Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Regulation Of Gene Expression > Lesson 20 > Flashcards

Lesson 20 Flashcards

1
Q

what does CAR-T stand for?

A

CAR stands for chimeric antigen receptor, a synthetic receptor that is expressed after engineering T lymphocytes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

what is the function of CAR-T?

A

is able to reprogram T lymphocytes to kill tumor cells after the recognition of specific target antigens on their surface

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

what is the most diffuse antigen targeted by CAR-T cells?

A

CD19 - a protein expressed in the vast majority of leukemia and lymphomas

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

how does the immune system fail in cancer?

A

the immune system and in particular T lymphocytes, responsible for the cytotoxic activity, fail to recognize
malignant cells

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

how fast do cells become malignant?

A

they need several mutations

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

what is the aim of immunotherapy?

A

to boost the immune system of the patients and make T lymphocytes able to recognize malignant cells

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

once T lymphocytes are collected from a patient, how can they be altered?

A

they can be engineered using gene transfer techniques to induce the expression on T lymphocytes of the chimeric antigen receptor

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

why is the chimeric antigen receptor unique?

A

it can recognize only the malignant cells and not normal ones

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

what occurs after these engineered antigen receptors are re-infused into the patient?

A

since the T lymphocytes are cytotoxic, they can destroy malignant cells thanks to the fact that the CAR-T cels have a high specificity for antigens present on malignant cells

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

what is one of the most important steps in CAR-T production?

A

the isolation of specific antigens expressed on malignant cells in order to design the chimeric receptor → otherwise CAR-T will act against healthy tissues as well

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

what was a problem that occurred during the production of CAR-T cells in terms of their expression?

A

If a new T cell receptor (TCR) is introduced in T lymphocytes, the resulting CAR-T cell will display the new chimeric antigen, but also the expression of the endogenous TCR →there will be competition between the new and the endogenous receptor

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

how is gene editing used to fix the TCR gene problem in the production of CAR-T cells?

A

KO of the endogenous TCR gene

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

describe the editing process to KO the endogenous TCR gene:

A

editing, we need a specific guide for the endogenous TCR genes, TCRa and TCRb, and Cas9. After disrupting the endogenous TCR locus, we can introduce the new CAR by viral vector nanoparticles or any other technique efficient for the transfer in T lymphocytes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

what is another important application of gene editing and KO in cancer therapy?

A

the knock out of PD-1

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

how can T-lymphocytes interact with PD-1?

A

tumor expresses PD-L1 (the ligand for PD-1) present on the surface of the T
lymphocyte, so the tumor cell can interact with a T lymphocyte through PD-1

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

what is PD-1?

A

one of the so-called immune checkpoint molecules → after the interaction with the ligand, this molecule gets activated and it represents one of the inhibitor signals for the cytotoxic activity of T lymphocytes.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

what occurs if we are able to disrupt PD-1 in cytotoxic T lymphocytes using CRISPR/Cas9 and other genome editing techniques?

A

PD-L1 won’t be able to interact with the receptor PD-1 anymore, because this is will not expressed in T lymphocytes → prevents the escape of the tumor from the IS

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

what are some possible delivery methods for there transfer of editing machinery to a cell?

A

electroporation and nanoparticles

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

what is off target activity?

A

can lead to oncogenesis if we disrupt important genes or other genes for proliferation

20
Q

why must we be careful when using Cas9?

A

Cas9 derives from bacteria and is immunogenic → we need to prevent the recognition of this protein or to engineer Cas9 to avoid
immunogenicity

21
Q

what is the limitation of gene replacement by HDR?

A

it occurs at a very low frequency

22
Q

what is smart targeted gene correction?

A

we can add a normal copy of the gene, like we do in gene addition, but in a specific targeted position → we have gene addition without the risk of insertional mutagenesis

23
Q

what disease can be treated with smart targeted gene correction?

A

Wiskott-Aldrich syndrome

24
Q

what is the advantage of having a native locus and an endogenous promotor?

A

since it is the native promoter, differently from viral or exogenous promoters, we have the optimal level of expression → we avoid the random insertion in the genome.

25
Q

when treating Wiskott-Aldrich syndrome, how did gene editing compare to the current treatment with lentiviral vectors?

