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4th year 2nd sem > BIOL 4507- Midterm > Flashcards

BIOL 4507- Midterm Flashcards

1
Q

translational medicine

A
  • “bench to bedside”
  • a network to connect people working in labs to people working in hospitals
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2
Q

describe the chain of people involved in translational medicine

A

PhD trained basic scientists -> MD, PhD trained clinician scientists -> regulatory, legal, and clinical trained specialists -> physicians

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3
Q

describe the process of translational medicine (8)

A

human disease -> hypothesis -> basic research funding -> innovation and discovery -> intellectual property (publishing and patenting) -> development pipeline (scaling and developing, pre-clinical assessments- animal models, etc.) -> clinical trials -> regulatory approval

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4
Q

regenerative medicine

A
  • developing and applying treatments to heal tissues and organs and restore function lost due to aging, disease, damage, or defects
  • encompasses multiple areas of scientific inquiries, each of which is complex, but produce a powerful combination of technologies
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5
Q

stem cell

A
  • undifferentiated or partially differentiated cells
  • retain the capacity to differentiate into various types of cells (“potency/potential”)
  • can proliferate indefinitely to produce more the same stem cell (clonal expansion)
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6
Q

what are the different levels of cells

A

totipotent, pluripotent, multipotent, unipotent, somatic

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7
Q

totipotent

A

zygote or morula cells; can contribute to all of the cell types of embryonic development, including extra embryonic tissues

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8
Q

name the extra embryonic tissues (5)

A

placenta, yolk sac, amnion, trophoblast, and extra embryonic endoderm lineages

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9
Q

pluripotent

A
  • have the ability to generate multiple classes of stem cells (e.g. embryonic stem cells can produce mesenchymal, hematopoietic, and neural stem cells) and give rise to all of the cell types that make up the body
  • more restricted than totipotent (can’t produce extra embryonic tissues)
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10
Q

multipotent

A

have the ability to differentiate into all the cell types within a particular lineage (more restricted)

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11
Q

unipotent

A

can produce only one cell type but have the property of self renewal that distinguishes them from non stem cells

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12
Q

somatic

A

body cells, can be reprogrammed into pluripotent SC (induced pluripotent SC)

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13
Q

what are the sources of stem cells

A
  • differentiated somatic cells
  • adult tissues
  • embryonic tissues
  • fetal stem cells
    • originally derived from miscarriages and abortions, restrictions on the use of fetal SC resulted in the development of human induced pluripotent SC
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14
Q

how is stem cell therapy administered

A

ICV transplantation, intravascular infusion, intranasal delivery

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15
Q

parkinson’s case study

A
  • 70 yr old patient with progressed Parkinson’s (lack of dopamine to coordinate fluid movements)
  • fetal ventral mesencephalon precursor from fetal SC were transplanted into the region of the brain that receives dopamine and gave rise to dopamine producing neurons at maturity
  • patient was able to coordinate fluid movement without medication
  • following research looked into deep brain stimulation due to the difficulty of use and controversy around fetal stem cells
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16
Q

research ethics

A
  • new technological treatments require an ethical backup plan for if the research doesn’t continue progressing
    • support for patients if the technology research does not continue
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17
Q

molecular organization of cells

A
  • multicellular tissues exist in one of 2 types of cellular arrangements:
    • epithelial
    • mesenchymal
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18
Q

epithelial (2)

A
  • adhere tightly to each other
  • produce a sheet of cells resting on a basal lamina with an apical surface
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19
Q

characteristics of epithelial cells (4)

A
  • regular columnar morphology
  • high degree of cell adhesion and cell-cell junctions
  • specialized apical membrane and underlying basement membrane
  • cells are relatively static
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20
Q

mesenchymal cells (2)

A
  • bipolar morphology
  • held together as a tissue within a 3D extracellular matrix (ECM)
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21
Q

characteristics of mesenchymal cells

A
  • irregular, rounded, and elongate morphology
  • loss of apico- basal polarity, front- back polarity
  • dynamic adhesions- lamellipoda and filopoda
  • cells are highly motile
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22
Q

epithelial sheets (3)

