lecture 12 - repair and regeneration Flashcards
What is the general principle of gene engineering and what are limitations of “traditional” gene therapy?
Limitations: size of cargo, transient effects, oncogenicity
Take T cells and engineer them to become CAR T cells
Engineer HSCs or iPSCs and edit the genome, then infuse the differentiated cells back into the patient
Huge number of disease states that can be cured using this approach
What are the general principles of CRISPR-Cas9?
Gene editing system based on components of the adaptive immune systems of certain bacteria and other microbes
Cas9 protein has endonuclease activity and can cleave the genome at specific sites
Design guide RNAs
Can insert a new gene into a specific region of the genome
How can CRISPR-Cas9 be used as a treatment for muscular dystrophy?
Mutations in DMD can lead to this disease
gene editing is being investigated to correct the defect in patient-derived cells
leads to premature ageing of muscle throughout the body - only cure is to correct the mutated dystrophin or to use stem cells to repopulate
engineer stem cells using CRISPR/Cas9 and correct the defect - stem cells contain the corrected dystrophin
How can dCas9 be used in regenerative medicine?
dCas9 has a mutation that deactivates nuclease activity and enables it to be used to control gene expression
Guide RNAs are designed to localise dCas9 to specific genes where transcription is activated or repressed
Can be used to up/downregulate gene expression
dead Cas9 - has a mutation in it that stops it cleaving DNA
potentially attach activators/repressors of gene expression
unregulated –> guide dCas9 to this point with an activator, to activate the gene
How can acellular scaffolds be used in regeneration?
Much more cost-effective than engineered tissues and cell-based therapies, easier for clinicians
provides scaffold and releases GFs, such as VEGF, PDGF and TGF-b
interest as possible treatment of type 2 diabetes, obesity and cancer
What is one way that regeneration can be used in diabetes treatment?
FMD (fasting mimicking diet) plays a role in beta-cell regeneration
When exposed to FMD (starving islets in cell culture) there is an increase in the expression of genes that promote the proliferation of beta islets
Potential for using FMD in humans for regenerating islets in the pancreas, to overcome type 1 and 2 diabetes
How can stem cells be used to treat chronic wounds?
E.g. diabetic ulcers that do not heal
Small molecules can be used to stimulate bone marrow cells to proliferate and home to the site of injury
Plerixafor and Tacrolimus can be used in combination to increase wound healing time by 25%
How can the epigenetic landscape be altered to reverse ageing?
There are several metabolic pathways that we can harness to potentially slow ageing
Mice have been engineered to express the 4 pluripotent genes - under control of the Tet-on system
In the absence of doxycycline, the cells do not express the genes
When doxycycline is present, the gene is switched on and proteins can be translated
the cells throughout these animals were turned into a younger state by these pluripotency genes
long-term partial reprogramming prevented or reversed epigenetic ageing in skin and kidney
How can newts/axolotls rapidly re-grow limbs?
If treated with myoseverin, the cytoskeleton breaks down and they differentiate
Myoseverin can be used to dedifferentiate them, and then differentiate them into different lineages
How can bioelectricity be used?
Bioelectric circuits have been shown to influence patterning, and it combines with the genetic code to determine the final shape/function of tissues and whole organisms
interplay between genetic and bioelectric, etc., that could potentially be used to achieve true regeneration.
