HMB204 Lecture 3: Modern Techniques in Translational Research PDF

HMB204 | Introduction to Human Biology Modern Techniques in Translational Research Lecture 3 James Hong, PhD Tested Translational research tools Unlike clinical research, our goal is to try to understand as much about human disease without using human samples To do this, we need to establish: Robust...

HMB204 | Introduction to Human Biology Modern Techniques in Translational Research Lecture 3 James Hong, PhD Tested Translational research tools Unlike clinical research, our goal is to try to understand as much about human disease without using human samples To do this, we need to establish: Robust models of human disease Genetic and pharmacological tools to test an intervention/therapeutic Imaging or functional readouts to evaluate the outcome of the therapeutic Tested Animal models of disease 1. Cellular models of disease 2. Animal models of disease Tested Cellular models of disease If you are interested in studying a particular cell population or even an organ in a controlled environment (like a cell culture dish, in vitro or isolated tissue in a dish ex vivo) Advent of induced pluripotent stem cells (Nobel prize 2012) allows you to derive cells from patients with the disease and revert them back to a stem cell state For instance: Neurons derived from an Alzheimer’s patient’s skin cells Tested Primer on iPS technology 1. Somatic Cell Isolation: Obtain adult cells from the patient 2. Reprogramming: Introduce specific factors (transcription factors) to the adult cells, which can reset their epigenetic state and induce pluripotency 3. Culture: Cultivate the reprogrammed cells in a laboratory setting, allowing them to multiply and form colonies Key is that it would retain the somatic mutations from the diseased patient Using iPSCs for disease mechanism and therapy Tested Not Tested Case study: iPSCs in ALS iPSCs from ALS patients can be efficiently induced into motor neurons MNs differentiated from iPSCs carry missense mutations in the FUS and TDP43 genes iPSCs recapitulated several neurodegenerative phenotypes, including mislocalization of RNA binding proteins FUS/TDP43 into the cytosol stress granules cellular vulnerability Okano et al. Trends in Pharmacological Science. 2020 Not Tested Case study: iPSCs in ALS High throughput drug screening A single drug target might not work for all forms of ALS Personalized medicine is necessary Okano et al. Trends in Pharmacological Science. 2020 Not Tested Case study: iPSCs in ALS Clinical trial for ALS drug: 1. Take cells from a patient 2. Generate iPSCs from the cells 3. Test whether or not the drug is effective in vitro prior to giving it to the patient Okano et al. Trends in Pharmacological Science. 2020 Tested iPSC-derived Organoids 3D miniaturized versions of organs or tissues grown in vitro from stem cells or tissue-specific progenitor cells They recapitulate key structural and functional characteristics of their respective organs Tested Advantage of organoids Human Relevance Disease Modeling Drug Discovery and Testing Precision Medicine Developmental Biology Tested Disadvantages of organoids 1.Complexity and Heterogeneity 2.Variability and Reproducibility 3.Limited Lifespan and Size 4.Lack of Systemic Interactions Not Tested Case study: Cerebral organoids iPSC derived from patient with microcephaly (A3842) induced by a nonsense mutation in the Ckd5rap2 gene Organoids show marked reduction in number of DCX+ neurons and smaller SOX2+ progenitor pools Lancaster et al. Nature. 