRCA 1/2 are examples of many genes that are implicated in cancer development when mutated - have tumor supressor activity - Both 1 + 2 are involved in homologous recombination of DNA repair pathways - Autosomal dominant - Carrying certain mutations in these genes can significantly increase risks, especially breast + ovarian - Varies massively between populations, not all mutations equal - Mutations have no affect on survival but change drug sensitivity

Answer 62

- Decision-analytical model developed using 29 different probablity values, combined with full costings of different scenarios + imoact on QALYs - It is cost-effective to screen

Answer 63

Dideoxy NTPs are "chain terminators" that sequence DNA one base at a time, producing highly accurate results for short DNA fragments (up to ~1000 base pairs). - Each labelled with different flurophores - Result is end-labelled sequences of DNA with different lengths - Readily detected by CGE, enabling sequence to be detected - Used for screening/confirming mutations, identifying single-nucleotide changes (insertions, deletions)

Answer 64

- Second generation sequencing techniques can allow more rapid sequencing - Method of specific chem depends on manufacturer - Millions of clusters, each with around 1,000 molecules of DNA can be sequenced in few hours - Tend to be flow based - Sequential processes and imaging

Answer 65

Illumina sequencing is a type of Next-Generation Sequencing (NGS) based on a method called sequencing by synthesis (SBS). It’s one of the most widely used technologies for high-throughput DNA and RNA sequencing. - using DNA polymerase to add complementary nucleotide depending on identity of nucleotide with different fluorescence - will give coloured dots based on DNA cluster present, colour dependent on first nucleotide - end of molecule cleaved off, removing fluorescent molecules too - DNA polymerase then adds complementary molecule then when washed away you get next nucelotide and on and on - pacific biosciences, small scale, real time A = green T = red C = blue G = yellow

Answer 66

- Sample Preparation: DNA/RNA is extracted and attached to a motor protein and adapter (amplification not needed) - Nanopore Device: the sample is added to a flow cell containing many nanopores embedded in a synthetic membrane and a voltage is applied across the membrane. - Translocation and Detection: the motor protein unwinds the DNA/RNA and feeds it through the nanopore. The current will fluctuate depending on which nucleotide is passing through. The system measures these changes in real-time. Messages decoded by computer.

Answer 67

- Immunodetection is a laboratory method used to detect specific proteins or antigens using antibodies. General term to describe detection of specific antigen using antibody binding technology - e.g. immunohistochemistry, immunocytology, western blotting, ELISA, flow cytometry - Antigen can be in situ, in solution, in a membrane plate, etc etc

Answer 68

A laboratory technique used to detect and quantify substances such as proteins, antibodies, antigens, or hormones, especially in blood or other biological fluids. - Used in research + clinically to detect infection of disease markers - Involves adsorption of antigen or antibody to multiwell plates

Answer 69

- A capture molecule is attached to a solid surface, typically the bottom of a 96-well plate. - The sample is added, if the target is present, it binds the capture molecule. - A detection antibody, linked to an enzyme (like HRP or alkaline phosphatase), is added and binds to the target (and incubate) - It is washed, and a substrate is added that the enzyme converts into a detectable signal usually a color change (usually AP or HRP). - The intensity of the color is measured using a spectrophotometer and is proportional to the amount of target present.

Answer 70

- If protein at a low concentration, adsorb capture antibody to bottom of plate before adding sample, wash away else - then add 1st + 2nd antibodies with colours

Answer 71

Diagnosing infections (e.g., HIV, Hepatitis B/C) Cancer biomarker detection (e.g., CA-125, PSA) Allergy testing (e.g., IgE levels) Autoimmune disease screening (e.g., anti-ANA) Cytokine quantification in immunology research

Answer 72

- PSA is secreted by prostate epithelial cells - High (>4ng/ml) levels in blood can indicate PC - Add sample to well - Region above has antibodies that recognise PSA and have dye attached (gold nanoparticle) which are free to move - Next window has more PSA recognising antibodies that will bind to PSAm while boudn to first antibody - Dyed antibodies will bind to tethered antibodies without PSA anyway, as the control line

