Ferrari: Lecture XX Flashcards
Applications of Gene Editing
What kind of cells can be used in cancer immunotherapy?
CAR-T cells
CAR_T cells with KO of the endogenous TCR
What is an important application in gene editing?
KO PD1 as it is one of the immune checkpoint molecules
What does CAR stand for?
chimeric antigen receptor
*it is a synthetic receptor that is expressed, after engineering, by T lymphocytes
What can the chimeric antigen receptor do?
reprogram T lymphocytes to kill tumor cells after the recognition of specific target antigens on their surface
What is the most diffuse antigen targeted by CAR-T cells?
CD19 (protein expressed by the majority of leukemias and lymphomas)
What is an engineered T lymphocyte that is currently sold in the market?
Kymriah, which is used to treat a particular leukemia
What is the aim of immunotherapy?
boost the IS of patient and make T lymphocytes able to recognize malignant cells
How do engineered T lymphocytes work?
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
How can we generate CAR-T cells to ensure they only target malignant cells?
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
What is smart targeted gene correction?
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
What is a genetic disease that can be treated using smart targeted gene correction?
Wiskott-Aldrich syndrome
What causes Wiskott-Aldrich syndrome?
mutations on a gene called WAS
How is Wiskott-Aldrich syndrome corrected using smart targeted gene correction?
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
What kind of genome editing approach was used for MucoPolySaccharidosis?
approach targeting lysosomal enzyme gene IDUA to the CCR5 locus since the CCD5 locus is a safe harbor for manipulation
What different functions can we obtain through the protein engineering of Cas9?
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.
What does CRISPRi (interfering) do?
prevents transcription when it is targeted to a transcription start site (TSS) with the right guide RNA (opposite is done in C)
What is base editing?
emerging technology that corrects a gene without inducing DNA ds breaks
What are the 2 types of base editors?
cytosine base editors (CBEs)
adenine base editors (ABEs)
What can CBEs do?
mediate the transition C → T through deamination by converting the dinucleotide C:G into T:A
What can ABEs do?
mediate the transition A→G through deamination by converting A:T into G:C
What can CBEs and ABEs mediate?
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
What is used for base editing?
Catalytic dead Cas9 since it is able to recognize the site to modify but cannot cut the site
dCas9
do not cut DNA
Cas9
mutated to cause a nick on one strand of DNA
What are the different versions of CBEs that have been developed?
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
How were ABEs engineered?
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
What do CBEs and ABEs rely on?
DNA repair system of the cell
What are DNA repair systems fundamental for?
function of the cell
What is a drawback of ABEs and CBEs?
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
What is needed in order for CBEs and ABEs to be used in therapy?
evaluation risk
