Novel Technologies 2 Flashcards
What is a cell therapy
Administration of live human cells to a patient for repair/replacement/regeneration of damaged tissue and/or cells
What are 2 well known cell therapies
Blood transfusion (1818), haematopoietic bone marrow transplantation (1957)
What disorders can cell therapies currently treat
Baldness, neurodegenerative disease, immune diseases, heart disease
What are the two categories of cell therapies in accordance to patient relation
Autologous and allogenic
What are the benefits and drawbacks of autologus cell therapies
Benefits
Immunological compatibility - no HLA required, and is an individualised treatment
Drawbacks
Heterogenous due to donor variability, imprecisely characterised thus not ideal for clinical trials
Stringent traceable logistics - collection, transport, manufacture/manipulation and administration
Manual process - high production work load, and is expensive
What are the benefits and drawbacks of allogenic cell therapies
Drawbacks - Risk of immune response/rejection/graft vs host disease, thus requiring immunosuppression
Benefits
Standardised product - granted dose, cell bank
Simplified supply, off-the shelf product
Automated process - less labour intensive, lower costs
What are the categories of cell therapies in accordance to the cell type
Terminally differentiated cells e.g. platelets, erythrocytes
Stem cells e.g. embryonic stem cells, induced pluripotent stem cells, adult/somatic stem cells
Genetically modified cells e.g. gene modified autologous stem cells, engineered T-cell therapies
Describe the use of blood platelet treatment
Donor platelet transfusions, for platelet deficiency (thrombocytopenia) due to :
Disease
Treatment related - chemotherapy
Injury or trauma
Autologous platelet rich plasma therapy
Why use platelets
Platelets are a rich source of growth factors - stimulate development of soft tissue/bone cells
What orthopaedic conditions can platelets treat
Osteoarthritis Tendonitis Tendon tears Nerve injury Professional athletes with muscle and ligament injuries
What are erythrocytes used for
RBC transfusions to treat anaemia due to
Heavy blood loss (trauma, surgery)
Bone marrow not producing enough RBC (chemotherapy, leukaemia, sickle cell)
Autologous erythrocyte encapsulated enzyme replacement therapy
Describe autologous erythrocyte encapsulated enzyme extract
Delivery of therapeutic enzymes encapsulated in patient erythrocytes ex vivo
This is i.v. administered to the patient, permitting elimination of pathological metabolites
Applies to disorders where pathologically increased metabolites permeate erythrocyte membrane
Increase enzyme activity half-life by living inside RBC, and decrease immunogenic reactions as the enzyme is hidden from the immune system
Clinically approved for MNGIE
What are stem cells, what do they do
Replicate itself to maintain stem cell pool and retain its undifferentiated state (unspecialised)
Differentiate into many cell types
Switch on specific genes in response to external/ internal chemical signals
Replace dead/damaged cells throughout life
What is the stem cell hirearchy/classifications
Totipotent - first few cell divisions in embryonic development, and can different into not only early embryonic tissue, but extra-embryonic tissue such as the placenta
Pluripotent - originate from 5-7 day old blastocyst, can differentiate into any embryonic cell type
Multipotent - organ specific stem cells, differentiating into limited range of cells e.g. haematopoietic
What are the three stem cells used therapeutically
Embryonic (pluripotent) stem cells - grown in laboratory from early embryonic cells
Induced pluripotent stem cells (iPSC) - made from adult specialised cells using laboratory techniques
Adult or somatic (multipotent) stem cells - found throughout the body
Where are embryonic stem cells derived from
Derived from inner cell mass (5-7 day old blastocyst, precursor to the embryo)
What conditions are ESC’s used to treat
ESC’s are being trialled for use in spinal cord injury, ischaemic heart disease, Parkinson’s disease, age-related macular degeneration and amyotrophic lateral sclerosis
These are directed to differentiate into specific progenitor cells to replace faulty cells
What are the drawbacks of ESC’s
Use is limited due to ethical issues, and can result in immune rejection from the host
How are iPSC’s made
Reprogramming of terminally differentiated cells and transfected with stem cell associated genes
Why are iPSC’s not widely used
Lack of in situ integration, genomic instability, immunological rejection, carcinogenicity or lack of QC
Investigated for use in age-related macular degeneration
What is age-related macular degeneration
Abnormal blood vessels growing under the macular leak blood, preventing retina function
Removal of unnecessary blood vessel and damaged pigment epithelium removed, with replacement of pigment epithelium grown by iPSC
What are adult somatic cells and where are they found
Multi-potent - limited to differentiating into specialised cell types within tissue of origin
Found in stem cell niches located organs and tissues e.g. skin, retina, brain, pancreas etc.