Answer 73

IHC is a laboratory technique used to visualize specific proteins in tissue sections by exploiting the high specificity of antibodies. IHC detects a specific antigen in a thin slice of tissue by using: - A primary antibody that binds the target protein. - A secondary antibody (often enzyme-linked) that binds the primary antibody. - A color-producing substrate that makes the antibody binding visible under a microscope. - Can also be used to incubate antibodies with tissue - Used to screen tissues for cancer eg HER2 (graded from 0-3+ from none to excess, FISH recommended if 2+)

Answer 74

- Flow cytometry is a powerful laboratory technique used to analyze the physical and chemical characteristics of cells or particles as they flow in a fluid stream through a laser beam. It can measure: - Cell size and granularity (physical characteristics) - Expression of surface or intracellular proteins using fluorescently labeled antibodies - DNA content (e.g., for cell cycle analysis) - Live/dead status of cells - Cell subtypes (e.g., T cells, B cells, cancer cells) Used for: - Detecting specific molecules on/within cells - Can also be used for sorting/isolating specific sub-populations of cells - Powerful analytical technique for cell bio research - Important for immuno-phenotying, diagnosing

Answer 75

- Cells are suspended in a fluid and funneled into a narrow stream. - Cells pass one-by-one through one or more laser beams, forward scatter measures cell size, side scatter measures internal complexity/granularity) - Antibodies tagged with fluorescent dyes bind specific cell markers. As labeled cells pass the laser, they emit fluorescent signals detected by sensors. - Each cell is analyzed individually. Results are displayed in dot plots or histograms

Answer 76

- Immunophenotyping - Diagnosis - Cell sorting/purification - Apoptosis & viability testing - Functional assays

Answer 77

- B-cell chronic lymphocytic leukaemia characterised by gradual accumulation of CD5+ and CD19+ malignant B cells - Analysis of peripheral blood mononuclear cells - Labelled with antibodies against CD5, CD19 + RTK ROR1/2

Answer 78

- Multiple analysis quantified per tube/well - Low volume - Sensitive/accurate

Answer 79

- Light emitted by a camera in presence of specific label, this is afetr ruthenium - Patient sample with POI - Antibody that recognises labeled with biotin - If particular analyte present in sample, will be sandwiched between this and another antibody that recognises it - Incubated with a streptavidin-coated paramagnetic bead, binds to biotic very strongly - Has 4 binding sites - These then sent through detector with magnet to capture beads, passes electrode to rutherium which emits light - More rutherium, more protein in sample

Answer 80

- Multiples immunoassay systemise many targets can be identified + quantified per well - Each bead type is flagged with a different coloured fluorescent marker + captures specific protein - Secondary antibody has phycoerythin tag - Beads are read in a flow-based system or layer at bottom of well - One laser classifies type of bead, the other quantifies PE signal

Answer 81

- Amplified by PCR, purified, then amplified again - Products from second round are sequence - Overall cancer detection using PCR based assay = 82% - Used samples from hundreds of patients with stage I-II cancers of ovary, liver, stomach, pancreas, oesophagus - Test predicted 20% of stage I oesophageal cancers but 100% of liver cancers - Cancer site localised to 2 sites in 83% of patients and 1 in 63%

Answer 82

- Patients were already diagnosed - Controls were healthy individuals - would other disease states change results

Answer 83

- cfDNA is normally present in serum from many cell types - Generally, cfDNA is unmethylated - Methylation patterns change in tumors + are characteristic of tumor location - Cancer patients will have a mixture of methylation patterns from healthy + tumor cells - Methylation analysis + machine learning can determine prescence and origin of cancer

Answer 84

- antigens - anti angiogenesis - antibodies - tumor suppressor genes - suicide genes - immunotherapy

Answer 85

- Aiming to deliver gene to overcome disease/infection - Disease can be acquired or underlying - Immunotherapy can reprogram patietns immune system to target disease - Patients cells can be transfected with a gene encoding a protein drug

Answer 86

Suicide genes are specially engineered genes introduced into cells (usually cancer cells or genetically modified immune cells) that can trigger the cell’s self-destruction when activated. - A suicide gene is inserted into the target cells via vectors (often viral). - The gene encodes an enzyme that can convert a non-toxic prodrug into a toxic compound that kills the cell. - If the modified cells start behaving dangerously (e.g., causing side effects or growing out of control), the patient is given the prodrug, and the suicide gene activates it, killing the modified cells. Also known as gene-directed enzyme producing therapy (GDEPT) Often uses a combination of HSV thymidine kinase and ganciclovir

