Lecture 8 - Modern Techniques in Translational Research

what is the goal of translational research?

what is the goal of translational research?

to try and understand as much about human disease without using human samples

how does translational research work?

it uses robust models of human disease, genetic and pharmalogical tools to test an intervention/therapeutic and imaging or functional readout to evaluate the outcome of the therapeutic

how do cellular models of disease work?

when studying a particular cell population or an organ in a controlled environment induced pluripotent stem cells can be used to derive cells from patients with the disease and revert them back to the stem cell state

in vitro

cell culture dish

ex vivo

isolated tissue in a dish

how does IPS technology work?

first you isolate the somatic cells, reprogram them to induce pluripotency and then culture them so they act as stem cells but still retain somatic mutations for the diseased patient

somatic cell isolation

obtain adult cells form the patient

reprogramming

induce specific factors (transcription factors) to the adult cells which can reset their epigenetic state and induce pluripotency

cell culture

cultivate the reprogrammed cells in a lab setting and allow them to multiply and form cell colonies

what are the short comings associated with IPS technology?

the further the target final cell type is from the original state the more different the epigenetic signature will be

how are IPSCs used for disease?

if you use the technology by taking a blood sample from a patient and deriving the IPS cells and reprogramming them through gene editing you can compare the genetically engineered cell to cells in a healthy donor to determine if gene editing was effective

how are IPSCs used for drug discovery?

you can take somatic cells and culture them so you can put a bunch of them in wells to screen them for different drugs to determine if they are effective in curing the target disease or not (allows testing of a bunch of different drugs at the same time

what are IPSC derived organoids?

3D miniaturized versions of organs or tissues grown in vitro from stem cells or tissue specific progenitor cells allowing the recapitulation of key structural and functional characteristics of their respective organs

what are the advantages of organoids?

they are relevant to humans, they are good for disease modelling and drug discovery + testing, they are precise in medicine because they are derived from a patient afflicted with the target disease, developmental biology

what are the disadvantages associated with organoids?

complexity and heterogeneity, variability and reproducibility since there is no way to control formation once it goes in the bioreactor and they lack systemic interactions because the organ of interest is model in an isolated manner

when are animal models of disease used?

if you are interested in recapitulating the symptoms of a disease in animals in vivo

in vivo

testing done in the body

what are animal inducible disease models?

that is when animals can be induced to have the disease

what are spontaneous animal models?

animals can be genetically modified to have the disease

what are the advantages of animal models?

you can understand the disease because it shows you the systemic interactions since its a full organism, you can do initial safety testing, good for pharmacokinetics and dosage, genetic manipulation

what are the disadvantages of animal models?

they have limited translational success because animals are still quite different from humans, there are species differences between animals and humans, it is unable to recapitulate the complexity of human lifestyle

in what instances have animal models failed?

in treating stroke and spinal cord injury

stroke treatment failure in animal models

neuroprotective drugs first showed promising results in animal models for stroke but failed to demonstrate similar efficacy in human clinical trials (ex: NXY-059, minocycline and tirilazad)

why is spinal cord injury considered the graveyard of translation?

because there has yet to be a single drug that has worked for SCI treatment, numerous promising therapies for SCI showed functional recovery in animal models but they didnt translate in humans (ex: chondroitinase ABC, neurotrophic factors and stem cell transplantation

what might be an explanation for why SCI treatment hasnt translated to humans from animal models?

since SCI is caused by blunt force trauma treating it is very hard in humans because it is usually accompanied by many more critical injuries at the same time vs an isolated animal model recovery is quick because it isnt as complex

what are examples of successes in animal modelling

the creation of antibiotic and vaccines that work because they target immunity which is usually pretty invariable

antibiotics

penicillin was first demonstrate in mice and rabbits leading to its successful use in humans

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 successful use in human populations

what are the different ways of creating induced animal models of traumatic brain injury (tbi)?

fluid percussion injury model (hammer to the brain), controlled cortical impact model (drop lever on the animal), weight drop model (drop weight on animal’s head), and blast injury model (animal placed near explosive inducing brain injury)

what are the different induced models of SCI?

impactor/contusion injury, clip (compression) and transection (cut spinal cord with a scalpel)

what are spontaneous models of CNS disease?