A
  • polarized
  • rest on a basal lamina (ECM that serves as a foundation, impenetrable to the cells at that state)
  • can bend to form an epithelial tube or vesicle
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23
Q

cell junction

A
  • bind epithelial cells robustly to one another and to the basal lamina
  • linker protein attaches to cadherin protein which will attach to another cell’s cadherin protein and link the cells together (dimerization)
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24
Q

cadherin protein

A

transmembrane protein that spans the entire cell membrane

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25
Q

adheren junction

A

initiation and stabilization of cell-cell adhesion

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26
Q

tight junction

A

continuous intercellular barrier between epithelial cells

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27
Q

retinal neuroepithelium

A
  • multipotent progenitors are located in the nueroblastic layer (NBL)
  • differentiating neurons and glial cells are located in the inner neuroblatic layer and ganglion cell layer
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28
Q

glial cell

A

provides physical and chemical support to neurons and maintains their environment

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29
Q

ganglion cells

A

project info perceived by the photoreceptors to the brain

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30
Q

vimentin

A
  • intermediate filament expressed in the mesenchymal cells that are differentiating
  • involved in the non-NBL
  • change the shape and polarity of the cell
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31
Q

epithelial mesenchymal transition (EMT)

A

rearrangement of cells to create additional morphological features

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32
Q

mesenchymal epithelial transition (MET)

A

the reverse process of EMT whereby cells coalesce into an epithelium

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33
Q

conversion of epithelial and mesenchymal cells

A
  • the early embryo is structured as one or more epithelia
  • in the adult organism, EMTs and METs occur during wound healing and tissue remodelling
  • requires the coordinated changes of many distinct families and molecules
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34
Q

what are the mechanisms that stimulate cells to transition into single migrating cells

A
  • changes in cell-cell adhesion
  • changes in cell- ECM adhesion
  • changes in cell polarity and stimulation of motility
  • inversion of the basal lamina
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35
Q

changes in cell-cell adhesion

A
  • cells must detach from the epithelium in order to migrate away
  • there are 2 main cadherins that mediate cell adhesion in epithelia:
    • E-cadherin (part of the epithelia layer)
    • N-cadherin (associated with mesenchymal cells, doesn’t dimerize with E cadherin allowing the cells to move)
  • often epithelia will down regulate E cadherin expression at the time of the EMT and express different cadherins, such as N cadherin to promote motility
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36
Q

integrin

A
  • transmembrane protein
  • 2 non covalently linked subunits that bind to ECM components (e.g. fibronectin, laminin, collagen)
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37
Q

changes in cell-ECM adhesion

A
  • clustering of integrins on the cell surface affects the overall strength (avidity) of integrin- ECM interactions
    • more integrins -> increased avidity and stronger ECM interactions
  • different ECM components allow you to manipulate cells differently
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38
Q

changes in celll polarity and stimulation of cell motility

A
  • epithelial polarity is characterized by cell-cell junctions:
    • apicolateral domain (non adhesive)
    • basal lamina (adhesive)
  • changes in cell polarity helps promote EMTs
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39
Q

inversion of the basal lamina

A
  • in most EMTs, the emerging mesenchymal cells must penetrate a basal lamina (consists of ECM substrates- collagen type IV, fibronectin, lamina)
  • mesenchymal cells may produce enzymes to degrade and breach the basal lamina (e.g. plasminogen activator)
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40
Q

how are EMTs controlled?

A
  • transcriptional control (transcription factors)
  • posttranscriptional regulation
  • molecular control (ligand receptor signalling, inflammatory signalling molecules, etc.)
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41
Q

transcription factors

A
  • regulate gene expression
  • act in concert with one another to create large circuits
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42
Q

transcriptome

A
  • a collection of transcription factors that define the cell and what it produces
  • e.g. a stem cell is expressing a certain transcriptome, when it transitions to another type of cell it will express a different transcriptome
  • requires a continuation of signalling that turns on specific profiles of transcription factors
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43
Q

posttranscriptional regulation of EMTs (5)

A
  • the activity of EMT transcription factors is regulated at the protein level
    • translational control
    • protein stability (targeting to the proteasome)
    • nucelar localization (in order for things to function in the cell, they have to be in the appropriate location)
    • non coding RNA (silences the ability of a gene to be expressed by binding just before the promoter and preventing transcription of the gene)
    • RNA binding protein
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44
Q

musashi-1

A
  • RNA binding protein (post transcription) expressed in progenitor cells
  • maintains control over genes that define the transition from multipotent progenitor to TD
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45
Q

ligand receptor signalling

A
  • ligands (receptor complexes) interact with target cell receptor and starts a signalling cascade
  • may be diffusible (floating) or expressed on the surface of another cell
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46
Q

hierarchical organization of stem cells (3)