A

it was just as powerful or even better in terms of effect of expression of corrected WAS in colony forming units (CFU) and other cells

26
Q

how did scientist try to perform gene editing for MucoPolySaccharidosis?

A

use a genome editing
approach targeting the missing lysosomal enzyme gene IDUA to the CCR5 locus → given that the CCR5 locus is a safe harbor for manipulation (mutants of CCR5 individuals are perfectly normal) there is no harm in targeting it by inserting a new gene

27
Q

what is a new emerging technique that has a lot of potential?

A

base editing → correction of a gene without inducing DNA double-stranded breaks

28
Q

how can we design Cas9 variants through protein engineering to get Cas9 with junctions different than doing DNA breaks?

A

Cas9 mutants can lose their DNA cleavage activity and be fused to a repressor or activating domain to regulate gene expression. CRISPRi (interfering) molecule prevents the transcription when it’s targeted to a Transcription Start Site with the right guide RNA, the opposite is done

29
Q

what are the two types of base editors?

A

cytosine base editors (CBEs) and adenine base editors (ABEs)

30
Q

how do cytosine base editors work?

A

are able to mediate the transition
C→T through a deamination, converting the dinucleotide C:G into T:A

31
Q

how do adenine base editors work?

A

are able to mediate the transition
A→G, through a deamination converting A:T into G:C bp

32
Q

what is the limitation of CBEs and ABEs?

A

they can mediate only base transitions → the field application is restricted to diseases treatable with only these two modifications

33
Q

what type of Cas9 are used for base editing?

A

catalytic dead Cas9 → they are still able to recognize the site to modify but are not able to cut it

34
Q

what are two characteristics of dCas9:

A
  • don’t cut DNA at all
  • mutated to just cause a nick on one strand of DNA
35
Q

how is dCas9 created?

A

a different Cas that recognize a
sequence → fused with a deaminase and the presence of the sgRNA that anneals to one of the two strands. Once there’s the unwinding of the dsDNA, the
strand not annealed to the sgRNA, is accessible to the deaminase and the desired base in the editing window is converted (A→G)

36
Q

what are the four types of CBEs?

A

BE1, BE2, BE3, BE4

37
Q

what is the function of BE1?

A

recognizes C at the target locus and converts it into U, nevertheless U:G bp is recognized as a mismatch by the cellular repair machinery that usually removes the U since it is not one of the four DNA bases (A-C-G-T), so the process stops here

38
Q

what is the function of BE2?

A

developed to protect the U from excision by fusing a uracil glycosylase
inhibitor, therefore U is not excited, the DNA repair system to match the U and the G, will replace the G with a A

39
Q

what is the function of BE3?

A

the nickase activity was added, so the dCas9 w snot replaced with a Cas ability to cut on one strand

40
Q

how does BE3 work?

A

A cut is done on the strand containing the C, thus inducing the DNA repair system. The U:A match is converted to T:A by the host repair machinery. As a final product we get the T, with all the intermediate steps of course (transition from C→T but the conversion is not direct)

41
Q

what is the function of BE4?

A

carries a second UGI conferring a higher editing efficiency and improved product purity

42
Q

what was generated to create ABE?

A

a synthetic enzyme was engineered, derived from the tRNA adenine deaminase (Tad) → these two chimeric proteins were fused to either Cas9 or dCas9

43
Q

what are the five steps of adenine base editing?

A

1) First, there’s the formation of the unwinding structure once the guide RNA joins the complex, so the
two strands are now separated.
2) The deamination of Adenine leads to the formation of Inosine as a product (A→I)
3) Nickase activity of Cas9 leads to the formation of a nick on the strand with the T
4) DNA repair is triggered, with the consequent presence of C as a complementary base to I
5) The C:I bp is seen as a mismatch and so the DNA repair system is triggered again to exchange I with G, which is complementary to C

44
Q

how are ABEs and CBEs different from CRISPR-Cas9?

A

they rely on the DNA repair system of the cell which is independent from the cell cycle phase

45
Q

what is a drawback to ABEs and CBEs?

A

the two systems can lead to bystander editing → even if there is the gRNA to guide the Cas9 and the editors to the right sequence, the other surrounding bases C and A inside the editing window can be modified too

sometimes have no effect, but can also lead to the presence of gene variants

Regulation Of Gene Expression (28 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions