BMS 3008: Integrated Biomedical Sciences Lecture 3: Experimental Design and Clinical Trials

Part 1: Hypothesis-Driven Laboratory Project

  • Growth Factors and Cell Differentiation:
    • Growth factors are peptides released by signaling cells to influence target cells.
    • They play a crucial role in controlling the differentiation of neural cells.
    • As organisms develop, stringent controls are placed on cells to prevent uncontrolled growth.
  • Regulation of Neural Cell Differentiation:
    • Gene expression changes are key for differentiation.
    • Two approaches to identify involved genes:
      • Candidate approach: Informed analysis using quantitative PCR but with limited output.
      • Global analysis: RNA-sequencing to interrogate all gene expression changes.

RNA-Sequencing Principles

  • RNA Sequencing Steps:
    1. Isolate RNA from samples.
    2. Fragment RNA into short segments.
    3. Convert RNA fragments into cDNA.
  • Transcriptome Analysis:
    • Align sequencing reads to the human genome to identify expressed transcripts, forming the transcriptome.
    • Compare transcriptomes between cell populations (e.g., with and without growth factor) to identify transcriptional changes during differentiation.
    • Heatmaps are used to display gene expression changes, with columns representing individual replicate samples, genes on the right, and expression level indicated by color.

PhD Project: Key Regulators of Neural Cell Differentiation

  • Experimental Design:
    • PhD student James treats undifferentiated neural cell populations with and without growth factor.
    • Performs RNA-seq analysis to identify differentially expressed genes.
    • One identified gene is Synaptophysin.
  • Validation using Quantitative RT-PCR:
    • Validates synaptophysin mRNA levels are elevated upon cell differentiation.
      1. Isolate RNA.
      2. Generate cDNA by reverse transcription.
      3. Determine cDNA level for sequences of interest by quantitative PCR.

Techniques to Assess Synaptophysin Protein Level Changes

  • Methods:
    1. Western blotting: Detect protein hybridized to a membrane by antibodies.
    2. Immunofluorescence: Detect proteins directly in intact cells by antibodies, providing cellular distribution information.
    3. ELISA: Quantifies proteins bound by antibodies on plastic.
    4. Flow cytometry: Quantifies proteins in cells using fluorescently-labeled antibodies.

Immunofluorescence

  • Indirect Immunofluorescence:
    • Detects native synaptophysin in cells.
    • Involves primary antibody binding followed by secondary antibody binding with fluorophore activation.
    • Advantages: Allows multi-color imaging; proteins are in physiological state.
    • Disadvantages: Protein of interest may not have suitable antibody; expensive and time-consuming.
    • 1o1^o Antibody Binding, Secondary Antibody Fluorophore activation, 2o2^o Antibody Binding
  • Direct Immunofluorescence:
    • Detects fluorescently-labeled Synaptophysin via transfection of cells with plasmid GFP-Synaptophysin fusion protein.
    • GFP fluorophore is activated.
    • Advantages: Quick, allows multi-color imaging, cheap.
    • Disadvantages: Signal variation between samples, relies upon cells being transfectable, fluorophore may affect normal protein activity.

Confirming Red Fluorescence is Synaptophysin

  • Methods:
    1. Perform experiment in cells lacking expression of synaptophysin.
    2. Block primary antibody binding with specific peptide/epitope.

