Gene therapy & editing Flashcards
cause of flu virus?
influenza A
name 2 stomach flu - viral gastroenteritis vomiting bug causing virus names
norovirus
retrovirus
what virus causes chickenpox?
VZV
or dormant VZV for shingles
what virus causes cold sore?
herpes simplex
what viruses may cause hand foot mouth disease?
coxsackievirus A16
enterovirus
What is gene therapy?
process of introducing foreign genomic materials (i.e. nucleic acids) into host cells to elicit a therapeutic benefit
What are the 2 nucleic acids that are delivered to treat disease in gene therapy?
DNA or siRNA
(using genetic material as drug, pack into DDS and deliver)
gene therapy = new tool to cure human disease and involves transfer of what?
genetic material into cells/tissue to prevent/ cure a disease
transfection
What are 3 gene alterations gene therapy is used to make?
- introduce a new gene
- replace defected/mutated gene
- silence gene not working properly (STOP protein expression)
What genetic material is introduced as a new gene or replacing a defected/mutated gene?
plasmid DNA
What genetic material is used to silence a gene not working properly?
- small interfering RNA (siRNA)
- short-hairpin (shRNA)
why do you need a delivery vector to deliver plasmid DNA?
else DNA will degrade
What are the 2 types of genetic diseases that gene therapy is used to treat?
- single gene defect disease
- polygenetic or non-hereditary
Give an example of a single gene defect disease.
haemophilia - a hereditary (genetic) disease affecting blood’s ability to clot
Give examples of polygenetic or non-hereditary diseases?
- cancer
- CVD
- hepatitis
polygenetic/nonhereditary conditions are harder to treat than single gene defect, why?
more than 1 gene involved, along w environmental factors
origin can be complex
gene therapy is used to treat but also prevent, as in the case of SARS-CoV-2 vaccines.
outline how these work
prevent infection by causing body to produce antibodies against SARS-CoV-2 spike proteins
elicit immune response
problem with influenza and sars-cov-2 vaccines?
the virus may change by nest year, body wont recognise -> infection
What are the 2 main classes of delivery vectors used in gene therapy? to induce transfection of cells
viral
non-viral
give examples of non-viral delivery vectors
(1st term)
liposomes
polymers
CNTs
What are 2 categories of transfection methods in gene therapy?
- in-vivo
- ex-vivo
What is meant by in-vivo gene therapy?
within living:
gene transferred to cells inside patient directly using vector
Which is more common: in-vivo or ex-vivo?
ex-vivo
Which is more risky: in-vivo or ex-vivo and why?
in-vivo
immune reaction possible from vectors used
could be rejected straight away
What are examples of in-vivo vectors? 3
viruses
bacterial plasmids
NPs
Methods used in in-vivo gene therapy 3
tissue injection- recombinant virus
systemic infusion- DNA liposome
(biolistic gene gun- plasmid DNA)
Outline the steps of how in vivo gene therapy works.
DNA (gene) encapsulated in vector
gene therapy- inject in
gene released into cell
gene expresses proteins
- receptor
- secreted
What is meant by ex-vivo gene therapy?
cells modified outside body then transplanted back in again
what does ex-vivo gene therapy allow?
targeting of specific cells
Explain how ex-vivo gene therapy works.
- cells from px blood/bone marrow removed and grown in lab
- cells exposed to gene therapy vector carrying desired gene (i.e. virus enters cells then inserts desired gene into cells DNA)
- cells grow in lab then returned by IV injection
What are the 3 limitations to ex-vivo gene therapy?
- complex as cells require maintenance in vitro
- indirect introduction: immune response
- modified/transfected cells may not fulfil function
In what ways can ex-vivo gene therapy transfected cells not fulfil function?
may:
- not function as desired due to patient rejection
- malfunction, triggering an immune response
- not work at all once transplanted
Diagram of ex-vivo gene therapy (retrovirus)
Summarise ex-vivo gene therapy….
slide 17
Summary of differences between in and ex-vivo gene therapy
slide 18
With viral drug delivery (virus as vectors), what is the basic essence of what we do?
how do they work?