2013 Tested Animal models of disease If you are interested in recapitulating the symptoms of a disease in animals (in vivo) Animals can be induced to have the disease (induced models) Animals can be genetically modified to have the disease (spontaneous models) Tested Advantages of animal models 1.Disease Understanding 2.Initial Safety Testing 3.Pharmacokinetics and Dosage 4.Genetic Manipulation Tested Disadvantages of animal models 1.Limited Translational Success 2.Species Differences 3.Unable to Model Complex Human Lifestyle Tested Examples of failures 1.Stroke Treatments: Neuroprotective drugs that showed promising results in animal models for stroke have failed to demonstrate similar efficacy in human clinical trials Examples include NXY-059, minocycline, and tirilazad 2.Spinal Cord Injury (considered by many as the “graveyard” of translation) Numerous promising therapies for spinal cord injury that showed functional recovery in animal models have not translated successfully to human patients Examples include treatments involving chondroitinase ABC, neurotrophic factors, and stem cell transplantation Tested Examples of successes 1.Antibiotics: Penicillin: Initially demonstrated in mice and rabbits, leading to its successful use in humans and revolutionizing the field of antimicrobial therapy 2.Vaccines: Animal studies played a critical role in the development and testing of vaccines for diseases like polio, measles, mumps, rubella, and hepatitis B, leading to their successful use in human populations Tested Induced animal models of TBI Tested Induced models of SCI Impactor (Contusion Injury) Clip (Compression) Transection Tested Spontaneous models of CNS disease 1.Parkinson's Disease (PD): The MPTP-treated primate model Administration of the neurotoxin MPTP to non-human primates results in the selective destruction of dopamine-producing neurons, leading to motor impairments Alzheimer's Disease (AD): The APP/PS1 transgenic mouse model Genetic mutations associated with early-onset familial AD, leading to the accumulation of amyloid-beta plaques and neurofibrillary tangles Some diseases can be modeled both ways Degenerative cervical myelopathy Ossification of the ligament of the spine that leads to spinal compression over time Twy mouse model (tiptoe-walking Yoshimura): mutation that needs to ossification in the C2/C3 spine leading to spinal cord compression Insertion of an ossifying material underneath the C5/C6 lamina compresses the cord over time Which is more clinically relevant? Tested 10-minute health break Ask me some questions! Tested What’s next? What happens after you recapitulate a human disease? You often try to understand two mechanisms in translational research: Pathobiological mechanism: How does it happen? What cells / molecules are involved? What is the sequence of events? Therapeutic mechanism: Is your therapy effective? If so, how does it work? What cells / molecules are involved? Is it effective across the entire disease trajectory? Understanding the pathobiology of the disease Understand what it looks like in humans Histopathology (tissue sections and staining) DNA/RNA/Epigenetic profiling Behavioural testing Recapitulate it in your model Investigate from bottom up, or top bottom (by scale): Bottom-up: you know the gene or molecule that is causing the problem, you want to find the cells involved Top-bottom: you only see the disease phenotype, but you don’t know the cells or molecule that is causing the problem Tested The top-bottom approach: an example with SCI PICO Framework Question: In spinal cord injury, (P) how do we remove the fibrotic scar? (I) Is it beneficial relative to no removal? (C) How does it impact functional recovery? (O) Model: A weight drop model of spinal cord injury in mouse What do you now need to know to answer this question? What is the cellular origin of the fibrotic scar? How can we silence its production? Does this silencing result in a functional change? Not Tested The bottom-up approach: an example with DCM PICO Framework Question: In degenerative cervical myelopathy (DCM), (P) Does having APOE4 carrier status, (I) Relative to APOE2/3 carrier status, (C) Have an impact on disease progression? (O) Model: An induced mouse DCM model What do you now need to know to answer this question? What is the impact of carrier status on the cells in the system? Is there a primary cell type in the spinal cord that is more effected by this than others? What is the impact of carrier status on the behavior and disease phenotype? Not Tested Example finding the cellular origin of the fibrotic scar To find the cellular origin of the fibrotic scar: Stain for fibronectin (FN1) FN1 Stain for some cell specific markers: PDGFRB (pericytes) ALDH1L1 (astrocytes) PDGFRB