A
  • self renewal (can clonal divide to self renew) vs terminally differentiated progeny (exited the cell cycle, no longer dividing, not considered a stem cell)
  • progressive differentiation states
  • vestige status
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47
Q

self renewal vs TD progeny

A

symmetrical division and asymmetrical division

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48
Q

symmetrical division

A
  • clonal expansion
  • SC -> 2 SC
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49
Q

asymmetrical division

A
  • can be clonal/TD or TD
  • regulation can be intrinsic (i.e. transcription factors) or extrinsic (e.g. growth factors)
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50
Q

progressive differentiation states

A

early stem cells, intermediate progenitors, terminally differentiated cells

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51
Q

early stem cells

A
  • long term renewal
  • lots of potential to divide and more likely to be a self renewal potential event
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52
Q

intermediate progenitor

A
  • limited renewal
  • more restricted in what it can generate
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53
Q

terminally differentiated (TD) cells

A

no renewal

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54
Q

vestige status (4)

A

quiescent, proliferative, intermediate, terminally differentiated

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55
Q

quiescent

A
  • in active but ready
  • not in the cell cycle but can reenter
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56
Q

proliferative

A
  • productive
  • engaged in the cell cycle
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57
Q

intermediate progenitor (vestige)

A
  • transient (between SC and TD)
  • usually migrating and sometimes dividing
  • lack pluripotency since they are not SC
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58
Q

terminally differentiated (vestige)

A

cannot divide unless cancerous or reprogrammed

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59
Q

criteria of pluripotent potential

A
  • expression of molecular markers
  • absence of molecular and morphological markers
  • the ability, upon indication of differentiation in vitro or in vivo, to form all 3 embryonic germ layers including the ectoderm (external), endoderm (internal), and mesoderm (middle)
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60
Q

how we test for pluripotency

A

stem cells are injected into an immunodeficient mouse and eventually a tumor is produced -> the tumour is removed and examined -> tumors from pluripotent SC will have all three embryonic germ layers

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61
Q

testing progenitor and SC heterogeneity

A
  • 2 general approaches:
    • transplantation protocols
    • in vitro expansion and differentiation protocols (bulk culturing and single cell colony formation)
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62
Q

bulk culturing

A

the activity of each cell of the population is not reflected by the population average (unable to capture the activity of rare and critical cells and transiently amplifying other cells

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63
Q

single cell colony formation

A

evaluate single cells with a high degree of comprehensiveness

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64
Q

stem cell sources (6)

A

single blastomere, morula, blastocyst, growth arrested embryo, somatic cell nuclear transfer, parthenogenesis

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65
Q

morula

A

totipotent SC at the 16 cell stage, the whole clump of cells is taken

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66
Q

blastocyst

A

larger mass of cells than a morula (100-200 cells), has a population of different types of pluripotent SC

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67
Q

growth arrested embryo

A
  • embryos are manipulated to guarantee they are only going to reach a certain stage of development
    • zona pellucida is removed (protection during early development)
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68
Q

somatic cell nuclear transfer

A

cloning using a donor egg and donor nucleus

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69
Q

parthenogenesis

A

egg can develop into an embryo without being fertilized with sperm, can result in a lack of genetic diversity

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70
Q

single blastomere

A

a single cell is moved from a blastocyst

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71
Q

stem cells from the amnion (fetal SCs)

A
  • from the placenta and fetal membrane (i.e. amnion and chorion)
  • less controversial source of SCs
  • usually discarded after deliver and accessible during pregnancy through amniocentesis and chorionic villus sampling
72
Q

amniocentesis

A
  • prenatal test that take amniotic fluid around the baby in the uterus
  • performed on women at higher risk of delivering a child with birth defects
73
Q

sources of FSC

A
  • amniotic epithelial cells (hAEC)- associated with the amniotic fluids/fluid membrane
  • amniotic mesenchymal cells (hAMSC)- associated with the amniotic fluids/fluid membrane
  • chorionic mesenchymal stromal cells- in the chorion
  • chorionic trophoblastic cells- in the chorion
    *all are candidates for use in transplantation
74
Q

Wharton’s jelly

A

a gelatinous connective tissue, insulates and protects the umbilical cord

75
Q

cord blood stem cells

A
  • unrelated donor source of hematopoietic SC and amniotic epithelial cells
  • accessing these cells allows you to modify them for later treatment
  • previous dark history
76
Q

clinical uses of CB (6)