- remove original disease-causing genes
- replace w genes needed to stop disease
- then insert altered viruses into px diseased cells in-vivo or ex-vivo
What are the 2 viral cycles?
lytic and lysogenic
What is lytic or virulent viral cycle?
virus enters host, replicates, lyses (burst open) host cell causing its death
(cell opens and releases stuff)
What is the lysogenic or latent viral cycle?
viruses become dormant in cell and integrate its own DNA into host cell DNA, later activating in response to external signal
2 examples of lysogenic/ latent viruses?
HIV and Herpes viruses
do you get integration of viral DNA into host chromosomes/DNA with lytic or lysogenic viral cycles?
lysogenic
What are examples of viral vectors?
- retroviruses
- adenoviruses
- adeno-associated viruses (AAV)
Structure of a retrovirus
viral DNA, enz, reverse transcriptase in core and around: envelope of proteins + lipids
What is the size of retroviruses?
nm diameter
100nm in diameter - considered nanomedicine
What genetic material is found within retroviruses?
RNA
How do retroviruses work?
uses reverse transcriptase to make DNA from RNA which can be inserted into the genome
What do retroviruses do when they infect host cells?
inject RNA and reverse transcriptase enzyme into cytoplasm of that cell
What is an example of a retrovirus?
HIV
what enzyme do retroviruses require to work? why?
reverse transcriptase
as want RNA later to be converted back to DNA
Are retroviruses used in in or ex-vivo gene therapy?
ex-vivo
What cell type do retroviral vectors affect?
divided
What is the therapeutic gene size that retroviruses can deliver?
8Kb
What genetic material is found within adenoviruses?
double-stranded DNA
What types of infections do adenoviruses cause in humans? 3
- respiratory
- intestinal
- eye
What is an example of an adenovirus?
common cold
whats the size range in nm of adenovirus?
90-100nm
What cell type do adenoviral vectors affect?
dividing and quiescent
eg cancer and dormant cells
What is the therapeutic gene size that adenoviral vectors can deliver?
37Kb
huge
what type of virus is AAV?
adeno-associated.
small DNA viruses
How do AAVs work?
non-pathogenic, small DNA viruses that integrate into chromosome 19 in humans w the helper virus (cannot replicate alone)
what helper virus may be used with AAV?
adeno!! or herpes virus
are AAV pathogenic or non?
non-pathogenic
not known to cause disease
What type of genetic material is found within AAVs?
linear single-stranded DNA
What cell type do AAVs infect?
dividing and non-dividing
therapeutic gene size of AAVs?
4.5kb
tiny
difference between viral vectors and CNTs (non-viral) in terms of genes you can incorporate.
CNT: can put multiple on surface but here limited
whats the easier choice out of the 3 viral vectors studied?
adenovirus. nothing req like reverse transcriptase (retro) or helper virus (AAV)
What is found within the AZ/Oxford vaccine (ChAdOx1)?
chimpanzee adenovirus vector containing spike protein of COVID-19
AZ/oxford vaccine can generate strong immune resoibse from 1 dose.
its been genetically changed so its impossible for it to do what?
grow in humans
How do non-viral delivery vectors change in change when complexed w therapeutic gene?
- lipoplex
- polyplex
- carboplex
What charge must non-viral delivery vectors have?
positive (cationic)
Why must non-viral delivery vectors be cationic?
DNA/RNA is negatively charged so it can increase its cellular uptake
with cationic DDS of non-viral vectors, hopefully no immune response so ->?
readministration can be acheived
What is the advantage of non-viral delivery vectors over viral vectors?
no limitation on size of therapeutic gene delivered
What is the advantage of viral delivery vectors over non-viral delivery vectors?
viral are more efficient at transfecting genetic material
in vivo efficiency higher than non-viral vectors
Explain how liposomes traffic genetic material.
trafficking: lipoplexes
1) complex liposome w gene to form lipoplex
2) lipoplex endocytosed into cell
3)a) if endosome used to transport in matures, DNA fragments as endosome forms lyosome/ degradation :(
b) if endosome mixes w lipid, gene escapes and reaches nucleus via nuclear pores
what do we want the non-viral vectors to escape in the cell?
endosome compartment
think low pH…
Which of the COVID-19 vaccines out of the Moderna, Pfizer and AZ/Oxford use mRNA?
non viral DDS
Moderna and Pfizer
What are the advantages of using mRNA as the therapeutic gene delivered?
