Crisp-Cas9 and Cancer

Audio Transcript Auto-generated

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  hi everyone from my project I will be focusing on

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  how CRISPR CAs nine is a tool for cancer research.

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  Um To begin I'm going to explain what is CRISPR CAS nine.

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  It is a genome editing tool that enables scientists to edit parts

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  of the genome by removing adding or altering sections of the D.

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  N. A. Sequence.

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  Um It is also known to be the most specific efficient, versatile,

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  simplest and precise method of genetic manipulation in living organisms.

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  It can also be found in the genomes

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  of programmatic organisms such as bacteria and archaea.

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  So how does CRISPR CAS nine work? I have inserted two different images.

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  The one on the left side is a more simplest former explaining um how this CRISPR CAs

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  nine works and then on the right side um It is a more in depth detail.

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  Um So you can just follow along for whichever one you like the most.

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  So the CRISPR CAS nine system consists of two

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  key molecules that introduce a change into the D.

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  N. A.

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  These two molecules are an enzyme called CAS nine and a piece of RNA.

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  Called guided RNA.

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  Um The enzyme CAS nine acts as a pair of molecular scissors that can cut the two

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  strands of DNA at a specific location in the genome so that it binds of D.

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  N. A.

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  Can be added or removed.

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  Um The

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  ARN a piece

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  um consists of a small piece of pre designed RNA sequence which is

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  located within a longer RNA scaffold the scaffold parts binds to the D.

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  N. A. And the pre designed sequences guides Cast nine to the right part of the genome.

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  This makes sure that the Cast nine enzyme cuts at the right point of the genome.

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  Um Then in the targeted sequences that guided our N. A.

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  Is designed to find and bind to a specific sequence in the DNA.

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  The guided RNA has an RNA bases that are complementary to

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  those of the targeted DNA sequences in the genome genome.

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  Um This means that at least in theory the guided RNA will only

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  bind to the target sequences and no other regions of the genome.

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  The Cast nine follows the guided RNA to the same location in

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  the DNA sequences and makes a cut across both strands of D.

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  N. A.

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  At this stage the cells recognizes that the D. N. A. Is damaged and tries to repair it.

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  So then scientists can use the D. N.

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  A repair machine machine eri to introduce changes to one

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  or more genes in the genome of the self interest.

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  In this case it will be in cancer.

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  So there are three main categories of genetic editing with CRISPR Cast nine.

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  The first one is disrupted editing which you can see on your forest left.

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  Um This happens when a single cut is made in a process called

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  non homologous and joint results in the addition or deletion of base pairs.

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  This disrupts the original DNA sequences and causes the gene inactivation.

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  The second one is deletion which is in the middle one.

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  Um It happens when a larger fragment of D. N. A. Is deleted.

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  By using two guided RNA is the target separate sites after privilege at each side.

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  Non homologous end, joining the separate ends,

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  deleting the intervening sequence.

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  Lastly there is a correction or insert,

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  there is correction or insertion which is to the farthest right.

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  Um ah This is an addition of DNA template

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  alongside the CRISPR CAs nine missionary which allows the cell

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  to correct the gene or even insert a new

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  gene gene using a process called homologous directed repair.

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  So how does CRISPR CAs and I help with cancer?

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  It does this by removing three genes that may interfere

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  with or limit the cell's ability to kill cancer.

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  Um It is able to edit genomes for exploring exploration of the mechanisms of

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  tumor tumor genesis and development.

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  Um It is also able to use to identify the cancer

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  heterogeneous he and it's therapeutic targets against different cancer cells.

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  So now we're going to go more into like the different clinical

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  trials that are done on cancer patients using CRISPR CAS nine.

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  The first clinical trial we're going to talk about is how

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  crisper um engine T cells in patients with refractory cancer.

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  So this clinical trial focuses on using human T cells using CRISPR

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  to fight cancer.

