Lecture 2-Vaccines
• Vaccines are biological compositions intended to stimulate and prepare the immune
system against infection or disease.
• The primary component of vaccines are antigens that are derived from the pathogen of
interest or bio-manufactured.
Ag: Most time protein, can also be glycoprotein, glycosylation, lipid, WHATEVER
TRIGGERS IMMUNE RESPONSE.
Made from pathogens or manufactured in lab: Trigger immune response to just Ag.
• Additional components may include preservatives, stabilizers, excipients, and traces of
products carried over during manufacturing.
• Often, adjuvants are added to improve immunogenicity (ability to induce an immune
response) and efficacy.
Adjuvants: trigger immune response, increase efficacy of vaccine.
Common vaccine components:
Active ingredient: Ag.
mRNA in Phizer
protein subunit folded into virus-like particle in HPV
Whole virus particle measles
Stabilisers
Sugar, gelatin
Preservatives:
Prevent bacterial and fungal growth inside vial.
Trace components:
Formaldehyde, residual cell culture material.
For whole virus particle, it has to grow in cell culture. Even if one purifies and removes cell
culture there will still be residual left.
Inactivated vaccine may be cultured in formaldehyde
Sometimes, other components:
Antibiotics:
In cases where vaccines are grown in cell cultures. Antibiotics suppresses bacteria growth.
Adjuvants:
Aluminium (salt), lipid nanoparticles (immune response would recognise ‘phospholipid ball’
as foreign)
How do vaccine work?
The main objective of a vaccine is to generate a long-lasting antigen-specific protective
immune memory.
Inoculated with vaccine:
Example: muscle injection: into arm or quadriceps (easier to administer), i. e. BIG
MUSCLES.
Pathogen Recognition Receptors in innate immune recognise non-self components
(example: single-strand DNA, lipopolysaccharides).
Recognition would cause downstream effects such as inflammation, where all immune
cells go to the site. (‘Warm, swelling’ triggered)
Dendritic (Ag-presenting cell) move into lymph-node area carrying non-self Ag, it will
present Ag to CD8+ or CD4+ T-cells. The CD4+ cells activate B-cell, which will differentiate
and proliferate. Memory B-cell would be a form of immune memory and will be re-activated
if the same Ag appears again. Plasma cells: form Ig.
In most vaccines, this is needed, for if it is a viral infection we do need to ‘kill-off’ the
invading cells, so we need CD8+ and CD4+ activation. But a lot of what we measure is Ig.
Importance of Ig/how do vaccines work:
They induce a “primed” state in the vaccinated subject so that following exposure to a
pathogen, a rapid secondary immune response is generated.
‘Fool immune system to think that it has been infected by pathogen’.
Ag will boost immune response, and Ag will also provide protection.
Accelerated elimination of the organism and protection from clinical diseases
History:
• Edward Jenner got credit for making the first vaccination in 1796:
• Observed that milkmaids who had been infected with cowpox virus were immune to
smallpox
• Took some material from cowpox lesions and inoculated into the skin of a young boy
• Inoculated the boy with infections materials containing deadly smallpox
• The boy did not develop smallpox and was protected
• The cowpox vaccine which Jenner initially documented was extremely successful in
controlling smallpox
• So much so that smallpox has now been eradicated from the human population
• The last natural case of smallpox was documented in Somalia in October 1978
Reason why this is successful: smallpox is HUMAN-SPECIFIC, and there is global support
and good isolation.
Importance of vaccines:
• Herd immunity:
Protection conferred to unvaccinated individuals in a population produced by vaccination
of others and reduction in the natural reservoir for infection.
If there is an infected individual in a group of susceptible individuals, disease would spread
rapidly.
Vaccinated individuals, no longer susceptible, reduces the likelihood of disease passing
on to susceptible individuals once the proportion of vaccinated individuals is high enough.
If entire population immunised: the number of people that can GET INFECTED would be
small, for there is no RESERVOIR for disease.
Viruses are inert particle and need living hosts. If there are no living hosts virus would die
out.
If majority of population vaccinated, it would also protect people who cannot be vaccinated
due to their special conditions.
• However, the herd immunity effect is seen only at relatively high levels of vaccination
within a population; for mumps, the necessary level is estimated to be around 80%, and
below this level sporadic epidemics can occur.
• This is illustrated by a marked increase in mumps in the United Kingdom in 2004-2005 in
young adults as a result of the variable use in the mid-1990s of a measles/ rubella vaccine
rather than the combined MMR, as the combined vaccine was in short supply at that time
Success of vaccine
Example:
polio:
Serious neurological disorder, lifelong effects.
From 7000-8000 annually to almost eradication
Measles:
Initial infection mild, long-term caused brain swelling and death
From Millions to almost eradication.
Features of successful vaccines:
Safe: must not cause illness or death
Protective: Vaccine must protect against illness resulting from exposure to live pathogen
Gives sustained protection: Protection against illness must last for several years
Induces neutralising Ag: Some pathogens (example: polio) infect cells that cannot be
replaced; thus, Neutralising Ag is essential to prevent infection of such cells.
Induces protective T-cells: pathogens such as viruses are more effectively eradicated by
ADAPTIVE IMMUNITY such as T-cell response.
Practical consideration:
Low cost per dose
Biologically stable
Ease of administration
Few side-effects
Vaccine types:
Traditional vaccines: derived from whole, inactivated or weakened pathogens.
example: smallpox: weakened pathogen.
Disadvantage:
DANGER: NEED CULTURE ORIGINAL PATHOGEN.
TIME CONSUMING TO PRODUCE
‘RISK OF REVERSION’: If one mutates, it may lead to NEW WILD-TYPE VIRUS.
That is:
• Vaccines can be classified based on their ability to replicate in the host (e.g., live versus
dead vaccines) and/or the technology/platform used in their manufacturing.
• Conventional vaccines use one or several antigens derived from inactivated or weakened
pathogens, or their components such as protein subunits or toxins to generate an immune
response.
• However, these technologies have been unsuccessful in creating vaccines against
complex pathogens that can evade the immune system, and/or display extensive variability.
• Conventional vaccines are also more time-consuming to produce, involve a greater risk
of reversion to virulence, and need more customized development against emerging or
rapidly evolving pathogens.
In terms of these vaccines:
Attenuated virus approved by FDA:
Example: Smallpox still exists but no longer in public use. Big virus particles used and are
easy to handle.
Inactivated viruses approved by FDA:
Adjuvant in such vaccines often ALUMINIUM SALT: WE KNOW THAT IT WILL ACTIVATE
IMMUNE RESPONSE.
Other vaccines:
Virus-like particles
synthetic peptides
NEW GENERATION VACCINES:
recombinant subunit, DNA, RNA, Recombinant viral vectors.
• New strategies for immunogen design and genetic engineering (including recombinant
DNA technologies) have contributed to the rise of next generation vaccine platforms that
enable a more potent immune response.
• Since next-generation vaccines rely on the genetic sequence of a pathogen, they can be
developed much faster than conventional technologies.
FDA approval became more rapid since Ebola and COVID. In COVID, they did not
complete PHASE I, PHASE II, PHASE III: manufacturing vaccines before PHASE I.
Details:
Virus-like particles (VLP):
Macromolecular assemblies designed to MIMIC MORPHOLOGY OF NATIVE VIRUS. This
includes SIZE, SHAPE AND SURFACE EPITOPES.
Can be SUBDIVIDED based on:
PRESENCE/ABSENCE OF LIPID ENVALOPE
NUMBER OF PROTEIN LAYERS FORMING THE CASPID
VIP vaccines are typically manufactured in BIOREACTORS following TRANSFECTION
OF INSECT, YEAST, BACTERIAL, PLANT or MAMMALIAN CELLS with ONE OR MORE
GENETIC CONSTRUCTS.
Details:
Example: HPV VACCINE
Protein on surface of HPV virus is growing recombinantly, and they assemble into ‘small
balls that look like viral particles.’ BUT THEY DO NOT ENCASE NUCLEIC ACIDS. THEY
ARE JUST PROTEIN CAPSULES.
These are designed by:
(1) Identify what the Ag is
(2) Ag is inserted into plasmid
(3) Plasmid is taken up by bacteria which will produce Ag proteins EXOGENOUSLY (That
is, through ‘secretion’)
(4) Proteins will spontaneously form into viral-like particles
(5) These are particles that ‘LOOKS LIKE A VIRUS BUT DOES NOT DO ANYTHING’. NO
NUCLEIC ACID CONTAINED.
Synthetic peptides:
Immune responses to pathogens are dominated by EFFECTOR CELLS that recognise
either one or more epitopes on Ag.
Identification and synthesis of immunodominant peptide sequences are used to develop
novel vaccine modalities.
Design involves extensive in vitro screening and modelling (atomic interactions) to identify
appropriate immunodominant peptides with suitable manufacturing characteristics.
Peptide vaccines are synthesized using fragment condensation techniques or solid-phase
synthesis, and are subject to stringent purification and characterisation.
Details:
Binding site on MHC and T-cell receptors are 4-12 amino acids. In Ig, there are around 2532 amino acids. If we know what those sequences are, WE MAY TRIGGER AN IMMUNE
RESPONSE WITH A MINIMAL PEPTIDE, PERHAPS OF ONLY 4 AMINO ACIDS IN
LENGTH!
Advantage:
It’s SAFE AND QUICK:
NO INFECTIOUS MATERIAL INJECTED.
We are not inserting any deleterious sequences with side effects that may cause other
diseases.
Quick onset.
Can be stored in PADDED FORM.
Disadvantage:
Too specialised. Only 2 peptide vaccines produced.
Too specific. If there are mutations it may not work.
We may or may not have conformational Igs: The peptide may not correctly fold to get
correct fold!
NONE OF THE VIRAL-LIKE PARTICLES or PEPTIDES HAVE BEEN USED IN COVID!
NEW GENERATION VACCINES:
Why not traditional vaccines, virus-like particles or synthetic peptides:
Development of vaccines against pathogens with PENDEMIC POTENTIAL is NOT
FEASIBLE using conventional technologies: inherent manufacturing issues
The development of viral vectors for gene therapy, and the use of nucleic acids to encode
antigens (such as mRNA vaccines), coupled with advances in molecular biology,
bioinformatics, and technologies such as NexGen sequencing, have enabled the
development of novel vaccine platforms.
HOWEVER:
Limitation of new-generation vaccine manufacturing: CAN ONLY PRODUCE NUCLEIC
ACID/PROTEIN RELATED VACCINES. If Ag is non-protein Ag such as lipid or
polysaccharide, these manufacturing platforms will not work, and we have to resort to
conventional technologies.
Details:
Subunit vaccines, mRNA vaccines, viral vector vaccines
NEW:
WE CAN REVERSE-GENERATE THEM FROM Ag.
Examples of new-generation vaccines:
VIRAL VECTORS:
Derived from viruses engineered to encode genes for one or several Ag CLONED INTO
VECTOR BACKBONE.
Can be engineered to be REPLICATION DEFICIENT/INCOMPETENT while maintaining
ability to INFECT CELLS and EXPRESS ENCODED Ag.
Replication-competent viruses are considered TRUE INFECTIONS akin to live-attenuated
vaccines.
Typically, this platform mimics natural infection to generate potent humoral and cellular
(CD4+ and CD8+) responses
Details:
Derived from viruses that we can manipulate.
We choose a virus that exist readily in population that DOES NOT CAUSE TOO MUCH
DISEASE. A lot of the time it would be ADENOVIRUS.
We manipulate the virus to EXPRESS PROTEINS THAT WE WANT or CARRY THE
GENES THAT WE WANT EXPRESSED.