- safer than whole virus/DNA delivery. not infectious
- processed directly IN cytosol (no need to reach nucleus like DNA)
- has a short half-life so can be regulated by molecular design
- not immunogenic
How is mRNA safer than whole virus or DNA delivery?
- not infectious
- cannot integrate into host genome
What does mRNA need to be used therapeutically?
a drug delivery system
What are the requirements for mRNA to be transported safely and efficiently in vivo?
- without degradation in circulation
- crossing plasma membrane: mRNA needs help
- reaching cytosol
What DDS has been used to deliver mRNA in the Moderna and Pfizer vaccine?
positively charged lipid nanoparticles
What are the advantages of the mRNA due to its DDS in Moderna and Pfizer vaccines?
- more stable
- resistant to RNase-mediated degradation
- nanoparticles promote endosomal escape into cytosol
- forms self-assembled virus-sized particles allowing various routes of administration
How does mRNA in the Pfizer/Moderna vaccines work against COVID-19?
mRNA translated into antigenic proteins causing immune system to produce neutralising antibodies
What is the disadvantage w mRNA used in Pfizer/Moderna vaccines?
long-term storage requires low temperatures leading to distribution logistic issues
need freezers… hot countried :/
oxford/ pfizer/ moderna vaccines use:
- viral DDS
- adenovirus
- double stranded DNA
oxford
oxford/ pfizer/ moderna vaccines use:
- mRNA
- have lipid based NPs in them +ve
pfizer and moderna
What are the main obstacles to gene therapy?
- immune response depletes after repeated doses
- viral vector issues
- difficult to integrate therapeutic DNA into genome
- polygenic disorders
- insertional mutagenesis
- expensive treatment
What are issues w viral vectors that form an obstacle to gene therapy?
risk of toxicity, inflammatory responses
What are problems w integrating therapeutic DNA that form an obstacle to gene therapy?
rapidly dividing cells means effect is short-term
need more doses
What is meant by insertional mutagenesis?
- integration of DNA in a sensitive place in the genome
- e.g. in place of tumour suppressor gene potentially leading to cancer :(((
What is an example of gene therapy used for cancer?
retrovirus encoding shRNA targeted ATP-binding domain to silence gene
Non-Viral (Nanomedicine) Gene Delivery……
amino based therapeutics involve proteins and peptides (<50 aa). name 2 peptides
insulin
cyclosporin
amino based therapeutics involve proteins and peptides (<50 aa). name examples of proteins used?
epo
vaccines
antibodies - targeting
3 examples of nucleic acid based therapeutics
siRNA
oligonucleotides
plasmid DNA
example of polysaccharide therapeutic
heparin
viral gene carriers have the natural ability to target what?
nucleus
but risky
2 types of non-viral gene carriers?
organic
inorganic - cationic
what interactions are used to form diff types of complexes + deliver genetic material to cell (non-viral carriers)
strong electrostatic attraction
how can you restore/ replace a defective gene?
increase protein expression
pDNA or mRNA
how can you silence a defective gene?
decrease protein expression
- antisence oligonucleotides
- miRNA
- RNA aptamers
- siRNA
- shRNA
RNA aptamers used for active targeting how?
act like antibodies recognise specific protein/ peptide sequence
to dec protein expression and silence defective gene
some formulation challenges?
degradation from enzymes
size
charge
hydrophilic
water soluble
- wont cross membrane on own+ too big to travel in between cells
What are examples of routes that non-viral vectors can be administered via?
