Novel Technologies 2 Flashcards

Question

What is haematopoietic stem cell therapy used to treat

Answer 1

Malignant/ non-malignant blood disorders | Genetic disorders of immune system

Answer 2

Starts with conditioning therapy - chemotherapy +/- radiation to kill leukaemia/tumour cells and eradicate bone marrow to create space for replacement and supress the immune system to reduce rejection

Answer 3

These are found in the bone marrow, placenta, umbilical cord, adipose tissue and peripheral blood They can differentiate into a wide range of cell types Fat, bone, muscle, skin, cartilage and CNS

Answer 4

Investigated for use in CVS, nervous system, autoimmune and osteoarthritis

Answer 5

Differentiate into various cell types Transdifferentiate - non-mesoderm Secrete soluble factors crucial for cell survival and proliferation Modulate immune response and regulate inflammation Migrate to sites of injury in response to cell signals - homing

Answer 6

Gene modified autologous stem cells Engineered T-cells

Answer 7

Stem cells collected from patient and genetically corrected prior to reinfusion

Answer 8

Haematological malignancies, inherited blood disorders (primary immunodeficiency, sickle cell anaemia)

Answer 9

Strimvelis - treats severe combined immunodeficiency disease due to adenosine deaminase deficiency ↓ immune responses, patients vulnerable to bacterial, viral, and fungal infections CD34+ cells genetically modified to replace defective adenosine deaminase gene

Answer 10

SC's isolated from bone marrow > CD34+ cells are isolated and expanded in cultures > transduced with retroviral vector expressing functional copy of defective gene (adenosine deaminase) > endogenous bone marrow progenitors are eliminated to favour engraftment > modified SC's infused This reconstitutes lymphoid lineages/restore immune function

Answer 11

One patient developed lymphoid T-cell leukaemia - due to insertional oncogenesis No longer used until an investigation is made

Answer 12

Therapeutic need: tumour cells often recognised as “self” → prevents T- cells from recognizing tumour proteins Solution - genetically alter T-cells to create recognition receptors unique to patient’s tumour

Answer 13

Altered T-cells expanded in culture→ infused into patient → seek out/destroy tumour cells

Answer 14

T-cell receptor (TCR) therapies Chimeric antigen receptor (CAR-T) therapies

Answer 15

CAR-T targets external antigens on the cancer cells Single chain antibody domain (scFv) forming dimers linked to various intracellular signalling domains Cell surface tumour antigen can be recognised TCR targets peptides processed from tumour proteins within cells (MHC) - No licensed treatments

Answer 16

Three therapies licensed - treatment of lymphoma or acute lymphoblastic leukaemia

Answer 17

Isolation of patients' lymphocytes from peripheral blood V ector encoding TCR or CAR is inserted into the cells These are expanded ex-vivo before transferred back

Answer 18

Usually use gamma retroviral vectors, however these can only target dividing cells thus these T cells are pushed into the cell cycle Lentiviral vectors can target most cell types, but human T cells can be fairly resistant

Answer 19

# Choose CD8+ T cells over bulk lymphocytes Memory T cells over Naïve

Answer 20

Co-stimulation/pre-activation with IL-7 and 15 can promote the expression of gene engineered T-cells with an early differentiated phenotype which allows greater expansion and prolonged in vivo expression Systemic administration of IL-2 can increase persistence of the transferred T cells This can cause toxicity and requires intensive care treatment Therefore certain inducible cytokine genes may be transfected for future local activation

Answer 21

Peripheral blood is removed from patient Cells are transduced with viral vectors containing construct encoding for tumour reactive TCR Expanded in culture and infused into patient -infused cells express modified TCR on cell surface These recognise tumour-specific proteins on the inside of the tumour cell through encountering tumour antigen peptides processed and presented on the cell surface

Answer 22

This single receptor has both T-cell activation and antigen binding domains Extracellular domain - antibody molecule, recognises target proteins expressed on cancer cell surface Transmembrane domain - links the components Intracellular domain - T-cell signalling machinery of the T-cell receptor

Answer 23

On binding to target it causes clonal expansion, secretion of cytokines to recruit immune system and destruction of the tumour cell

Answer 24

Peripheral blood lymphocytes removed from patient Cells are transduced with viral vector containing CAR construct CAR is expressed in the cell membrane This is expanded in culture and infused into patient Expressed CAR recognises external antigen Clonal expansion Cytokine release, which recruits the immune system leading to death of targeted cancer cells

Answer 25

Pre-clinical trial animal model and dosage choices Manufacturing Clinical development and trail - patient and dosage selections Regulatory affairs - time and costs Commercialisation - reimbursement and widespread adoption

Answer 26

Conduction the right animal toxicity studies to support clinical programme Choosing the correct animal model and dose level

Answer 27

Cell selection - tissue source, autologous/allogenic Move from small scale to large scale - can affect cells No terminal sterilisation Limited shelf-life (cell viability) Manufacture process - manual or automated Distribution logistics - centralised/decentralised manufacture model High production costs

Answer 28

Selecting the right patient population (patient variability) Dosing, number of cell, single/repeated Immunological responses/immunosuppression End point measures (change from baseline/survival)

Answer 29

Understanding and navigating complex process (academic institutions) Cost of approval process

