Ferrari: Lecture XX

Question 1

What kind of cells can be used in cancer immunotherapy?

Answer 1

CAR-T cells

CAR_T cells with KO of the endogenous TCR

Question 2

What is an important application in gene editing?

Answer 2

KO PD1 as it is one of the immune checkpoint molecules

Question 3

What does CAR stand for?

Answer 3

chimeric antigen receptor

*it is a synthetic receptor that is expressed, after engineering, by T lymphocytes

Question 4

What can the chimeric antigen receptor do?

Answer 4

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

Question 5

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

Answer 5

CD19 (protein expressed by the majority of leukemias and lymphomas)

Question 6

What is an engineered T lymphocyte that is currently sold in the market?

Answer 6

Kymriah, which is used to treat a particular leukemia

Question 7

What is the aim of immunotherapy?

Answer 7

boost the IS of patient and make T lymphocytes able to recognize malignant cells

Question 8

How do engineered T lymphocytes work?

Answer 8

they are able to recognize a specific target expressed on the surface of malignant cells and destroy them since the T lymphocytes are cytotoxic and CAR-T cells have high specificity for antigens present on malignant cells

Question 9

How can we generate CAR-T cells to ensure they only target malignant cells?

Answer 9

isolate T lymphocytes

KO endogenous T cell receptor (TCR) using gene therapy (using TCR𝛼, TCRβ and Cas9 as guides for endogenous TCR)

CAR is introduced by viral vector nanoparticles or another technique efficient for the transfer of T lymphocytes

use library to select the clone able to recognize malignant cells

isolate specific antigens expressed on malignant cells

design chimeric antigen receptor

Question 10

What is smart targeted gene correction?

Answer 10

normal copy of the gene is added in a specific tageted position

*prevents the risk of insertional mutagenesis using a guide RNA and donor template

Question 11

What is a genetic disease that can be treated using smart targeted gene correction?

Answer 11

Wiskott-Aldrich syndrome

Question 12

What causes Wiskott-Aldrich syndrome?

Answer 12

mutations on a gene called WAS

Question 13

How is Wiskott-Aldrich syndrome corrected using smart targeted gene correction?

Answer 13

engineered single guide RNA (sgRNA) recognizes the 1st exon of the gene

a normal copy is then inserted (donor WAS gene) directly in locus by HDR

homology is present since the sgRNA has a homology arm, using the AT of the endogenous gene

*happens in the native locus using the endogenous, native promoter

Question 14

What kind of genome editing approach was used for MucoPolySaccharidosis?

Answer 14

approach targeting lysosomal enzyme gene IDUA to the CCR5 locus since the CCD5 locus is a safe harbor for manipulation

Question 15

What different functions can we obtain through the protein engineering of Cas9?

Answer 15

we can have Cas9 mutants lose the DNA cleavage activity and be fused with a:

repressor (B)
or activating domain (C)

to regulate gene expression.

to regulate gene expression.

Question 16

What does CRISPRi (interfering) do?

Answer 16

prevents transcription when it is targeted to a transcription start site (TSS) with the right guide RNA (opposite is done in C)

Question 17

What is base editing?

Answer 17

emerging technology that corrects a gene without inducing DNA ds breaks

Question 18

What are the 2 types of base editors?

Answer 18

cytosine base editors (CBEs)

adenine base editors (ABEs)

Question 19

What can CBEs do?

Answer 19

mediate the transition C → T through deamination by converting the dinucleotide C:G into T:A

Question 20

What can ABEs do?

Answer 20

mediate the transition A→G through deamination by converting A:T into G:C

Question 21

What can CBEs and ABEs mediate?

Answer 21

ONLY base transitions (purines become purine & pyrimidines become pyrimidines)

*this is a limitation as the field of application is restricted to dieases treatable with these 2 modifications

Question 22

What is used for base editing?

Answer 22

Catalytic dead Cas9 since it is able to recognize the site to modify but cannot cut the site

Question 23

dCas9

Answer 23

do not cut DNA

Question 24

Cas9

Answer 24

mutated to cause a nick on one strand of DNA

Question 25

What are the different versions of CBEs that have been developed?

Answer 25

BE1: recognizes target locus and converts it to U, U:G is recognized as a mismatch and U is removed

BE2: RNA repair system matches U and the will replace G with A

BE3: nickase activity was added so dCas9 was replaced with Cas so a cut was induced on the strand containing C and DNA repair system was induced… U:A match was converted to T:A by host repair machinery

BE4: one of the last products carries a 2nd UGI conferring a higher editing efficiency and improved product purity

Question 26

How were ABEs engineered?

Answer 26

synthetic enzyme, derived from tRNA adenine deaminase from E. coli, was engineered

these chimeric diameric enzymes were generated from Tad and were fused to Cas9 or dead (catalytically inactive) Cas9

A converts to T

Question 27

What do CBEs and ABEs rely on?

Answer 27

DNA repair system of the cell

Question 28

What are DNA repair systems fundamental for?

Answer 28

function of the cell

Question 29

What is a drawback of ABEs and CBEs?

Answer 29

bystander editing can take place even if there is a gRNA to help Cas9, other C & A bases inside the window can be modified to

Question 30

What is needed in order for CBEs and ABEs to be used in therapy?

Answer 30

evaluation risk