A
  • transplantation for hematological and non-hematological malignancies
  • inherited metabolic disorders
  • postnatal hypoxic ischemic encephalopathy
  • cerebral palsy
  • stroke
  • autism (inflammatory theory)
77
Q

induced pluripotent stem cells

A

introduction of 4 transcription factors can allow any somatic cell to be reprogrammed into an iPSC

78
Q

4 transcription factors (yamanaka)

A

Oct 4, sox 2, Klf4, l-myc

79
Q

describe the process of producing iPSC

A
  • retrieve patient’s somatic cells
  • turn on yamanka transcription factors
  • different protocols are used to induce the different tissue types
    *positive feedback loop- transcription factors regulate the expression of a network of pluripotent associated genes
80
Q

what are the 4 iPSC reprogramming techniques

A

retroviral transduction, Sendai virus, episomal vectors, introduction of synthetic mRNAs

81
Q

retroviral transduction

A
  • retroviruses have the ability to transform their single-stranded RNA genome into a double stranded DNA molecule that stably integrates into the genome of dividing target cells
  • Myc is a proto-oncogene (can mutate into an oncogene)
82
Q

sendai virus

A
  • enveloped, single stranded RNA virus
  • replicates directly into the cytoplasm of the host cell without any DNA intermediary
83
Q

sendai virus in iPSC reprogramming (3)

A
  • non integrating (replicates independent of the cell cycle)
  • produces high copy numbers of the target gene (efficient)
  • does not translocate to the nucleus and is diluted with each cell
84
Q

episomal (bacterial) vectors

A
  • non viral and non integrating
  • plasmids are replicated and partitioned into daughter cells
85
Q

advantages of episomal vector (2)

A
  • generation of iPSC in a single transfection
  • plasmids are lost over time resulting in transgene free reprogrammed cell lines
86
Q

disadvantages of episomal vectors

A

can sometimes be integrated into the host genome, so the iPSC clones must be subject to genomic analysis

87
Q

introduction of synthetic messenger RNAs

A
  • mRNA is modified to overexpress pluripotent factors in somatic cells to reprogram them into iPSC
    • substantial number of pluripotent factor expressions needed to drive cell transformation
88
Q

disadvantages of the introduction of synthetic mRNA

A

requires repeated transfections to induce over expression of pluripotent protein and further promote reprogramming due to transient (short lasting) expression

89
Q

epigenetic

A
  • changes in cell function that happen without changes in DNA sequence
  • methylation state of an adult somatic cell must be remodelled during the reprogramming process to unlock access to the pluripotent associated gene network
  • many genes are hypermethylated in donor cell types but hypomethylated in iPSCs
90
Q

epigenetic memory

A
  • ability of cells to stably remain and transmit thier unique gene expression patterns to daughter cells
  • epigenetic memories of iPSCs are short term and easily lost; time in culture reduces epigenetic differences among iPSCs
91
Q

methylation

A

suppresses gene expression

92
Q

acetylation

A

encourages gene expression

93
Q

induced transdifferentiation

A
  • direct transformation from one cell type to another
  • a major mechanism in fish for regrowth
94
Q

advantages of transdifferentiation

A
  • relatively fast in comparison to iPSC
  • less likely than iPSC derivatives to contain residual undifferentiated cells that could produce teratomas
95
Q

disadvantages of transdifferentiation

A
  • mature cell types have a limited capacity to divide
  • require a large input population- cells are not going into the cell cycle, you’re pushing them from one population to another
    • finish the process with less cells than you started with
  • population isclonally derived so genetic manipulation and characterization is difficult/not feasible
96
Q

application of SCs

A

disease modelling, personalized medicine, cell therapy, conservation of endangered species

97
Q

disease modelling

A
  • positive results in animal models do not always translate to human studies
    • not surprising since rodents diverged evolutionarily from humans 60 mya
  • iPSCs allow scientists to study human diseases using human cells and dieases for which there is evidence of inheritance but no specific mutations identified
  • iPSCS can be derived from patients with complex diseases that lack a clear genetic basis and preserve their genome
98
Q

personalized medicine

A
  • many research methods tend to be similar ethnic backgrounds
    • genetic background of an individual can determine the success or adverse effects of a particular drug
  • can be used to create a customized medical plan or personalized regenerative medicine
99
Q

cell therapy

A
  • autologous transplantation of iPSCs and their derivative is expected to be tolerated by the immune system
  • banks of iPSCs that match a large percentage of the population have been created
  • delivery of therapeutic cell types must be targeted to a region of interest
    • IV injection is simple and easy, but often results in the cells being captured by the lungs or liver
100
Q

conservation of endangered species

A
  • endangered organisms can be rederived to increase their population
  • if the population is extinct and you have a cell sample or their genome, you could induce pluripotent SCs and create an embryo to implant into another species
101
Q

the hematopoietic system is a self renewal system

A
  • divide and differentiate to produce maturing progeny
  • divide to self renew to maintain a pool of SCs
102
Q

the site of hematopoiesis changes during development

A
  • placental
  • fetal liver
  • bone marrow (adults)
103
Q

hematopoietic SC therapies (6)

A
  • bone marrow transplantation
  • autologous/allogenic peripheral blood SC transplantation
  • CB transplantation
  • HSCs for severe combined immunodeficiency
  • HSCs for autoimmune diseases
  • HSCs for tolerance induction post transplant
104
Q

HSCs for tolerance induction post transplant

A
  • used to prevent the reaction of transplanted organs/cells
  • allogenic cells are mixed with host cells and injected months before the transplant to integrate them into the immune system
105
Q

mesenchymal SC sources (6)

A
  • bone marrow compartment (best known source)
  • adipose (accessible)
  • dermal
  • placenta
  • CB and peripheral blood
  • intervertebral disc (remnants of the notochord)
    *high degree of potential
106
Q

multipotent adult progenitor cells

A

typically quiescent but possess some primordial potential to recapitulate SC activity

107
Q

types of ASCs

A
  • hematopoietic/immunomodulatory, neural, bone, cardiac
  • certain parts of the organ will have quiescent compartments
  • brain creates new SC for the olfactory area (neurons are exposed and need to be replaced often and for the hippocampus (requrie new neurons for new memories and refreshing memories and synaptic connections))
108
Q

biomaterials

A
  • any material, natural or synthetic, that comprises a whole or part of a living structure or biomedical device which performs, amplifies, or replaces the function that has been lost by injury or degenerative condition
  • synthetically and pharmacologically inert
  • used in a variety of sub disciplines (medicine, surgery, dentistry, vet med)
109
Q

conventional examples of biomaterials

A
  • substitute heart valves (synthetic, xenotranspants)
  • artificial joints
  • dental implants
  • fracture fixes
  • skin regeneration templates
  • contact lens
  • kidney dialyzers
  • blood vessel angioplasty
  • cochlear implants
110
Q

3 main classes of biomaterials

A
  • metals (e.g. orthopaedic screws)
  • ceramics (e.g. dental implants)
  • polymers (e.g. drug delivery)
  • can be combined into composites
111
Q

properties of biomaterials (6)

A
  • surface properties (rough, coarse, porous, reaction with water)
  • corrosion resistance (electrical signals within the body may be corrosive)
  • innate degradation (material will naturally break down over time)
  • mechanical properties
  • biochemical reactivity (is it going to react with the chemistry around it)
  • radiation
112
Q

related applications (4)

A
  • implantation (is it inside or outside the body)
  • hemocompatibility (a lot of chemistry occurs in the circulatory system- oxygen carrying capacity, coagulation, etc.)
  • biodegration (body will break down the material- enzymes)
  • immune surveillance (immune system will be reactive towards any foreign material)
113
Q

applications for biomaterials in regenerative medicine (5)

A
  • cardiopulmonary organ replacements
  • orthopaedic and dental implants
  • surgical sutures and adhesives
  • biological scaffolds
  • nerve regeneration
114
Q

cardiopulmonary organ replacements

A
  • cardiovascular implants and devices
  • extracorporeal artificial organs
  • artificial erythrocyte substitutes
115
Q

cardiovascular devices and implants

A
  • artificial heart
  • synthetic heart valves- used for training, potential future use for treatment
  • coronary artery bypass surgery
116
Q

what do you need to consider when producing synthetic heart valves/vessels

A
  • kinetic energy (heat)
  • stretch
  • diameter
117
Q

coronary artery bypass surgery (CABG)

A

artery in the heart is blocked, disrupting functioning and rhythm of the heart -> artery is harvested from another part of the body -> donor artery is used to bypass the blocked section

118
Q

extracorporeal artificial organs

A
  • provide mass transfer operations to support failing/impaired organ systems
  • e.g. pace maker, insulin pump
119
Q

artificial erythrocyte substitutes

A
  • perflurocarbon emulsions (lipid based liquid to contain perflurocarbon)/ microcapsules (more elaborate with a membrane wall)- molecules that can bind O2 (similar to hemoglobin- not good at removing CO2)
  • encapsulated hemoglobin- requires hemoglobin source
120
Q

dental implants

A

retropharyngeal space is linked directly to the pericardium- may introduce bacteria to the rest of the body