When do adult somatic cells differentiate
Remain undifferentiated until activated
To maintain tissue homeostasis
To recover from disease and/or tissue injury
What is haematopoietic stem cells
These are sourced from umbilical cord blood, bone marrow and peripheral blood
Differentiate into all types of blood cells
Autologous and allogeneic cells used
What is haematopoietic stem cell therapy used to treat
Malignant/ non-malignant blood disorders
Genetic disorders of immune system
What do you need to do in preparation for hematopoietic stem cell therapy
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
What are mesenchymal stem cells
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
What diseases might mesenchymal stem cells be used in
Investigated for use in CVS, nervous system, autoimmune and osteoarthritis
What is the therapeutic potentials of mesenchymal stem cells
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
What are the two types of genetically modified cells
Gene modified autologous stem cells
Engineered T-cells
Describe gene modified autologous stem cells
Stem cells collected from patient and genetically corrected prior to reinfusion
What are therapy targets for gene modified autologous stem cells
Haematological malignancies, inherited blood disorders (primary immunodeficiency, sickle cell anaemia)
What is Strimvelis treating
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
What is the strimvelis procedure
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
Why is strimvelis treatment under investigation
One patient developed lymphoid T-cell leukaemia - due to insertional oncogenesis
No longer used until an investigation is made
Why engineer T cells
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
What is the general procedure of expanding engineered T cells
Altered T-cells expanded in culture→ infused into patient → seek out/destroy tumour cells
What are the two types of engineered T cells
T-cell receptor (TCR) therapies
Chimeric antigen receptor (CAR-T) therapies
What is the difference between T-cell receptor therapies and CAR-T cells
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
What can CAR-T cells currently treat
Three therapies licensed - treatment of lymphoma or acute lymphoblastic leukaemia
What are the steps of engineering T cells
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
What vectors are used to insert TCR or CAR into engineered T cells
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
How are engineered T-cells preselected for transferring back into patients
Choose
CD8+ T cells over bulk lymphocytes
Memory T cells over Naïve
What methods can prolong the expansion/expression/persistence of engineered T-cells
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
Describe T cell receptor therapy
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
What is the chimeric antigen receptor and its parts
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
What actions does CAR promote
On binding to target it causes clonal expansion, secretion of cytokines to recruit immune system and destruction of the tumour cell
Outline CAR-T cells therapy
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
What are the 5 challenges with cell based therapy commercialisation
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
What issues in the preclinical stage cause challenges in commercialising cell based therapies
Conduction the right animal toxicity studies to support clinical programme
Choosing the correct animal model and dose level
What issues in manufacturing cause challenges in commercialising cell based therapies
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
What issues in clinical development cause challenges in commercialising cell based therapies
Selecting the right patient population (patient variability)
Dosing, number of cell, single/repeated
Immunological responses/immunosuppression
End point measures (change from baseline/survival)
What issues in regulatory affairs cause challenges in commercialising cell based therapies
Understanding and navigating complex process (academic institutions)
Cost of approval process
What issues in general commercialisation cause challenges in commercialising cell based therapies
Securing reasonable reimbursement
Encouraging adoption - NHS slow to adopt novel biotechnologies
What is cystic fibrosis and what does CFTR stand for/what does it do
AR disease affecting the cystic fibrosis transmembrane regulator which influences movement of Cl-, HCO3- and other anions
This can also affect water movement
Why is the CFTR important
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
What are the common CF-causing variants
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
What are the classes of CF
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
Which classes of CF does the DELTAF508 variant fall under
Class 2/3/ defects can be caused by DELTAF508
What is the treatments for CF
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
How may cDNA be potentially used as a CF treatment
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
How can CFTR be edited by CRISPR-Cas9
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
What are the types of CRISPR editing
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
What are the conditions for HDR CRISPR targeting
Need donor template
Only works in dividing cells as the repair machinery must be present
Describe HDR Precision editing
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
How has HDR been used in CF research
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
Describe NHEJ targeted deletion/disruption
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
Describe the HDR super exon method
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
What is the main issue with HDR
Low efficiency as it only works for dividing cells
The other half is resynthesised
Describe NHEJ targeted insertion method
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
What is base editing
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
Describe the Cas9 molecule used in base editing
Catalytically disabled Cas9 fused to deaminase
This catalyses the removal of an amine group from adenosine to convert A to G for base editing
Describe the process of base editing
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
What are the benefits and restrictions of base editing
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
What is prime editing
Higher efficiency than base editing using a reverse transcriptase
Catalytically impaired Cas9 + gRNA + RT and a priming editing guide (pegRNA)
Describe how prime editing works
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
What are the problems with gene editing
‘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
How has gene editing been used in the clinic
Sickle cell disease treatment to transfect the healthy gene into blood ex vivo and reinfuse