- Parenteral
- Intrathecal (spine)
- Intravitreal (eye)
- Intradermal
- Intratumoural (tumour)
why is immunogenicity not wanted with this therapy?
some of these gene materials produced using bacteria: can have lot of endotoxins left - problem if inject to px
why is mucosal delivery for gene therapy not wanted?
mucosal sites have high enzymatic activity + RNA esp: suceptible to enzymatic degradation
strong risk of not getting enough therapeutic macromols at site of action
usually what prevents gene therapy crossing mucosal barrier?
too big: cant cross sbetween cells
too hydrophobic: cant cross memb either
What are the 3 routes of mucosal delivery possible?
- transcellular
- paracellular
- receptor/carrier-mediated
What types of molecules go through the transcellular route?
small hydrophobic molecules (lipid bilayer forms membrane)
What type of molecules go through the paracellular route?
small water-soluble (limited by tight junctions)
what is paracellular route?
and whats it therefore limited by?
in between cells
limited by tight junctions
what is the transcellular route?
through membrane
apical + basolateral side
What is the main obstacle to mucosal drug delivery?
enzymatic degradation activity
What are techniques for improving transport across mucosal membranes?
- increase hydrophobicity
- add permeation enhancers
- overcome enzymatic degradation (chemical modification)
- co-administration inhibitors
- encapsulation via nanomedicines
How can we increase hydrophobicity to improve transport across a mucosal membrane?
lipidisation of peptides and oligonucleotides
What are examples of permeation enhancers we can add to improve transport across a mucosal membrane and how do they work?
- surfactants
- cationic polymers
- calcium chelators (e.g. EDTA)
- opens tight junctions of cell
What enzymes are responsible for the enzymatic activity within the mucosal membranes?
endo and exonucleases
How do endonucleases work?
cut INSIDE DNA/RNA
How do exonucleases work?
cut ENDS of DNA/RNA
restriction enzymes recognise specific sequences and are useful for what?
molecular cloning, restriction fragment length polymorphism testing
restriction enzymes recognise specific sequences and are useful for what?
molecular cloning, restriction fragment length polymorphism testing
How does the enzymatic degradation activity change across the mucosal departments?
oral
buccal
nasal
rectal
transdermal
pulmonary
Oral > rectal > buccal = nasal > transdermal > pulmonary
For a drug highly susceptible to enzymatic degradation, which mucosal compartment will it be degraded in the most?
oral
For a drug highly susceptible to enzymatic degradation, which mucosal compartment will it be degraded in the least?
pulmonary
which has highest -> lowest surface for absorption from…
oral
buccal
nasal
rectal
transdermal
pulmonary
rectal
oral
nasal
buccal
pulmonary
transdermal
What are examples of chemical modifications that can be made to overcome enzymatic degradation?
- replace O w S in oligonucleotides so enzyme can’t recognise where to cut (phosphorothioate)
- PEGylation
what does replacing O w S (phosphorothiodating oligonucleotides) do the molecule?
makes it harder for enz to recognise where to cut
inc stability of ON
What is the danger of co-administration inhibitors?
can’t degrade RNA/DNA long-term causing toxicity
(body cant fight infections)
why effect does encapsulating gene material in nanameds have?
enz wont see it so wont be degraded
if using plasmid RNA/ shRNA.. getting to nucleus vs being in cytoplasm has impact bc if DNA in nucleus, what happens when cell divides?
DNA will be in cells produced, diff generations will still have that DNA
will activity be longer or shorter ig gene therapy agent in nucelus and why?
bc will still exist there when cell divides
longevity
What are examples of nanomedicines used as non-viral vectors?
polymers
liposomes
carbon nanotubes
metal nanoparticles
dendrimers
with a polyplex etc what is it formed of?
= complex between +ve DDS and drug (gene material)
once you administer non-viral gene therapy, what so you hope about the complex?
that its strong enough to prevent premature release of the gene therapy agent
what happens if gene therpay agent is prematurely released afteer admin?
gene therapy agent: DNA/ RNA, would become susceptible to attack by nucleases + would be degraded quickly in systemic circ
formation of the complexes relies on electrostatic interaction. So anything that could change the XXX could mask or hide or interact with any of the components of the complexes -> potential to change system PKs.