ELISA: Quantifying Synaptophysin Levels

  • Enzyme-linked immunosorbent assay (ELISA): Useful technique to quantify the level of a specific protein in a complex protein mixture (cell lysate).
  • Sandwich ELISA Steps:
    1. Cell lysate is added to a vessel containing capture antibody.
    2. Target protein (analyte) specifically binds to capture antibody.
    3. Vessel is washed, and primary antibody is added, binding target protein.
    4. Secondary antibody is added, selectively binding to primary antibody.
    5. Secondary antibody-conjugated enzyme is activated to generate a color change.
    • The intensity of color change is proportional to the quantity of target protein in the mixture.
  • ELISA Procedure:
    • Synaptophysin capture, synaptophysin labeling with 1o1^o Ab, activate 2o2^o Ab conjugate with substrate and read color change.
      Important controls include No cell control, - Growth Factor, + Growth Factor, and Neural stem cell.
      Conc. (ng/well) O.D Mean O.D
      0 0.02 0.03 0.01 0.02
      2 0.04 0.03 0.05 0.04
      20 0.35 0.39 0.34 0.36
      40 0.56 0.62 0.68 0.59
      60 0.98 0.93 0.97 0.96
      80 1.2 1.24 1.19 1.21
      100 1.31 1.25 1.16 1.24
      Sample O.D. Mean O.D.
  • cytokine 0.07 0.03 0.05
  • cytokine 1.18 1.06 1.12
  • Preparing for ELISA:
    • Collect cell lysate and measure protein concentration.
    • Student requires 50μg50 \mu g total protein per well in a total volume of 50μl50 \mu l.
  • Calculations Example:
    • Concentration of untreated cell lysate = 2.3mg/ml2.3 mg/ml
    • Calculate dilution factor: 2.3mg50μg=2300μg50μg=46x\frac{2.3 mg}{50 \mu g} = \frac{2300 \mu g}{50 \mu g} = 46x
    • Calculate volume for 50μg50 \mu g: 1ml46=0.0217ml\frac{1 ml}{46} = 0.0217 ml or 21.7μl21.7 \mu l
    • Add 28.3μl28.3 \mu l H2OH_2O to generate 50μl50 \mu l solution.

Generating a Standard Curve for ELISA

  • Standard Curve:
    • Wells contain a range of known concentrations of synaptophysin.
    • Quantifying unknown samples requires the generation of a standard curve.
      *Treated ELISA plate set-up, Untreated
      Note: each sample, including standards, are plated in triplicate
      Why?

ELISA Plate Reading and Data Analysis

  • Spectrophotometer: The ELISA plate is read using a spectrophotometer for each unknown and standard samples.
  • Optical Density: Optical density is directly proportional to protein concentration.
  • Calculating Synaptophysin Concentration:
    1. Plot curve of standard samples (O.D on y-axis; [Protein] on x- axis).
    2. Estimate synaptophysin concentration in the lysates (0.05 and 1.12 for untreated and treated cells respectively).
      *Draw a line of best fit.
      TreatedCells=71ng/wellTreatedCells = 71 ng/well and UntreatedCells=3ng/wellUntreatedCells = 3 ng/well

Determining Synaptophysin Amount per mg of Total Protein

  • Calculations:
    • Amount of total protein per well in untreated sample was 50μg50 \mu g.
    • In 50μg50 \mu g total protein, we have 3 ng synaptophysin.
    • Amount of synaptophysin in 1 mg of total protein is: 1mg50μg\frac{1 mg}{50 \mu g} or 1000μg50μg=20\frac{1000 \mu g}{50 \mu g} = 20
    • 3 ng x 20 = 60 ng/mg in untreated cells.
  • Fold Change Calculation:
    • James has discovered that during neural cell differentiation, synaptophysin gene expression increases, that results in a 23.7 fold increase in cellular levels of the protein.
  • Fold change in synaptophysin level = Growth factor treated cells / Control cells can be calculated as: 142060=23.7foldincreaseinexpression\frac{1420}{60} = 23.7 fold increase in expression

Using siRNA to Assess Synaptophysin Activity

  • siRNAs:
    • Short double-stranded RNA molecules (~21 base-pairs).
    • Specific hybridization of siRNA to complementary mRNA causes degradation of mRNA.
    • This reduces protein levels encoded by that degraded mRNA.
  • Synaptophysin levels are depleted in cells transfected with a synaptophysin- targeting siRNA.
    *Experimental controls
    scrambled siRNA:
    Synaptophysin siRNA:
    1o/2o Antibody Binding
    Synaptophysin
    Anti-Synaptophysin Ab

Clinical Trials

  • Aims of Clinical Trials:
    • To improve current treatments available to patients
    • To prevent disease.
    • To better screening and diagnoses techniques for diseases.