Perform colocalization studies (merge) ALDH1L1 Merge Not Tested Tested How to manipulate the cell Cre-lox toolbox FLP-FRT toolbox Tested Cre-lox: A Primer The system involves the use of two key components: the Cre recombinase enzyme (usually downstream of a cell specific promoter) specific DNA sequences called loxP site that are the Cre target sites (usually flanking a gene of interest or a stop codon) The Cre recombinase enzyme is derived from the bacteriophage P1 and is capable of catalyzing recombination events between DNA sequences flanked by loxP sites The loxP sites are short DNA sequences, typically 34 base pairs long, that contain specific recognition sites for the Cre recombinase The activity of the Cre recombinase enzyme is controlled by the substrate tamoxifen (Tam) Tested Cre-lox recombination Not Tested Example scenario You order one animal that carries the Cre recombinase gene downstream of a cell specific promoter such as Pdgfrb You order another animal that carries the Fn1 gene that is flanked by loxP sites in a cis manner Tested Conditional deletion at a specific time Conditional deletion of Fn1 in Pdgfrb+ pericytes – timing is controlled by tamoxifen administration: Cre Pdgfrb Cross loxP Fn1 loxP loxP Fn1 loxP homo loxP loxP Fn1 loxP loxP Fn1 loxP Cre het Pdgfrb loxP Cre Fn1 Backcross loxP loxP homo Cre het, loxP het Pdgfrb Cre loxP Fn1 loxP loxP Fn1 loxP Cre het, loxP homo – final product Rosa26 and conditional gene expression Abbreviated R26 A commonly used genetic locus (location) in mice that has become a popular site for targeted gene expression and genetic manipulation Often the target of Crispr-Cas9 insertion of transgenes due to its conservation across species/strains In expression-based approaches, a strong promoter like CAG is inserted in front of the transgene Tested Conditional expression at a specific time Conditional expression of DTR in Pdgfrb+ pericytes – timing is controlled by tamoxifen administration: Without excision, STOP would arrest CAG driven DTR expression Pdgfrb Cre Cross R26 loxP STOP loxP CAG DTR R26 loxP STOP loxP CAG DTR Cre het loxP homo Cre Pdgfrb R26 loxP STOP loxP CAG DTR Cre het, loxP het – if one copy of expression is enough Tested Tested Conditional ablation with DTR Cre Pdgfrb R26 loxP STOP loxP CAG DTR Diphtheria toxin receptor (DTR) is a receptor that is not endogenous to the mouse When expressed, the administration of diphtheria toxin (DT) results in the death of the DTR-expressing cell Cre-dependent DTR expression allows for selective ablation of cell types Tested Flp-Frt: A primer Basically identical to the Cre-lox system, but there is no chemical trigger (no tam) for the enzyme to work There is a version that is also chemically triggered called Flpo Usually the FLP recombinase is downstream of a temporal-sensitive developmental trigger The FLP recombinase recognizes the FRT sites and catalyzes recombination events between them Combining Cre-lox/Flp-Frt for dual manipulations If you wanted to investigate the specific roles of dopamine D1 receptor (D1R)-expressing and D2 receptor (D2R)-expressing neurons in the striatum, a brain region involved in reward processing and addiction Use a dual recombinase strategy combining Cre-lox and FLP-FRT systems to selectively manipulate these two neuronal populations (this is called “intersectional” genetics) The researchers generated two different mouse lines: 1. D1R-Cre 2. D2R-Flp These lines were crossed to generate mice with D1R-Cre;D2R-Flp double transgenic offspring Tested Conditionally expressing fluorescent tags Termed “reporter lines” as they can report on the location of cells By crossing these double transgenic mice with: Double transgenic tdTomato-LoxP; eGFP-Frt mice The D1R cells would be red after tam administration, and the D2R cells would be green A “dual” reporter! Tested Tested Summary of lines and nomenclature Conditional expression lines: Promoter-Cre-R26-CAG-Transgene-loxP Ablation: Promoter-Cre-R26-CAG-DTR-loxP Reporter: Promoter-Cre-R26-CAG-FluorescentTag-loxP Conditional deletion lines: Promoter-Cre-Gene-loxP Example: stopping pericytes from proliferating Not