+ve charge on carrier
anything that can change +ve charge on carrier could do what 3 things thus having potential to change system PKs?
mask
hide
interact w any complex components
why do proteins like albumin have possibility of interacting w gene carrier and displacing gene therapy agent -> premature release + quick elim by enzymatic degradation?
Albumin is also -ve charged so therapy may interact with albumin and not genetic material.
cationic +ve gene carrier
(SoM1 colloids): salt concentration (ionic strength) could be a problem w gene therapy why?
salt could mask charge on cationic gene carrier, making interaction w gene therapy agent weaker.. premature release, quick elim
systems in lab, usually made in simple solutions where ionic strength not too high, no proteins.
So the 1st time 2 systems will be exposed to proteins and higher ionic strength - might be in body, could ->?
destabilisation of system.
what can protein adsorption do to gene therapy agent in body?
potentially displacing gene therapy agent
could -> increase in size, increased opsonisation, = complexes removed faster from systemic circulation :(
why do we need to ensure at pH in systemic circulation the charge will be +ve?
if not, interaction between carrier and therapy agent will be a lot weaker -> premature release of gene therapy agent.
as using +ve gene delivery systems, able to interact with negatively charged membranes e.g. of RBC = those gene delivery systems could cause some X
haemolysis! = 1 of toxicities that we are worried about when we are working with +ve charged delivery systems.
polyplexes have diff ways to use endocytosis to get into cell, and depends a lot on what?
size
once inside cell, whatll happen depends on what?
gene therapy agent used
polymers used often contain amino +ve function.
but what is done so we can escape the endosome eventually?
prefer mix of primary, sec, tert amines
What is the proton sponge effect?
theory of what happens when polyplex inside endosome.
Protonation causes an osmotic gradient.
Swelling and an increase in osmotic pressure causes a rupture.
the endosome inside goes through a process of acidification which is what?
conc of H+ inside increases
if have polyplex, can accept H+ and act as buffer… get influx of protons into endosome
will the pH inside change or not?
no
as these wont be free protons, but attached to polymer form polyplex
why does endosome eventually erupt in protonge sponge effect?
too many ions inside
water enters to try and dilute + maintain OP on either side of memb. swells then explode, releasing complex in process
whats an alternative theory to proton sponge that better describes liposomes, lipids, lipoplexes after they enter endosome. 3 steps
protonation + polymer memb interaction
local hole formation
protons can be used for coating what?
gold NPs (-ve) will complex w +ve polymers and conform hybrid gold NP polyplex for delivery
Describe the endosomal release of lipoplexes?
- lipoplex made w cationic lipid that can interact w negative membrane
- phospholipids rearrange so +ve goes w negative lipids
- Formation of neutral-phospholipid pairs.
- DNA displaced and released into cell
Lipofectamine 3000 main one used
What are the 2 pathways a lipoplex can undergo once inside an endosome?
- endosome matures to lysosome leading to DNA fragmentation
- liposome escapes from endosome
the theory of endosomal release depends on type of lipids used, if using purely permanently +ve lipids, what theory is likely?
fusion + arrangement
(liposome membrane fusion
cel penetrating peptides)
How can we exploit the way which DNA is released from a lipoplex?
- use pH sensitive liposomes that become charged in acidic endosome -> memb disruption + endosomal degradation
- helper lipids (DOPE) that enter charge conformation when endosome is acidic enough to trigger endosomal escape process
give an example drug lipoplex that is PEGylated and using interaction with ApoE to -> hepatocytes in liver :)
Onpattro
Why is PEG used with lipoplexes?
Used to hide negative charge
else complex way too toxic and would cause lot of haemolysis after admin
What is the limitation of using charged lipids for lipoplexes?
can be too toxic causing embolisms in blood or activating immune system - need PEG
why do we use corticosteroids before amdinistering Taxoter?
because of CARPA
interaction w complement = causing severe pseudoallergic reaction and we can admin corticosteroids/ antihistamines before to dec risk of interactn
with PEG still not 100% sure as has some issues. Can use it but what must be carefully selected?
type of PEG
What are the pros of non-viral gene delivery?