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  They work in the first in human phase one

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  clinical trial to test the safety and the possibility of

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  CRISPR CAS nine Using some editing to engineer T

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  T cells in three patients with the refractory cancer.

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  This can be done because gene editing offers the potential to correct DNA

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  mutations that can also offer to treat or eliminate countless human diseases.

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  The goal of gene editing is to change the

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  DNA of cells with single base pair precision.

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  Most cancers are recognized and attacked by

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  the immune system but as cancer progresses

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  with time it becomes harder for the immune system to fight it off.

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  This clinical trial focuses on how the infusion

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  of ex vivo engineering T cells and termed

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  adoptive T cell therapy can increase the natural

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  anti tumor immune response of the patient.

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  Gene therapy combined with gene editing has the potential to improve the

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  effectiveness and increase the safety nous of the engineered T cells.

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  So in the end their hypothesis was um that removing the

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  endogenous T cell receptor T. C. R. And the immune checkpoint molecule programmed

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  cell death protein one pd one

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  would improve the function and persistence of engineered T cells.

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  So now we're gonna go into the methods of this clinical trial.

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  In their methods they chose to target trans genetic T. C. R. T cells which are the T. R.

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  A C T. R. B. C. N. P. B. C. D.

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  Because it allowed them to increase the trc expression and reduce the potential for

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  mixed hetero diamond formation and to limit the development of T cell exhaustion.

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  The two genes encoding the T. T cell receptor TC. Our chains are T. C. R. A. And T. CRB.

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  Which were deleted in T cells. To reduce the T. C. R.

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  MS peering into enhance,

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  enhance the expression of a synthetic cancer specific trans gene.

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  Um There were over all six patients that

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  were gathered for this specific um clinical trial.

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  And of the six patients who were initially enrolled for patients had their cells.

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  Um So sex fully engineered using T cells of the four patients.

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  One picture experience rapid clinical progression with the cancer cells.

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  Since one of the requirement was to have advanced cancer,

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  this patient was no longer eligible for infusion due to

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  the inability to meet the protocol mandates safety criteria.

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  Thus leaving three patients to be infused with CRISPR cast nine

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  engineered T cells.

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  The three pensions with advanced refractory cancer

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  were given infusions of the CRISPR cast nine

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  engineered T cells.

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  The infusions were well tolerated by the patient bodies with

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  no serious adverse events and there were no cases of cytokine

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  cytokine release syndrome.

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  Um This syndrome is a life threatening systematic inflammatory

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  response that has been associated with cancer immuno therapies,

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  immuno therapies.

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  The patients were giving um lipo the abolition chemotherapy with

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  um cyclo

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  cyclophosphamide Mind and

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  Flow. There have been four or 5 days.

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  Um No psychokinesis were administered to the patients after having T cells

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  that were manufactured by electro operations or rival nuclear protein complexes.

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  Um So now I want to go more in depth

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  on how this part of the methods um happened specifically.

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  I want to focus on how they manufacture the T

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  cells and how it was replaced into the humans.

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  So I have inserted this image to help um kinda guide you and follow as I am explaining.

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  So we're gonna start on how the T cells were engineered the t cells that you can see um

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  in the Petri dish um in dark blue um were

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  isolated from the blood of a patient with a cancer.

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  Um They make the crisper they were able to make the crispy or edited T

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  cells in the lab and they were able to do this by inserting A.

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  N. Y. Es. 01 receptor which interferes with

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  that crispy are using causing a delusion of three genes

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  which are P. C. D. One T. R. A. C. And T. R. B. C.

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  Then they grow tons of crispy are edited T cells and infuse them into the patient.

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  Once it's inside the patient the edited T cells

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  binds to the cancer cells and eventually killed them.

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  Um And as you can see um this image kind of goes step by step on how they did this.

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  So the results were that they found that the highest efficiency observed of the T. C.

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  R.

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  Um

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  oh sorry. Um They observed that the highest efficiency observed for the T. C.

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  Are exchanging TC. R. And the lowest efficiency for the T. C. R. Was a be changed gene.