121
Q

bone and cartilage implants

A
  • must consider the space between the impact (can facilitate bacterial growth)
  • must consider porosity of the implant (is it rough enough for the bone to adhere to the implant)
122
Q

titanium implants

A
  • titanium is relatively non-reactive, lasts a long time, lightweight
  • technology to detect substances improved (liquid chromatography mass spectrometry)- found that more titanium was leaching from implants than first thought
  • titanium reduces osteogenic differentiation (bone growth), increases peri-implantitis (inflammation around the implant), increases osteoclast differentiation (bone breakdown), and decreases epithelial homeostasis
123
Q

surgical sutures and adhesives (cyanoacrylate)

A
  • accidentally invented by Harry Coover in 1942 (WWII) while he was working on making plastic lenses for firearm optics
  • observed an acrylic resin that rapidly polymerized when reacted with hydrogen and strengthened into a long chain, poly-cyanoacrylate when exposed to water
  • quickly and easily used to close lacerations
124
Q

issues of cyanoacrylate

A
  • curing is exothermic (produces heat) and could cause damage to surrounding tissues
  • curing creates formaldehyde, which irritates skin, eyes, and respiratory systems
125
Q

alternatives to cyanoacrylate

A
  • 2-octyl-cyanoacrylate and n-butyl-2-cyanoacrylate
  • both are improved and less irritating medical adhesives
126
Q

biological scaffolds

A
  • 2d and 3d scaffolds for in vitro growth
  • 3d scaffold for mesenchymal SC tissue growth
  • skeletal and cardiac muscle repair
  • techniques include electrospinning, natural tissues, 3d printing, self assembly, and 3d bioprinting
127
Q

nerve regeneration

A
  • peripheral NS can regenerate nerves relatively proficiently, but not always
  • grafts can be used to bridge nerves together and reestablish connection and function
  • chemotaxis and pathfinding
    • when a neuron is injured and trying to regrow, the pathfinding molecule that it developed surrounded by are no longer present and the cell will likely grow in the wrong location (preferential motor nerve regeneration)
128
Q

bioengineered bridging

A

synthetic material used to bridge the ends of nerves together and facilitate chemotaxis, pathfinding, regrowth, and cell survival mechanisms

129
Q

issues to consider when trialing biomaterials for prosthesis, implants, tissues, and fluids/gels

A
  • biocompatibility- harm, dissolve, get encapsulated, bond with tissue
  • hydrogels-cross linked polymers
  • drug delivery, cell delivery, tamponade, wound healing
130
Q

good manufacturing practice standards

A
  • is the material you’re putting in the body endotoxin (toxin anchored to the cell) free?
  • dalkon shield IUDs
    • composite biomaterial was created, the process of synthesis created endotoxins
    • nylon retrieval string wicked in bacteria
    • resulted in 18 deaths
131
Q

cell-substrate interactions

A
  • ECM, substrates, physical properties
    • crystallinity, morphology, stiffness, compliance
    • surface wet ability, surface charge, cell response
    • can it be engineered/modified
    • is it electrically conductive
    • can its composition be altered
  • cell adhesion, motility, proliferation/differentiation
    • effects of topography and diffusible factor signalling
132
Q

tissue clearing

A

transforms 3d tissue into a 3d nonporous hydrogel-hybridize form that is optically transparent and macromolecule permeable

133
Q

CRISPR

A

clustered regularly interspaced short palindromic repeats

134
Q

describe the origin story of CRISPR

A
  • CRISPRs were first identified in E coli in 1987, when a series of repeated sequences interspaced with spacer sequences was accidently cloned
  • in 1993, JD van Emden discovered that different strains of mycobacteria tuberculosis also had CRISPRs and other genomes were eventually discovered- thought to be a DNA repair mechanism at this point
  • in the early 2000s, Mojica found similar spacer sequences in bacteriophages, viruses, and plasmids
135
Q