- complexation
- internalisation
- protein interaction
What are the cons of non-viral gene delivery?
- toxicity
- protein interaction
- biocompatibility issues
why is toxicity an issue w non-viral gene delivery?
using +ve systems. can have intercactions w membrane -> issues w immune system…
whats the use of SCR (scrambled sequence) in research?
to check for non-specific effects
genotoxicity of carrier
- same conposition as therapeutic macromols, diff order
technique used for gene expression
PCR
important in diagnostics too inc COVID19 before LFTs
technique used for protein expression - assessing efficacy by looking at protein levels
western blot
- -therapeutic macromols mini lectures on word—–
Gene editing techniques….
what is the word for: genetic material that defines an organism?
genome
a complete set of nucleic acids sequences encoded as DNA within what?
23 chromosomes
How many genes from the 25,000 have been linked to diseases from the human genome?
3,000
name 2 examples of well established diseases w genetic link
SC anaemia
haemophilia
what has limited the efficacy of drugs esp those in cancer treatment eg kinase resistance and combination therapy
genetic mutation
T/F: if we fix faulty gene at top, will fix 100% of problem
False
What is gene editing?
process in which engineered nucleases are inserted, replaced, or removed DNA from a genome.
How do the types of gene editing technologies differ?
by 3 different types of nucleases
What are the 3 main gene editing technologies developed?
- zinc finger nucleases (ZFNs)
- transcription activator like effector nucleases (TALENs)
- Clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas-based RNA-guided DNA endonucleases (CRISPR/Cas-9).
What is the general process of gene editing?
1) double strand break at nuclease target site
2) either homologous directed repair or non-homologous end-joining
What are the 2 gene alterations that are made w homologous directed repair?
gene insertion and correction
whats the difficulty w homologous directed repair?
want template to figure out WHERE double strand break happened
what is the first step of gene editing?
double strand break at nuclease target site
What are the gene alterations that are made w non-homologous end-joining?
gene disruption
Describe the basic structure of a ZFN.
- DNA cleaving domain (Fok1)
- DNA binding domain
How do zinc finger nucleases create their DNA double strand break?
recognise 3 base-pair sequences and cleave DNA here
why do we engineer ZFN before put in?
so knows exact sequence to recognise
(directed in proper place, not random)
what type of gene editing:
targeted editing of genome by creating double strand breaks in DNA at user specified locations
ZFN
Describe the basic structure of a TALEN.
- DNA cleaving domain (Fok1)
- DNA binding domains
How do TALENS create their DNA double strand break?
recognise single bases
How do TALEN and ZFN compare?
TALEN more sensitive however more difficult to generate as v precise
What is CRISPR-Cas 9 based on?
bacteria DNA defence mechanism against virus DNA: RNA-guided DNA cleavage
What types of genetic errors can CRISPR be used to target?
double strand break at nuclease target site
…cells translate + process sequence to give crRNA
these anneal to transactivating crRNAs (tracRNAs) - recruit + direct Cas proteins -> direct seq-specific cleavage (+ silence pathogenic RNA)
How is CRISPR used in drug discovery?
Target identification and validation.
Why is CRISPR better for screening than traditional methods?
CRISPR can screen for multiple cells at once.
genes are involved in drug sensitivity in CRISPR/Cas9 positive or negative screening?
positive
describie CRISPR Cas9 positive screening
put cells w crispr
use drug then study which cells alive after drug tretament
has potential to target and kill specific cells
spot several genes/ targets at same time :)
describe CRISPR Cas9 negative screening
got 2 pools of cells
treat 1 w drug, keep 1 untreated
at end compare the 2 (which cells are now dead)
T/F with crispr cas9 -ve screening, you get same results as +ve screening
true
give example of crispr/cas9 screening
cells w/out p53
still alive after pt as cant kill
will survive Pt drugs = link between presence of p53 and killing w Pt
main difference between crispr cas 9 +ve and -ve screening?
+ve: 1 pool of cell
-ve: 2 pools of cells