They can be REPLICATION COMPETENT OR REPLICATION INCOMPETENT:
The replication-competent ones: LIKE REAL INFECTION, BUT DO NOT CAUSE
DISEASE.
When adenovirus replicated, we have Ag replicated.
Example: SARS, Ebola, etc.
Manufacturing:
(1) We find the Ag: the protein on the surface of the virus triggering immune response,
(2) We insert it into plasmid, and plasmid is used to transfect cells
(3) Transfected cells are purified: transfection is with vaccine immunogen and also genes
required to make adenovirus. (Example: Influenza: 8-segmented genome. We can put
8 plasmids with one fragment in each into the same cell and we can make the virus.)
(4) Cells will produce proteins associated with sequences to produce viral particles.
(5) We purify ‘viruses’ to make vaccine.
Synthetic DNA vaccines:
• DNA vaccines are large, polyanionic, sensitive to nucleases, and exhibit less efficient
passive entry into cells.
• Delivery methods such as gene gun, jet, electroporation (EP), and nanoparticle-based
systems have increased synDNA uptake in vivo.
• SynDNA vaccines can induce both humoral and the cellular components of the immune
responses with several preclinical and clinical studies demonstrating potent antigenspecific CD4+ and CD8+ T cell responses.
• Compared with the conventional inactivated, attenuated, and recombinant subunit
vaccine platforms, synDNA vaccines are faster, cheaper, and easier to manufacture.
• They are also amenable to lyophilization, thermostable, and display high pharmaceutical
stability (long-term storage).
Details:
LARGE POLYANIONIC CONSTRUCTS
Disadvantage:
SENSITIVE TO NUCLEASES
CANNOT ENTER INTO CELL THROUGH PASSIVE ENTRY:
We need: transfection reagents, gene gun, electroporation
(Cells do not pick up dsDNA)
Advantage:
If they get into cell:
They can generate good B and T cell responses, which are even better than those of other
viruses.
We can store them in ?padded form: DNA is very thermostable, nice with long-term storage.
In comparison: RNA VACCINES ARE NOT STABLE AND NEED TO BE STORED AT 80°C.
To generate response in a mouse, we may use amounts equivalent to 1kg DNA for
humans…
(Costly…)
Design:
(1) We get sequence.
(2) Synthesize the genes (cloning)
(3) Purify, get purified plasmids, just inoculate.
mRNA Based Vaccines
• Immune responses to the mRNA vaccines rely greatly on the delivery system, the
immunogenicity of the encoded antigen, and the longevity and subcellular localization of
antigen expression.
• Intramuscular and intradermal administration of mRNA vaccines is highly immunogenic
and induces local cytokine and chemokine production that initiates prompt recruitment of
neutrophils, monocytes, and other cells to prime the immune response/s.
Details:
There are a lot of different constructs in RNA vaccine:
(1) Unmodified or modified RNA: 5’ to 3’
(2) Self-amplifying: add promoter to sequence to cause amplification on coding region.
INCLUDE REPLICASE GENES IN THE CONSTRUCT.
(3) Trans-amplifying: REPLICASE GENES AND CODING SEQUENCE ARE ON
SEPARATE GENES.
(4) Circular RNA ‘plasmid’.
We inoculate them intramuscular or intradermal. RNA uptaken by neutrophils, monocytes
and other cells, RNA will generate protein.
Design:
(1) We still need to GO THROUGH THE STEPS USED TO MANUFACTURE A DNA
VACCINE TO GET PLASMID, but from here, once we get plasmid, we can CONVERT
IT ACROSS, and we inoculate with only RNA.
Disadvantage:
Not stable, degrades quickly. In Pfizer: use lipid nanoparticles to contain mRNA to stabilise
mRNA for long enough to be uptaken.
Challenges:
• One of the most significant challenges in public health is posed by the emergence of new
pathogens with higher transmissibility, fatality rate, or immune evasion potential.