Answer 87

Oligonucleotides are short strands of nucleic acids, typically made up of around 15 to 30 nucleotides. - If gene that produces protein to cause disease state is present, expression can be reduced by including decoy oligonucleotides that interfere with gene transcription - Often will bind to promoter region of genen and will stop expression - Alternatively can be delivered to block translation of mRNA for particular protein - Will bind to complementary sequence in mRNA - Easier to deliver than genes Antisense oligonucleotides (ASOs): Bind mRNA to prevent it from making protein. siRNA (small interfering RNA): Trigger degradation of target mRNA. Aptamers: Fold into shapes to bind proteins, blocking their function.

Answer 88

- >50% of cancers have reduced ability of miRNA 34a - It can regulate signalling pathways which control cell proliferation, migration and invasion, resistance to apoptosis + immune invasion - Targets include androgen receptor, PDL-1, C-MYC + CD44 - miRNA therapy limited by stability, immunogenicity + non-specific delivery - Chemically modified miRNA -34a which overcomes these limitations - Folate receptor over-expressed in cancers including colorectal, lung, ovarian + breast

Answer 89

- Delivering miRNA to cells in reducing growth and survival (only fully modified) - Used as combination therapy

Answer 90

- Directly into plasmid/naked gene like a nucelotide and inject into patient hoping it reches target tissue - Cell based ex-vivo approach - Take pt's own cells , in vitro - Replace into patient, treating via haematopoietic system

Answer 91

- Nucelotides are very large and very hydrophilic - Poor stability in serum and when in cells they get released into endosome - Getting to the nucleus, have to get past all obstacles, cytoskeletons etc, nuclear pores very small, only < 40kDa for pore transport, genes are ~3-5MDa

Answer 92

- Most vectors are viruses - This is due to viruses evolving to deliver nucleic acids into cells - Theory: take a wild-type virus, remove viral genome and replace with therapeutic gene, virus able to infect cell, deliver cargo but unable to duplicate

Answer 93

e.g HIV - ssRNA genome, 8-11kb, 2 copies of genome - ~100nm diameter, covered in glycoproteins - some infect only dividing cells (not lentivirus) - can tagret specifc cells, possible immune response, risk of tummor development

Answer 94

- HIV binds to cell, inserts genome - genome hitches ride on cytoskeleton straight into nuclear pores - viral genome randomly inserted into genome, dormant until transcribed into viral messenger RNA - umRNA assembled into proteins of virus - lentivirus enter randomly and if inserted next to proto-oncogene it can activate it (if inserted into TSG it can suppress action) - modern techniques reduces copy number of deisgning viruses more effectively

Answer 95

- common cold - dsDNA genome - 60-90nm diameter - infects dividing + non-dividing cells - doesn't integrate its genome into ours - can tagret specific cell types - possible immune response

Answer 96

- Recombinant DNA virus p53 - Works in combination with oncorine - Genetically modified virus - Not delivering a gene, but produces E1B blocking p53 so th evirus can't replicate + prevents cell death

Answer 97

- ssDNA genome (genome ~5kbp) - ~20nm diameter - Infects dividing + non-dividing cells - Specific integration - Can be used to target specific cell types - Little immune response - Glybera developed by uniQure approved in Nov 12 - For treatment of lipoprotein lipase deficiency - Rare - affects 1-2 people per million - causes dramatically increased fat in blood - One-time series of small infections in leg - AAV serotype 1 used as vector

Answer 98

- High transfer efficiency - Some inherant tissue tropisms

Answer 99

- Limited DNA/RNA capacity - Difficult to produce at scale - Can be immunogenic

Answer 100

- direct injection - gene gun - ultrasound - elctroporation

Answer 101

- encapsulation - complexation - nanoscale particulars

Answer 102

- liposomes, dendrimers, cationic polymers

Answer 103

- easy to prepare and modify - reduced immunogenicity - large capacity

Answer 104

- poor efficiency - transient

Answer 105

- A mixture of cationic liposome and DNA - Can interect with +ve charge cell surface + be taken up into cell - Ideally avoid lysosome formation