CRISPR and adaptive immunity

A
  • viruses cannot infect bacteria harbouring homologous spacer sequences
  • suggests that these sequences play a role in the adaptive immune system in prokaryotes
136
Q

natural mechanism for CRISPR based adaptive immunity

A
  • virus infects a bacteria and inserts its own genome, hijacking the genetic expression mechanism and making all the components needed to reassemble itself
  • if the viral genome is exposed to Cas protein, the cas protein will fragment the spacer sequences of the genome and clone them back into the bacterial genome creating a genetic record of immunization
  • during a subsequent attack, the spacer sequences in CRISPR arrays are transcribed to generate CRISPR RNA which forms an association with a Cas protein. the crRNA will guide the cas to bind to and cleave complementary DNA or RNA viral sequences, munching them up
137
Q

describe the CRISPR-cas system

A
  • CRISPR cas systems can regulate DNA (Cas9) and RNA (cas13)
    • important for viruses, which can be made of DNA or RNA
  • an endonuclease complex- has the ability to bind and cleave DNA
  • requires a guide strand- RNA species that is going to associate with the Cas complex and act as a template to direct it to the target sequence
138
Q

protospacer adjacent motifs (PAM)

A
  • fundamentally required for the cas complex to cleave DNA
  • specifically expressed on all things other than bacteria - cas cannot cleaves DNA without PAMs- ensures that only foreign viral nucleic acids are cleaved
  • short (2-6 base pairs)
139
Q

can CRISPR be used without PAMs

A
  • no, not at the moment
  • alternative variations on CRISPR editing innovations help add flexibility to this limitation
    • different cas proteins or guide RNA species
140
Q

improving CRISPR

A
  • engineered CRIPSR systems to contain 2 components:
    • CRISPR associated endonuclease
    • synthetic guide RNA (sgRNA/gRNA)- short (20 nucleotides, targets specific regions of the genome
141
Q

site specific endonuclease activity

A
  • endonuclease activity- breaking DNA into 2 fragments
  • the genomic locations that can be targets for editing by CRISPR are limited by the presence of nuclease specific PAM sequences
142
Q

vector

A
  • plasmid DNA ring
  • DNA is continuous with itself
143
Q

blunt end cloning

A
  • insert from a vector is removed and ligase is used to ligate that insert into another vector
  • ends of stand are straight across and may result in inversion
    • the wrong direction is typically non functional
144
Q

sticky end cloning

A
  • insert and vector have overlapping ends (4-6 base pairs)
  • prevents inversion and is more precise
145
Q

restriction enzymes

A
  • recognize specific sequences on the genome
  • might make stick or blunt end cuts
146
Q

non homologous end joining

A
  • DNA is cleaved apart, may result in an insertion, deletion, or frameshift mutation
  • more error prone and less precise
147
Q

homology directed repair

A
  • repair template telling the mechanism what to assemble
  • less error prone and more precise
  • create variations in the template sequence and create whatever mutations you want
148
Q

how is DNA inserted into cells? (5)

A

microinjection, balistique gene gun, in vitro/in vivo electroporation, cationic lipids, viruses

149
Q

microinjection

A

teeny, tiny micropipette is used to inject individual cells using electric pulses

150
Q

ballistic gene gun

A

gold particles are coated with the DNA of interest, packed into a bullet, and shot into cells- particles are coat in the cells and the coating is released

151
Q

in vitro/in vivo electroporation

A

opens pores in the cell membrane or envelope allowing the material into the cell

152
Q

cationic lipids

A
  • mini membrane is formed in the presence of DNA/RNA
  • membrane will capture DNA/RNA resulting in a loaded micro particle
  • membrane will integrate efficiently into the target cell’s membrane
153
Q

viruses

A
  • lentivirus- robust and indiscriminate, not worried about the long term outcome of the organism
  • adenovirus- transient gene expression in both dividing and non dividing cells
  • adeno-associated virus (AAV)- transient transgene expression in both dividing and non dividing cells
154
Q

challenges of CRISPR

A

delivery, ethics, off targeting, gene polymorphism in cancer, autoimmune response

155
Q

delivery of CRISPR

A
  • intramuscular
  • systemic (circulatory system)- hitting more organ systems, may be too many (off target effect)
156
Q

phase 1 clinical trial

A
  • evaluates safety, treatment side effects, and best dosage levels
  • 1-2 dozen participants, typically less
157
Q

phase 2 clinical trial

A
  • evaluates safety and efficacy of the treatment (does it actually relieve, reverse, or stop a condition)
  • hundreds of participants
158
Q

phase 3 clinical trial

A
  • evaluates long term adverse effects, how effective the treatment is compared to other standard treatments
  • hundreds to thousands of participants
159
Q

phase 4 clinical trail

A
  • evaluating the safety of the treatment in the broader population, long term risks and benefits
  • thousands of participants
160
Q