MPTP treated primate model and the APP/PS1 transgenic mouse model

what is the MPTP treated primate model for?

used for parkinson’s disease

how does the MPTP treated primate model work?

administration of the neurotoxin MPTP to non human primates results in the selective destruction of dopamine producing neurons leading to motor impairments

what is the APP/PS1 transgenic mouse model for?

used for alzheimer’s disease

how does the APP/PS1 model work?

genetic mutations associated with early onset familial AD, leading to the accumulation of amyloid beta plaques and neurofibrillary tangles

what is degenerative cervical myelopathy?

ossification (bone growth) of the ligament of the spine that leads to spinal compression over time

what is the twy mouse model?

it is a spontaneous model for degenerative cervical myelopathy that is made by a mutation that leads to the ossification in the C2/C3 spined leading to spinal cord compression

what is the induced version of the degenerative cervical myelopathy model?

modelled by the insertion of an ossifying material underneath the C5/C6 lamina compressing the spinal cord over time

is the induced or spontaneous mouse model more clinically relevant?

induced is preferred because it reduced the likelyhood of the acquisition of off target effects

what are the effects caused by degenerative cervical myelopathy?

it causes the mice to tip toe around their cage because of the immense neuropathic pain they had in their paws which is why the model is typically referred to as the tip toe walking Yoshimura model

pathobiological mechanism of translational research

addresses the questions: how does it happen?, what cells/mutations are involved and what is the sequence of events of the human disease?

therapeutic mechanism of translational research?

asks the questions: is your therapy effective?, if so how does it work?, what cells/molecules are involved? and is it effective across the entire disease trajectory

how do we understand what a disease looks like in human?

by investigating the histopathology of the disease through tissue sectioning and staining, DNA/RNA/Epigenetic profiling and behavioral testing and using this to recapitulate the disease in your model

bottom up approach

used when you know the gene or molecule that is causing the problem and you want to find the cells involved (sees how induction of the gene or molecule affects the rest of the cell system)

top down approach

used when you can see the disease phenotype but you don’t know the cells or molecules that is causing the problem (tries to narrow down the cause of the disease

what are the 2 key components involved in the Cre/lox toolbox

the cre recombinase enzyme and the loxP sites

cre recombinase enzyme

usually situated downstream of the cell specific promoter derived from the bacteriophage P1 and is capable of catalyzing recombination events between DNA sequences flanked by loxP sites

loxP sites

short DNA sequences typically 34 base pairs long that contain specific recognition sites for the Cre recombinase, acting as a guie marking where the recombination events happen

tamoxifen (Tam)

substrate that controls the activity of the Cre recombinase enzyme

what are the different types of Cre-lox recombinations?

excision, inversion and translocation

cre lox excision recombination

2 loxP sites are placed on either side of a section of the genome on the same allele and everything between those two sites are cut out

cre lox inversion recombination

2 loxP sites are placed on the same allele in opposite directions that either turns ON expression of the gene or turns it OFF (genetic flip for functional activity of the gene)

cre lox translocation recombination

trans placement of the 2 loxP sites switching genetic material from one allele to another

cre het animal nomenclature

marker - cre

what is conditional deletion of a gene at a specific time?

the permanent excision of a gene controlled by the administration of tamoxifen (Tam) when using the Cre/lox toolbox

lox P homozygous animal nomenclature

loxp - gene - loxP (x2)

cre heterozygous, lox p heterozygous animal nomenclature

marker - cre + loxp-gene-loxp

cre het loxP homozygous animal nomenclature

marker - cre + ( lox P - gene - lox P x 2)

how does conditional deletion of a gene work?

a cre heterozygous animal is crossed with a loxp homozygous animal creating cre heterozygous, loxP heterozygous offspring but this isnt enough for gene deletion because there is still a functional copy of the fn1 gene allele meaning it is knocked down not knocked out so this animal gets backcrossed into a loxP homozygous animal creating a cre heterozygous, lox P homozygous animal knocking out the gene permanently once tamoxifen is administered

what is Rosa 26 (R26)?

a commonly used genetic locus in mice that has become a popular site for target gene expression and genetic manipulation, usually the target of Crispr-Cas 9 insertion of transgenes due to its conservation across species/strains

how do expression based approaches work?

by using a stop codon and flanking it with loxP sites you can regulate when expression by controlled tamoxifen administration so when administered the STOP codon is excised and only the promoter and transgene on the R26 locus remain causing expression of the gene

lox P homozygous conditional expression nomenclature

R26 - loxP - STOP - loxP - CAG - DTR (x2)

how does condition expression at a specific time work?

a cre heterozygous animal is crossed with a loxP resulting in cre het loxP het offspring so when Tam is administered expression driven by the promoter occurs

cre heterozygous loxP heterozygous condition expression nomenclature

marker - cre, R26 - loxP - STOP - loxP - CAG - DTR

what is DTR (diphtheria toxin receptor)?

it is a receptor that is not endogenous to the mouse so when itis expressed DT administration results in the death of the DTR expression cell meaning that Cre-dependent DTR expression mediated by Tam allows for selective ablation of cell types

what does ablation mean?

means that the gene isnt just deleted but the cell that the gene is expressed on is also killed

what are the components of the Flp-Frt toolbox?

Flp recombinase and Frt sites

how does the Flp/Frt recombination system work?

