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Personalised Medicine > Novel Technologies 2 > Flashcards

Novel Technologies 2 Flashcards

1
Q

What is a cell therapy

A

Administration of live human cells to a patient for repair/replacement/regeneration of damaged tissue and/or cells

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2
Q

What are 2 well known cell therapies

A

Blood transfusion (1818), haematopoietic bone marrow transplantation (1957)

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3
Q

What disorders can cell therapies currently treat

A

Baldness, neurodegenerative disease, immune diseases, heart disease

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4
Q

What are the two categories of cell therapies in accordance to patient relation

A

Autologous and allogenic

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5
Q

What are the benefits and drawbacks of autologus cell therapies

A

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

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6
Q

What are the benefits and drawbacks of allogenic cell therapies

A

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

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7
Q

What are the categories of cell therapies in accordance to the cell type

A

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

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8
Q

Describe the use of blood platelet treatment

A

Donor platelet transfusions, for platelet deficiency (thrombocytopenia) due to :

Disease
Treatment related - chemotherapy
Injury or trauma

Autologous platelet rich plasma therapy

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9
Q

Why use platelets

A

Platelets are a rich source of growth factors - stimulate development of soft tissue/bone cells

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10
Q

What orthopaedic conditions can platelets treat

A 
Osteoarthritis
Tendonitis
Tendon tears
Nerve injury
Professional athletes with muscle and ligament injuries
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11
Q

What are erythrocytes used for

A

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

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12
Q

Describe autologous erythrocyte encapsulated enzyme extract

A

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

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13
Q

What are stem cells, what do they do

A

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

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14
Q

What is the stem cell hirearchy/classifications

A

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

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15
Q

What are the three stem cells used therapeutically

A

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

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16
Q

Where are embryonic stem cells derived from

A

Derived from inner cell mass (5-7 day old blastocyst, precursor to the embryo)

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17
Q

What conditions are ESC’s used to treat

A

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

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18
Q

What are the drawbacks of ESC’s

A

Use is limited due to ethical issues, and can result in immune rejection from the host

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19
Q

How are iPSC’s made

A

Reprogramming of terminally differentiated cells and transfected with stem cell associated genes

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20
Q

Why are iPSC’s not widely used

A

Lack of in situ integration, genomic instability, immunological rejection, carcinogenicity or lack of QC

Investigated for use in age-related macular degeneration

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21
Q

What is age-related macular degeneration

A

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

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22
Q

What are adult somatic cells and where are they found

A

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.

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23
Q

When do adult somatic cells differentiate

A

Remain undifferentiated until activated

To maintain tissue homeostasis
To recover from disease and/or tissue injury

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24
Q

What is haematopoietic stem cells

A

These are sourced from umbilical cord blood, bone marrow and peripheral blood

Differentiate into all types of blood cells

Autologous and allogeneic cells used

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25
Q

What is haematopoietic stem cell therapy used to treat

A

Malignant/ non-malignant blood disorders

Genetic disorders of immune system

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26
Q

What do you need to do in preparation for hematopoietic stem cell therapy

A

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

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27
Q

What are mesenchymal stem cells

A

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

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28
Q

What diseases might mesenchymal stem cells be used in

A

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

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29
Q

What is the therapeutic potentials of mesenchymal stem cells

A

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

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30
Q

What are the two types of genetically modified cells

A

Gene modified autologous stem cells

Engineered T-cells

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31
Q

Describe gene modified autologous stem cells

A

Stem cells collected from patient and genetically corrected prior to reinfusion

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32
Q

What are therapy targets for gene modified autologous stem cells

A

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

33
Q

What is Strimvelis treating

A

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

34
Q

What is the strimvelis procedure

A

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

35
Q

Why is strimvelis treatment under investigation

A

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

36
Q

Why engineer T cells

A

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

37
Q

What is the general procedure of expanding engineered T cells

A

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

38
Q

What are the two types of engineered T cells

A

T-cell receptor (TCR) therapies

Chimeric antigen receptor (CAR-T) therapies

39
Q

What is the difference between T-cell receptor therapies and CAR-T cells

A

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

40
Q

What can CAR-T cells currently treat

A

Three therapies licensed - treatment of lymphoma or acute lymphoblastic leukaemia

41
Q

What are the steps of engineering T cells

A

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

42
Q

What vectors are used to insert TCR or CAR into engineered T cells

A

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

43
Q

How are engineered T-cells preselected for transferring back into patients

A

Choose

CD8+ T cells over bulk lymphocytes

Memory T cells over Naïve

44
Q

What methods can prolong the expansion/expression/persistence of engineered T-cells

A

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

45
Q

Describe T cell receptor therapy

A

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

46
Q

What is the chimeric antigen receptor and its parts

A

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

47
Q

What actions does CAR promote

A

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

48
Q

Outline CAR-T cells therapy

A

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

49
Q

What are the 5 challenges with cell based therapy commercialisation

A

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

50
Q

What issues in the preclinical stage cause challenges in commercialising cell based therapies

A

Conduction the right animal toxicity studies to support clinical programme

Choosing the correct animal model and dose level

51
Q

What issues in manufacturing cause challenges in commercialising cell based therapies

A

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

52
Q

What issues in clinical development cause challenges in commercialising cell based therapies

A

Selecting the right patient population (patient variability)

Dosing, number of cell, single/repeated

Immunological responses/immunosuppression

End point measures (change from baseline/survival)

53
Q

What issues in regulatory affairs cause challenges in commercialising cell based therapies

A

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

54
Q

What issues in general commercialisation cause challenges in commercialising cell based therapies

A

Securing reasonable reimbursement

Encouraging adoption - NHS slow to adopt novel biotechnologies

55
Q

What is cystic fibrosis and what does CFTR stand for/what does it do

A

AR disease affecting the cystic fibrosis transmembrane regulator which influences movement of Cl-, HCO3- and other anions

This can also affect water movement

56
Q

Why is the CFTR important

A

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

57
Q

What are the common CF-causing variants

A

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

58
Q

What are the classes of CF

A 
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
59
Q

Which classes of CF does the DELTAF508 variant fall under

A

Class 2/3/ defects can be caused by DELTAF508

60
Q

What is the treatments for CF

A

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

61
Q

How may cDNA be potentially used as a CF treatment

A

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

62
Q

How can CFTR be edited by CRISPR-Cas9

A

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

63
Q

What are the types of CRISPR editing

A

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

64
Q

What are the conditions for HDR CRISPR targeting

A

Need donor template

Only works in dividing cells as the repair machinery must be present

65
Q

Describe HDR Precision editing

A

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

66
Q

How has HDR been used in CF research

A

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

67
Q

Describe NHEJ targeted deletion/disruption

A

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

68
Q

Describe the HDR super exon method

A

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

69
Q

What is the main issue with HDR

A

Low efficiency as it only works for dividing cells

The other half is resynthesised

70
Q

Describe NHEJ targeted insertion method

A

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

71
Q

What is base editing

A

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

72
Q

Describe the Cas9 molecule used in base editing

A

Catalytically disabled Cas9 fused to deaminase

This catalyses the removal of an amine group from adenosine to convert A to G for base editing

73
Q

Describe the process of base editing

A

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

74
Q

What are the benefits and restrictions of base editing

A

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

75
Q

What is prime editing

A

Higher efficiency than base editing using a reverse transcriptase

Catalytically impaired Cas9 + gRNA + RT and a priming editing guide (pegRNA)

76
Q

Describe how prime editing works

A

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

77
Q

What are the problems with gene editing

A

‘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

78
Q

How has gene editing been used in the clinic

A

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

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