Tested Conditional deletion of cell cycling gene KRas in GLAST+ pericytes – timing is controlled by tamoxifen administration: Cre GLAST Cross loxP KRas loxP loxP KRas loxP homo loxP loxP KRas loxP loxP KRas loxP Cre het GLAST loxP Cre KRas Backcross loxP loxP homo Cre het, loxP het GLAST Cre loxP KRas loxP loxP KRas loxP Cre het, loxP homo – final product Preventing pericyte proliferation = scar is gone Not Tested Viral approaches to label and deliver transgenes What if you wanted localized Cre/Flp dependent expression? You can consider using viral delivery of the loxP/Frt constructs, works for expression but also for siRNA/shRNA mediated knockdowns Viruses can also be restricted in their spread and in the case of CNS, in their directionality as well AAV9 (not shown below) is well known to infect the entire CNS system in both directions Tested Tested Region specific ablation of pericytes AAV2-CAG-DTR-loxP Spinal cord of Pdgfrb-Cre mice Tested Tracing of neurons Let’s say you wanted to know the circuitry responsible for forelimb extensor movement PRV-CAG-tdTomato Expressing a transgene in excitatory neurons that innervate the forelimb Excitatory neurons are VGlut2-expressing PRV-VGlut2-tdTomato Tested Tested Combining Cre mouse + virus Express the gene KCC2 only in VGlut2+ neurons that innervate the forelimb PRV-CAG-KCC2-loxP VGlut2-Cre mouse Tested Can also be dual viral approach AAV2-VGlut2-Cre PRV-CAG-KCC2-loxP Optogenetics and light mediated control of neuronal activity These are collectively termed “opsins” Blue Light Red Light Only need to know NpHR Tested Optogenetic control of predatory behaviour Not Tested Chemogenetics and drug mediated control of neuronal activity Tested Selective labeling silencing of forelimb neurons Tested Optogenetics PRV-VGlut2-NpHR-GFP + yellow / red light to the spinal cord Chemogenetics PRV-VGlut2-hM4Di-GFP + CNO Tested Viral intersectional techniques Goal: Concurrent light activation of excitatory VGlut2 neurons and drug silencing of inhibitory Vgat neurons VGlut2-Flp; Vgat-Cre double transgenic mouse Optogenetics - ACTIVATION PRV-CAG-ChR2-GFP-Frt + Tam + blue light to the spinal cord Chemogenetics - INHIBITION PRV-CAG-hM4Di-tdTomato-loxP + Tam + CNO Tested Viral Nomenclature Generally, VirusSerotype-Promoter-Transgene Cre-lox conditional viruses: VirusSerotype-Promoter-Cre VirusSerotype-Promoter-Transgene-loxP Similar nomenclature for Flp-Frt viruses Not Tested Let’s go back to this example now PICO Framework Question: In spinal cord injury, how do we remove the fibrotic scar? Is it beneficial relative to no removal? How does it impact functional recovery? What do you now need to know to answer this question? What is the cellular origin of the fibrotic scar? Immunohistochemistry with Fn1 and cellular markers, RNA-sequencing of all cells and see which ones are responsible for Fn1 production. How can we silence its production? Conditional deletion of Fn1, conditional ablation of pericytes, conditional deletion of cell cycle gene in pericytes Does this silencing result in a functional change? The animals run faster on the gait analyses system. Which neurons are responsible for this improvement? Retrograde viral tracing of hindlimb muscles, see if there is more neurons after scar removal. Not Tested What about this one? PICO Framework Question: In degenerative cervical myelopathy (DCM), does having APOE4 carrier status, relative to APOE2/3 carrier status, have an impact on disease progression? Model: An induced mouse DCM model What do you now need to know to answer this question? What is the impact of carrier status on the behavior and disease phenotype? Use ApoE4-KI and ApoE3-KI mouse, look at behaviour when walking, memory tasks etc. What is the impact of carrier status on the cells in the system? Use ApoE4-KI and ApoE3-KI mouse, and immunohistochemistry to look at cell morphology, RNA sequencing to look at transcriptomic changes. Is there a primary cell type in the spinal cord that is more effected by this than others? Evaluate conditional KI of ApoE3 and ApoE4 in the cell type that changed the most and see if phenotype is the same as the total knock-in.