Flp recombinase acts the same as Cre recombinase but it doesn’t need a chemical trigger for it to activate so expression cant be timed, it will recognize the recombination events between them

what is FlpO?

version of Flp recombinase that is also chemically triggered by Tam administration (inducible form of Flp)

when are dual manipulations combining Cre-lox and Flp-Frt used?

used when wanting the investigate specific roles of 2 different cell populations, for example roles of the dopamine D1 receptor expressing and D2 receptor expressing neurons in the striatum (called intersectional genetics)

what were the two mouse lines used in the striatum double intersectional genetic model?

D1R-Cre and D2R-Flp

what are conditionally expressing fluorescent tags?

they are termed reporter lines cause they can report on the location of cells by fluorescing different colors

• ex: in the striatum mode, tdtomato is used to tag D1R cells so they fluoresce red when tam is administered and DR2 cells are GFp tagged so they fluoresce green since they are associated with the Flp/Frt toolbox (dual reporter)

conditional expression lines nomenclature

promoter-cre-R26-CAG-transgene-loxp

condition ablation line nomenclature

promoter-cre-R26-CAG-DTR-loxP

conditional reporter line nomenclature

promoter - CRE - R26 - CAG - FluorescentTag - loxP

conditional deletion line nomenclature

promoter - Cre- Gene - loxP

how would you approach localization of Cre/Flp dependent expression?

using viral delivery of the loxP/Frt sites works, can also use it for siRNa/shRNA mediated knockdown

AAV9

known to infect the entire CNS system in both directions

AAV1 and AAV2

allow infection to be localized so only site of injection is afflicted

anterograde virus

used when you have a group of neurons that are damaged post SCI and want to know what role that group of neurons play (downstream infection)

retrograde virus

injected at the site of the muscles and if the virus is polysynaptic it jumps through the synapses tracing back to the CNS to tell you all the different neurons involved in the target muscle group (upstream infection)

how does region specific ablation of pericytes work?

AAV2-CAG-DTR-loxP is injected into the spinal cord of a Pdgfrb-Cre mice so when Cre is activated by Tam its clears the stop codon so that DTR can be expressed to induced ablation which is region specific due to the use of AAV2

how would you investigate the circuitry responsible for forelimb extensor movement?

used PRV-CAG-tdtomato because PRV is a retrograde virus so upon injection into the forelimb it’ll trace back all the way up the CNS highlighting all the neurons involved in this movement due to fluorescent tag

how would you express a transgene in excitatory neurons that innervate the forelimb?

you would use PRV-VGlut2-tdtomato because the PRV retrograde virus will trace back and highlight all the Vglut2 expressing neurons marking them as excitatory due to the addition of the fluorescent tag

how would you combine Cre mouse and virus to express excitatory neurons innervating the forelimb?

you would use a Vglut2-Cre-mouse so upon Cre activation by Tam only Vglut2 marked neurons are expressed and then inject PRV-CAG-KCC2-loxP into the forelimb for the retrograde virus to trace back neurons that have the KCC2 transgene

how would you use a dual viral approach to investigate excitatory neurons that innervate the forelimb?

inject AAV2-Vglut2-Cre and PRV-CAG-KCC2-loxP into the forelimb so that neurons that are localized at the injection site being the forelimb will trigger KCC2 expression determining if this location has a particular function involved in the forelimb extension movement

gain of function experiment

studying something where a certain function is lost and you want to give that function back (rescue)

loss of function experiment

something is functional but you want to prove that only one target variable is responsible for it working so when you knock it out this function shouldnt work anymore

what is the point of optogenetics?

it allows you to do gain and loss of function experiments at the same time

what is the role of blue light in the activation channel of optogenetics?

it stimulates the activation of ChR2 which causes Ca2+ to enter the neurons causing them to fire cause it depolarizes the celll

NpHR

causes repolarization response in optogenetic inhibitory channel inhibiting the neuron

what is the role of red light in optogenetics

it stimulates the inhibitory channel by activation of NpHR causing repolarization response by Cl- influx

what are the benefits of optogenetics?

its regionally specific because expression or inhibition only occurs where the blue or red light is shined and activation is transient so mechanism can be turned off and on by light

hM3Dq

activating dreadd

hM4Di

inhibitory dreadd

CNO

substrate for inhibitory and activating dreads in chemogenetics

what limitation is associated with optogenetics?

cross activation can occur if the wavelengths for the inhibitory and activation channels are too close together + invasive

what is chemogenetics?

uses specifically designed ligands that are activating specifically designed receptors

what is the limitation associated with dreads?

you cant use a dual dreads approach cause it uses the same substrate for activation and inhibition

what is the benefit with using dreads/chemogenetics?

drugs are transient so you can turn them off and on and its a non invasive measure