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  So the T cells revealed that 30% of the cells

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  had no detectable mutation where areas 40% had a single mutation

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  and 20 and 10% of the engineer T cells were doubled and tripled mutated

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  T cells um expressing the Engineer T. C. R.

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  Was much more durable and there were no

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  clinical toxicities toxicities associated with engineered T cells.

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  So overall the trial demonstrated that the

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  multiplex human genome engineer is safe and

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  festival using crispy crass nine.

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  Now we're gonna move on into the second trial um that I'll be talking about in this

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  um presentation. So the second child focused on how CRISPR

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  mediated direct mutation of cancer genes in the mouse

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  liver.

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  It's a little introduction on this.

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  Um It is that they described a new method of

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  cancer model using the CRISPR CAS nine system in vivo.

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  In wild type mice. They also use hydrodynamic injection to deliver the CRISPR cast

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  nine plasma DNA expressing a single guided RNA to

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  deliver that directly targeted the tumor suppressor genes.

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  P. 10 and P. 53

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  alone and in combination

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  this study demonstrates the feasibility of direct mutation of tumor suppressor

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  genes and encode genes in the liver using CRISPR CAS nine system

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  which represses a new avenue for rapid development of liver cancer models

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  by using the insertion or deletion of the tumor suppressor genes.

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  Um The clinical trial also offers sequences specific direct editing of the DNA.

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  Um rather than using an RNA interference based approach.

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  Um This allows it to achieve a complete loss of function of the encoded protein.

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  Um They also wanted to know how the generation of somatic

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  cancer mutation works in adult animals using CRISPR CAS nine.

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  So overall this trial wants to investigate the potential of the crisper

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  system to directly induced loss of function mutations in rebel organisms.

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  They chose to target the tumor suppressor gene P.

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  10, which is a negative regulatory of the P. I three K. And the A. K. T pathway.

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  So for this trial I will be using figures to describe the process and

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  the results um findings for each step that happened in this clinical trial.

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  So it will be a little different from the previous clinical trial that I explained

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  in this trial. They wanted to investigate the potential of CRISPR

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  system to directly in those loss of function mutations in vivo.

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  They chose to target the tumor suppressor gene P.

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  10, which is a negative regulatory of the

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  um P 13 canines and a cape bathroom I stated um in the previous slide

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  um it was specifically chosen to do the P 10 because of the mutation.

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  And genomic loss of P 10 has been identified in many types of human cancer.

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  There are liver specific knockouts of Peten and mice

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  that induces lipid accumulation in um liver cancer.

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  So they cloned a P. X. 3 30 factor nine um co expressing an S. G. RNA targeting P.

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  10 and CAs nine.

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  It was first show that um SGP 10 could induce P. 10 mutations in mouse with three T.

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  Three cells following transfer action.

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  Um To deliver crisper

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  to deliver CRISPR to deliver in adult mice. The plasmid expression cast nine.

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  And the S. G. RNA targeting P.

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  10 were hydro dynamically tail vein injected into the wild type F.

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  We we might

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  to express the CRISPR components in the HEP two sites

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  Um which can deliver DNA to about 20% of um

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  hypothesis sides for transgenic expression. Um A showing N. B.

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  Or you can see this in figure one of how they injected.

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  Um

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  That's how they dynamically injected

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  this into the wild type animals

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  um mouth.

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  Um So then as shown in Figure one B.

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  Hydrodynamic injection of a luciferase,

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  plasma DNA resulted in liver specific expression of luciferase and mice.

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  Um As you can see it is the red spot that is

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  on the left side of the mouse by like the stomach.

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  Um

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  So then they next decided to inject a cohort of wild type F.

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  BB mice with the SGP 10 and an equal number with a P. XB.

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  30 plasmid encoding and SG RNA targeting GPS. As a control

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  they also genetically deleted Peyton in the liver of the Peten flux mice.

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  Um via tell vein injection two weeks later that