Answer 106

- Commercialised DNA transfection reagents available

Answer 107

- High +ve charge, toxic, short circulation, large particle size, unstable

Answer 108

- Used to deliver both genes and oligonucleotides - Including: liposomes, drug-loaded liposomes, targeted liposomes, stealth liposomes

Answer 109

- Built upon a central core - +vely charged so complex well with -ve charged nucleic acids - Wide range of chemical approaches + functional groups - Surface groups can be functionalised for targeting stealth etc - Can be designed to release under specfic conditions

Answer 110

- Cationic polymer and nucleic acid combined - Forms a toroid, a circular complex - Commonly generated by polyerlysine etc

Answer 111

- HIV TAT protein shown to translocate cell membranes - Short sequences of theses able to cross membranes - Low cytotoxicity, dose dependent + can be conjugted to carriers - Tend to arginine rich - Various uptake mechanism including direct translocation - Promising for oligonucleotides, packaging and delivery

Answer 112

Ideally - small, stable in serum - low toxicity, specific cell targeting - cytosolic release of cargo - nuclear targetting - high transfection efficiency

Answer 113

- blocking angiogenesis can halt or limit tumor progression, achieved through genes or oligonucelotides - antisense RNA or siRNA to block translationof GF or its receptor - oligonucleotides to block transcription of GF or its receptor - gene therapy to induce production of soluble receptor or mAb - eg glioblastoma multiforme - VEGF highly up-regulated in GBM tissue and levels correlate to disease progression - gene for soluble VEGF-receptor delivered in murine glioma model mops up VEGF and reduces angiogenesis - siRNA against VEGF-mediated angiogenesis in tumors, 120 nm 'nanoplexes', effective 'in vivo'

Answer 114

- can deliver directly into systemic circulation or into tissue - uses a vector (such as AAV) cell based delivery - correcting a defect/transforming the cells in some way that makes them appplicable - these added to a harmless retrovirus or lentivirus and combined with pt stem cells - stem cells with corrected gene returned to patient

Answer 115

- Bone marrow transplant - Basically wipes out patients existing haematopoietic system - Cells collected from donor, aren't changed - Drawbacks - reconstitution of immune system isnt great

Answer 116

TK cell therapy - allogenic T cells transfected with HSV-TK - zalmoxis approved in 8/16 - oncraft and help reconstitute the immune system alongside HCST from same donor to improve overall survival + non-relapse mortality Cytotoxic T cells are activated by antigen presenting cells such as dendritic cells which prime them with specific antigens for target cells - T cellls recognise and kill cells which express these antigens via release of perforin and grandzyme, inducing apoptosis - efficacy process can be improveed with cell engineering approaches

Answer 117

- Artificial APCs are an emerging approach - decorate cells/microparticles with antigens to prime T cells - Off the shelf - no reconstitution required

Answer 118

- T cells genetically engineered to express a Chimeric Antigen Receptor - CAR T cells proliferate and kill tumor cells upon contact with antigen receptor - Continued generations have increased signalling

Answer 119

- White blood cells obtained from pt - Cells sent to manufacturer using antibody coated beads to activate T cells - Activated T cells reprogrammed using retroviruses to express CARs - CAR T cells are expanded ex vivo - Cells sent back to treatment centre - Patient recieves lympodepleting chemo prior - CAR T cells tranfused to pt

Answer 120

MSCs have a number of characteristics making them suitable for therapeutic cells - Ability to home to site of injury - Anti-inflammairy properties - Hypoimmunogenic - Off the shelf However some pro-tumorgenic effects

Answer 121

- Current approaches to nucleic acid therapy are often transient + have varying degrees of success - Permenant gene editing in a specific way is promising eg correlating an effect, knok=cking down a specific gene or activating

Answer 122

- A gene editing system based on components of adaptive immune system of certain bacteria of other microbe - Clustered Regularly Interspaced Short Palindromic Repeats - Could use virus to deliver CAS9 genes - Could also use for generating CAR T cells or modifying T cells - Influencing angiogenesis, correct genes, enzyme engineering

