Lecture 23: Therapeutic CRISPR Modifications of Stem Cells

Question 1

Describe the Cas-9 mediated double strand break

Answer 1

Cas9 cleaves targeted DNA sequence and causes a double strand break (DSB)

Question 2

What enzyme was first discovered when studying/forming CRISPR technology?

Answer 2

Cas9 enzyme

Question 3

How does Cas9 work and how does this relate to CRISPR technology?

Answer 3

Cas9 cuts both strands of the DNA. This causes the cell to freak out and the DNA tries to glue its genome back together which can result in frameshift, deletions, or insertions. However, if the cell is given a template, then the cell will bind to the template and make a copy of its own genome of whatever it is given. This can be used in CRISPR technology to randomly cut segments of DNA (turn it off) or activate segments of DNA (turn on) to see what happens to cells in response to being turned on and off.

Question 4

Explain CRISPR Knock-outs

Answer 4

This involves Cas9, guide RNA. When DNA is changed then the central dogma (DNA> RNA>Protein) changes in response meaning that the protein gets coded differently. This may cause a frameshift mutation that produces a useless protein that is then recycled resulting in a “knockout”.

Question 5

What helps genes know whether to stop or start?

Answer 5

Start codons and stop codons determine whether a gene needs to produce more proteins or not.

Question 6

What is involved in the motility of microglia in the brain?

Answer 6

TREM2

Question 7

Explain CRISPR Knock-ins

Answer 7

This involves Cas9, the guide RNA, and a template. This can be used to either correct a mutation or take a healthy line of cells and knock in a mutation. Looking at these side by side can be good for verification of functions of the genes in the cells.

Question 8

Explain CRISPRa

Answer 8

Cas9 “scissors” are knocked out so the enzyme is mutated in a way where it doesn’t have the ability to mutate anymore. It can still bind to the DNA using the guide. This can be used to piggyback a ton of other transcription factors in vitro in the cell to turn genes on.

Question 9

Explain CRISPRi

Answer 9

This involves a similar idea to CRISPRa where the Cas9 scissors are knocked out and a bunch of different transcription factors are added in however this is used to turn genes off.

Question 10

Knock-in and Knock-out CRISPR techniques are _____

Answer 10

permanent

Question 11

CRISPRa and CRISPRi techniques are _____

Answer 11

reversible

Question 12

What are CRISPR screens?

Answer 12

This involves genome wide sequencing (GWA) which is when you take biopsies of patients with a specific condition and see what mutations align/are shared to see what contributes to the disease. Different combinations of genes can be turned on and off to see how this affects the cells and how this affects the pathology of the disease.

Question 13

What are some applications of CRISPR?

Answer 13

Gene editing/therapies
Diagnostics
Agriculture
Bioenergy

Question 14

Could iPSC-microglial transplantation also be developed for therapeutic applications?

Answer 14

Cell therapies for neurological diseases should ideally
-Use cells that belong in and can integrate within the nervous system.
-Use cells that can migrate toward and/or respond appropriately to pathology.
Enable regulated delivery of a therapeutic cargo (proteins, antibodies, etc).
-Provide long-lasting and safe engraftment (no tumor formation) (MOST IMPORTANT)

Question 15

iPSC-microglia can be safely transplanted into immunodefiecent mice with no evidence of ___

Answer 15

neoplasms (n>920)

Question 16

What did >970 transplantations of iPSC microglial progenitors into immune deficient mice aged from 2-16 months post transplant result in?

Answer 16

No evidence of human derived tumors, human cells outside the CNS, or non-myeloid differentiation.

Question 17

What was the primary goal of the CRISPR engineering of iPSC-microglia for regulated payload delivery?

Answer 17

The primary goal was to perform a proof of principle experiment to determine whether human microglia can be used to deliver a protein of interest to the brain.

Question 18

What is neprilysin?

Answer 18

It is an Alpha Beta degrading enzyme that decreases with an age.

Question 19

What was the result when CD9-NEP and CD0-sNEP iPSC microglia was transplanted into mice?

Answer 19

Alpha Beta pathology induced CD-9 regulated NEP expression in vivo.

Question 20

Microglial delivery of Neprilysin reduces ____ plaques and ___

Answer 20

Alpha Beta plaques and oligomers.

Question 21

Microglial delivery of S-Neprilysin restores 5XFAD synaptic proteins to ____ levels

Answer 21

wild-type

Question 22

Are there off target effects of pathology driven Neprilysin delivery?

Answer 22

It can also degrade Bradykinin and Somatostatin

Question 23

What is the problem with scale up of microglial payload delivery?

Answer 23

Engraftment of therapeutic microglia is regulated by the endogenous microglial niche. Adult transplantation into an occupied niche provides limited engraftment.
We can clear the niche by killing microglia with CSF1R antagonists but as soon as the drug is removed, remaining microglial rapidly proliferate to refill the niche. Scaling up to a human brain would be even more challenging.

Question 24

Recent studies have combined ___ _____ transplantation with microglial depletion to promote recruitment of _____ _____ to the mouse brain

Answer 24

bone marrow
bone marrow derived macrophage

Question 25

Appraoches such as bone marrow transplantation also require the use of ____ or ____ of the host bone marrow which also disrupts the BBB.

Answer 25

irradiation
chemoblation

Question 26

However, bone-marrow derived macrophages remain transcriptionally and functionally distinct from microglial even ___ months post engraftment.

Answer 26

9 months

Question 27

What type of pathology do human blood monocytes engrafted in adult FIRE mice exhibit?

Answer 27

They exhibit differing pathology. 3 months post engraftment monocytes remain less ramified.

Question 28

True or false we can instead develop an approach to achieve robust engraftment of human iPSC microglia within the adult brain.

Answer 28

True

Question 29

True or False Antagonist resistant CSF1R mutations have no significant effect on iPSC microglial gene expression.

Answer 29

True

Question 30

What were the results of of the robust engraftment experiment?

Answer 30

After 60 days, PLX-3397 treatment, G795A human microglia have expanded from their initial hippocampal injection site to completely fill the adult mouse brain.

Question 31

What happens when the PLX-3397 treatment is removed?

Answer 31

G795A human microglia engraftment remains stable.

Question 32

What are the main key points of this lecture?

Answer 32

-Chimeric models can be used to examine the interactions between human microglia and disease pathology in vivo.
-The CD9 promoter can drive AB induced production of Neprilysin reducing AB pathology protecting against synaptic loss, and reducing astrogliosis.
Ongoing studies are examining microglial delivery of her therapeutic genes including CAR-Microglia strategies protective AD genes, and lysosomal enzymes.
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