CRISPR clinical trials

A
  • trial phases may be combined to expedite testing a treatment
  • all current CRISPR clinical trials target specific cells or tissues in individuals without affecting germ cells
161
Q

CRISPR treatment areas (6)

A

blood disorders, cancer, inherited eye disorders, diabetes, infectious diseases, protein folding disorder

162
Q

blood disorders and CRISPR

A
  • SCD patients have sickle shaped RBCs which clog vessels and cause pain
  • beta thalassemia (BT) patients lack sufficient hemoglobin levels
  • CRISPR can deactivate the gene that prevents the formation of fetal hemoglobin, allowing cells to produce fetal hemoglobin and normal shaped cells
163
Q

cancers and CRISPR

A
  • leukemia and lymphoma
  • Car-T immunotherapy involves genetically modifying a patient’s T cells to have a receptor that allows them to recognize and attack cancer cells
164
Q

car-t immunotherapy trial results

A
  • treatment was safe to administer and had acceptable side effects
  • edited T cells took up residence in the bone marrow and remained at stable levels for the 9 months of the study
  • T cells were able to find and infiltrate tumours
165
Q

off target effects of car-t immunotherapy

A
  • unintended edits at the target site
    • 70% of cells showed at least one mutation at or near the target site during the T cell manufacturing process
  • over time, the percentage of cells with mutations decreased
166
Q

inherited genetic eye diseases and CRISPR

A
  • Leber congenital amaurosis (LEB) is the most common cause of inherited childhood blindness
  • LCA10 gene is truncated, creating a mutant photoreceptor gene
  • CRISPR replaces short gene with full length gene
  • clinical injection of AAV with photoreceptor tropism- restores gene function
167
Q

diagnostic challenge of LEB CRISPR treatment

A
  • difficult to evaluate attenuated loss of vision as a positive treatment- how do you tell if someone is getting les blind
  • not restoring vision, just stopping the progression of blindness
168
Q

diabetes and CRISPR

A
  • type 1 is an endocrine disorder that occurs when pancreatic beta cells are destroyed, usually by an autoimmune reaction
  • CRISPR is used to edit the immune related genes of these cells so the patient’s immune system does not attack them
  • edited cells are implanted into the patient’s body in a special pouch and blood vessels grow along the outside of the pouch, bringing oxygen and nutrients and taking up insulin
  • results- only in phase 1, no efficacy data yet, cells need to be isolated to prevent tumour growth
169
Q

infectious diseases and CRISPR

A
  • UTIs
  • cocktail of 3 bacteriophages combined with CRISPR-cas3 designed to attack the genome of the three strains of e coli responsible for 95% of UTIs (destruction of genome kills bacteria)
170
Q

results of CRISPR UTI treatment

A
  • pending
  • trial supported the safety and tolerability of the new therapy with no drug related adverse effects
171
Q

inflammatory diseases and CRISPR

A
  • hereditary angioedema- patient has severe attacks of inflammation and swelling
  • bradykinin regulates vasodilation (introduces more cells to an area, exacerbating inflammation) and is balanced by c1 inhibitor
  • HAE patients have too much bradykinin, so reducing bradykinin restores the balance of C1: bradykinin
  • results- pending, trial supported the safety and tolerability of the new therapy with no adverse drug effects
172
Q

protein folding disorder and CRISPR

A
  • Hereditary transthyretin amyloidosis (hATTR) is a fatal disease causes by mutations in a single DNA base in the TTR gene
  • mutated TTR gene encodes a misfolded protein that is sticky (amyloid fibrils- clump), and aggregates in organs and tissues
  • results- most adverse effects were midl, all patients showed a reduction in TTR protein levels
173
Q

prion

A
  • pathogenic agents that are transmissible and induce abnormal protein folding
  • prion diseases are fatal degenerative brain disorder
174
Q

mad cow disease

A
  • a progressive neurological disorder of cattle that results from infection by a prion
  • industrial neocannibalism- dead diseased cows are steam treated and refed to cows
    • if you continue reintroducing prions into a cattle community, they will eventually accumulate and cause disease
175
Q

future of gene editing

A
  • “pasting”- inserting DNA to repair or replace harmful DNA
  • multiplex editing- therapy to edit multiple genes at the same time
  • treatment that uses base or prime editing- using CRISPR to directly change single DNA base pairs without making double stranded breaks
  • turning on or off genes without changing DNA sequences

4th year 2nd sem (6 decks)

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