Clinical Trial Design

Once you have a drug that has shown great promise in mammalian cell culture and animal model systems you may be in a position to conduct
a clinical trial
However, the process of clinical trial design is complex and you must be aware of numerous criteria before embarking:
i. What research questions are being addressed in the trial?
ii. Why should the trial be conducted?
iii. Is the trial adequately designed to answer the questions it addresses?

Controlling Bias in Clinical Trials

  • Methods to prevent bias:
    • Randomisation: Assigns patients to treatment arms by chance.
      Example, patients will be assigned to a single group by tossing a coin!
    • Blinding: Ensures that neither patients, doctors, nor researchers know to which group each patient has been assigned.

Clinical Trial Phases

  • Phase 1:
    • Evaluate safety and determine safe dose range, identify side effects.
    • Small groups of healthy volunteers (but cancer drugs use patients who failed conventional treatments).
    • Identifies appropriate route for administering drug.
    • Gradual increasing doses are generally given to successive individuals (dose escalation).
      *3+3 model is used to identify Maximal Tolerated Dose
      Maximal tolerated dose (MTD) is defined as the highest dose of drug that does not elicit significant toxicity
      Dose-limiting toxicity (DLT) is where the patient experiences any number of unacceptable adverse events (reactions) to the drug
      *During the course of the trial, patient are monitored routinely to assess:
      i. Drug levels in blood: information on drug kinetics in the body
      ii. Predicted effects of drug on patient: biomarker analysis (e.g. blood pressure is measured for an anti-hypertensive drug, prostate specific antigen measured for a prostate cancer drug).
      iii. Toxicity - i.e. unwanted side effects
      *PK and PD measurements
      Pharmacokinetic studies. What the body does to the drug. e.g. what concentrations are achieved in blood? How long are they maintained?
      Pharmacodynamic studies. What the drug does to the body. e.g. measure change in enzyme activity to show the drug acts on planned target.
      Toxicity.
  • Phase 2:
    • See if the new drug or treatment is effective using defined biomarkers/treatment response in patients
    • Further evaluate its safety
    • Larger groups of patients (40-100).
      *Cancer trials, they normally occur in one specific cancer type (unlike Phase I)
  • Phase 3:
    • Further determine effectiveness and side-effects of the new drug or treatment
    • Strict eligibility criteria; comparison with standard treatments.
    • Large groups of people (more than 200) for statistical validity
      *Phase III trial has strict and well-defined eligibility criteria (i.e. inclusion and exclusion criteria); patients enrolled in the trial must meet these criteria
      Generally by comparison with standard treatments: Superiority Trial.

also to collect information that will allow it to be used safely
Generally involve large groups of people (more than 200) for statistical validity etc

  • Phase 4:
    • Continued testing after the drug or treatment has been approved or marketed. e.g. to collect information about their effect in specific populations and side effects from long-term use.

Ethical Considerations for Clinical Trials

  • 7 Ethical Requirements:
    1. Collaborative partnership
    2. Social Value
    3. Scientific Validity
    4. Fair subject selection
    5. Favourable risk-benefit ratio
    6. Independent review
    7. Informed consent
  • Informed consent consists of 4 elements
    Competence of the subject
    Disclosure of information to the subject
    Understanding or comprehension by the subject
    Voluntariness of the decision
    Ethics committees
    Every clinical trial is covered by regulations that protect the health, safety and dignity of the people taking part.
    All medical research involving people in the UK, whether in the NHS or the private sector, has to be approved by an independent research ethics committee.
    The committees are often based at local hospitals and are formed of local people, such as health professionals, patients, lawyers and members of the public.
    They have to include members who are not health professionals.
    All clinical trials of medicines need to be authorised by the Medicines and Healthcare products Regulatory Agency (MHRA)