Answer 30

Securing reasonable reimbursement | Encouraging adoption - NHS slow to adopt novel biotechnologies

Answer 31

AR disease affecting the cystic fibrosis transmembrane regulator which influences movement of Cl-, HCO3- and other anions This can also affect water movement

Answer 32

Airways - hydrated airway surface liquid, allowing for a low viscosity environment for the cilia to beat Decrease in CFTR = dehydration = thick mucus = colonisation with pathogens Pathogens = chronic inflammation, airway remodelling and respiratory failure Systemic so all epithelial secretory tissue affected - pancreas, GI tract

Answer 33

Exonic R117H F508del, G542X, R553X, G551D W1282X, N1303X Intronic 621+1 G>t = splice donor 1717-1 G>A = splice acceptor 3849+10kb C>T = deep intronic

Answer 34

``` 7 classes 1 = most severe, no synthesis 2 = blocking processing/folding/trafficking 3 = block regulation/channel opening 4 = reduced conductance 5 = reduced synthesis 7 = less severe, functional abnormality ```

Answer 35

Class 2/3/ defects can be caused by DELTAF508

Answer 36

Small molecules to correct CFTR function - Ivacaftor, not a cure as lung function still decreases overtime Lung transplant - most ideal, but this also has decreasing function

Answer 37

Insertion of WT CFTR cDNA into CF cells increased function and restored ion channel activity Putting cDNA in patients instead of just CF cells = fairly negligible effect

Answer 38

Hypothetical process Find the mutation within the gene Then you cut this region of mutation out Copy wildtype and insert into mutated cell CRISR-Cas9 = RNA guided endonuclease Guide RNA = find mutation Cas9 = cuts the mutation Template RNA with HDR = reconstructed WT CFTR

Answer 39

HDR - homology directed repair Precision editing - targets one mutation in the gene Super-exon - targets all mutations in the gene NHEJ - non-homologous end joining Targeted deletion - targets one mutation in the gene Targeted insertion - targets all mutations in the gene Prime editing

Answer 40

Need donor template | Only works in dividing cells as the repair machinery must be present

Answer 41

gRNA binds 3' DNA (OPPOSITE 5' PAM sequence) Cas9 cuts within the matched site, with cellular endonucleases removing the DNA prior to cut (and mutation!) on 3' strand and removing the DNA after the cut (including mutation!) on the 5' strand up to and including PAM - this removes the single base mutation on the 5' DNA Donor template binds 5' therefore the mutation region and PAM are reconstructed, with the 3' strand DNA polymerase proof reads and corrects the mutation on the 3' end

Answer 42

Intestinal stem cell organoids made, with donor template encoding WT CFTR + puromycin resistance gene This enabled for selection of successfully engineered cells The functional proven by showing the movement of water into the organoids and seeing them swell Non-functional cells did not swell, while in the edited group swelling was similar to that of WT

Answer 43

Doesn't require template, and works at any point of the cycle gRNA finds genes, makes ds-breaks and cell mechanisms ligate the ends with random insertions/deletions Two endonucleases and gRNA = larger deletion to remove deep intronic mutations Less control, higher efficiency

Answer 44

Incorporation of really big exons, via dsbreak in an intron 99.3% of mutations occur between exons 11 and 27 in X disease Thus by inserting a super exon of all of the WT coding sequences you can fix a majority of the gene The super exon is a cDNA only of exons, with splice acceptor upstream and a polyA site downstream

Answer 45

Low efficiency as it only works for dividing cells The other half is resynthesised

Answer 46

Uses a superexon flanked with two guide sequences These flanking guide sequences also ensure it is inserted in the correct orientation Does not require cell to be dividing

Answer 47

Converts a nucleotide base or base pair into another Works in non-dividing cells Does not create ds break by using a catalytically disabled Cas9

Answer 48

Catalytically disabled Cas9 fused to deaminase This catalyses the removal of an amine group from adenosine to convert A to G for base editing

Answer 49

gRNA target is adjacent to the PAM Helicase unzips the DNA, but only a nick is made in one strand On the 5' strand with PAM, an A is deaminated to a G The catalytic dead site prevents the double strand break DNA polymerase changes the gRNA bound strand amino acid to correct it to a C

Answer 50

More efficient and precise No ds-breaks, less off target effects, less dangerous No needed for donor template / super exon Can be readministered without danger However, it is restricted by need for NGG PAM with target base 12-16 places Different Cas9 have been developed

Answer 51

Higher efficiency than base editing using a reverse transcriptase Catalytically impaired Cas9 + gRNA + RT and a priming editing guide (pegRNA)

Answer 52

pegRNA identifies the target site and provides the genetic info to replace the target DNA, using primer binding site which allows the 3' to hybridise the pegRNA for the reverse transcriptase to create cDNA from the template portion The normal gRNA guides the Cas9 to the non-edited DNA strand Mediates targeted insertions, deletions and base-to-base conversions

Answer 53

'Vandalism' - worry that there will be off target cut sites and crossover events Selects for cancer prone cells with a mutated DNA damage response Especially if he cell taken ex vivo, proliferated then inserted - you are selecting for cancer cells Cas9 immunity as Cas9 taken from bacteria to which we have been already exposed to

Answer 54

Sickle cell disease treatment to transfect the healthy gene into blood ex vivo and reinfuse