Answer 123

- Head and neck cancer - SOX2 overexpression in cancer stem cells promotes growth and resistance to apoptosis - Specifically knocking down SOX2 in tumor cells using CRISPR-CAS9 should cause tumor regression - CAS9 mRNA and sgRNA packaged in LNPs targeting EGFR - Mice with xenograft tumor treated with this LNP - 90% inhibition of tumor growth and 90% survival

Answer 124

- Can be engineered to increase specificity or change MoA eg dCAS9 has a mutation which deviates nulcease activity and enable it to be used to control gene expression - Precise CAS 9 targeting enables genomic mutation prevention - Method developed to tune guide RNA so that two target sites can be detected - Current work focuses in gain of function mutations mutations which can lead to resistance - Genes which develop the mutation can be cleaved, inactivated them - This 'mutation prevention' system could be used to prevent disease - First US trial of a CRISPR derived cancer therapy - Knockout of TCR and PD-1 genes plus transfection with TCR which recognises a cancer antigen - Therapy is safe and well tolerated - Only 10% of T cells used in trial had all 4 genetic mutations - Mixed response - tumors in 2/3 patients stopped then resumed

Answer 125

- Prime editing of cells used to reate >1000 variations observed in >40,000 cancer patients - Cells were often used to identify new pathogenic p53 variations

Answer 126

- Small, cheap, specifc, versatile - Developments in base + prime editing offer more control

Answer 127

- Off-target effects - unintentional editing similar genomic regions to target - oncogenic? - Autoimmune risks, possible unintended side effects on healthy tissue - Efficient delivery to all target cells or specific subset remains challenging - Ethical concerns - Limited simultaneous change - Abuse

Answer 128

The tip of the Y in the antibody Usually for the antibody to bind to its target - for normal immunity this would be an antigen - for drugs in a disease marker (eg cancer, a sepcfic or selective antigen for cancer to be recognised)

Answer 129

The tail of the Y - The typical functions of the antibody - contains the effector functions of the antibody and the binding sites that recognise the complement system

Answer 130

- two heavy chains, two light chains joined by disulphide bond between conserved cysteine residues at hinge region - lage molecules confined to IV admin only

Answer 131

- overall structure/isotope - antigen binding variable region - hinge region (aa sequence) - glycans - Fc receptor

Answer 132

- immunogenicity - selectivity/specificity - efficacy - MoA - serum t1/2 - stability - PK + PD

Answer 133

- Made from mouse tissues, and very immunogenic - They were continuoulsy mixed with human cells, becoming less and less immunogenic (safer)

Answer 134

extra cellular activity - antibody-directed cell killing - solid tumor growth requires angiogenesis - T-lymphocytes function to kill intruding cells that don't have protective surface markers - radiation is mutagenic - toxins can be redirected - effectors cells can be brought in proximity of cancer - CAR T cell technology - outgrowth of bio-pharmaceuticals can target and kill

Answer 135

- next gen antibodies - antibody attached to toxic agents is immunoconjugate - if warhead used is sm cytotoxic, is specifically an ADC - antibody is responsible for targeting - linker responisble for release - warhead responsible for apoptosis

Answer 136

- less off target affects than conventional chemo - better tolerated than alternatives - phase II study compared Trastuzumab Emtansine (an ADC) to conventional mAbs and found that it caused fewer and milder ADRs

Answer 137

Cysteine modification requires step approach - initial reduction of interchain disulfides followed by chemical conjugation More specific sites through drug, antibody ration still variable Alternatives - glycan modified approach - Thio-mab approach

Answer 138

- Initiated by antigen in combination with major histocompatibility proteins on the surface of antigen presenting cells - These bind to T cell antigen receptor to propagate inrecellular signals in T cells - Full T cell activation requires second 'co-stimulatory' signal - Some receptors however serve to limit T cell activation (immune checkpoints) which can be blocked by mAbs to boost weaker anti-tumor responses

Answer 139

Cancer cells can over express inhibitory receptors on their surface, thereby stopping T cell activation, blocking immune responses

Answer 140

- Sometimes the Fc effector function must be reduced - IgG4 more suitable than IgG1 - But IgG4 more prone to disulfide exchange - IgG4 can be genetically engineered to become more like IgG1s

Answer 141

- Afucosylated IgG has enhanced affinity for certain things, increasing antibody-dependent effector function

Drug Delivery Flashcards

(166 cards)