• Vaccine development against pathogens that evade the immune response (e.g., HIV,
Tuberculosis, and malaria) has not been very successful and continues to be an on-going
challenge.
• Genomic variability of certain pathogens, and their ability to rapidly mutate creates
challenges for vaccine development, and could lead to evasion, making current vaccines
less effective.
• Depending upon the platform used, next-generation platforms that can be quickly adapted
to emerging variants could help resolve this issue to a certain extent.
• Global surveillance and monitoring efforts are key factors in gaining quick and effective
control over pathogens with epidemic and pandemic potential.
• Conventional manufacturing technologies have defined the past century of vaccine
development, effectively protecting against diseases with high disability and fatality rates
such as smallpox, polio, measles, etc.
• The infrastructure and resources needed for these technologies have been well
established and the development costs have been amortized.
• Though well understood and effective, these technologies are limited by slow, empirical,
and expensive development in addition to short-lived protection against several pathogens.
• Technological advances such as genetic engineering and superior cell-culture techniques
could help reduce cost, improve production output, augment knowhow, and enhance their
ability to respond faster to emerging threats
• Next-generation platforms such as mRNA and DNA derived vaccines offer an exciting
and promising avenue for vaccine development owing to low costs, safety, high potency,
and rapid mass deployment.
• These platforms are especially relevant for complex pathogens with immune evasion
potential. • Moreover, unlike conventionally derived vaccines, these platforms are also
likely to offer successful solutions for non-infectious diseases such a scancer.
• Prototype pathogen preparedness can significantly improve response time in the event
of a pandemic.
• Undoubtedly, further funding and effective monitoring of new data will help define a new
era of vaccinology and vaccinomics to mitigate present and emerging public health threats
Lecture 4-Synthetic Biology
-
‘How to exploit biology to our own means’
-
CREATE SYNTHETIC LIFE
-
DNA is at the base of phenotype, underpins synthetic biology.
If we can change genome in major ways, that fits into the field of synthetic biology.
Definitions:
‘Design of new biological parts, devices and systems’
‘Redesign of existing natural biological system for useful purposes’
” … a) the design and construction of new biological parts, devices and systems and b)
the re-design of existing natural biological systems for useful purposes.” Synthetic
Biology.org
… an emerging area of research that can broadly be described as the design and
construction of novel artificial biological pathways, organisms or devices, or the redesign
of existing natural biological systems.” UK Royal Society
”… a maturing scientific discipline that combines science and engineering in order to
design and build novel biological functions and systems. This includes the design and
construction of new biological parts, devices, and systems (e.g., tumor-seeking microbes
for cancer treatment), as well as the re-design of existing, natural biological systems for
useful purposes (e.g., photosynthetic systems to produce energy). As envisioned by
SynBERC, synthetic biology is perhaps best defined by some of its hallmark characteristics:
predictable, off-the-shelf parts and devices with standard connections, robust biological
chassis (such as yeast and E. coli) that readily accept those parts and devices, standards
for assembling components into increasingly sophisticated and functional systems and
open-source availability and development of parts, devices, and chassis.” SynBERC
… the engineering of biology: the synthesis of complex, biologically based (or inspired)
systems which display functions that do not exist in nature. This engineering perspective
may be applied at all levels of the hierarchy of biological structures – from individual
molecules to whole cells, tissues and organisms. In essence, synthetic biology will enable
the design of ‘biological systems’ in a rational and systematic way.” High-level Expert
Group European Commission
This is still an evolving area. Perhaps its basic element is APPLICATION OF
ENGINEERING PRINCIPLES TO FUNDAMENTAL BIOLOGICAL MOLECULES.
History:
April 24th 2000 – Chemical & Engineering News “using the synthetic capability of organic
and biological chemistry to design nonnatural, synthetic molecules that nevertheless
function in biological systems”
2009 - Nature Biotechnology - the design and construction of new biological “parts”,
“devices”, and “systems”
2013 - Microbiology - the re-design of existing, natural biological systems for useful
purposes
THERE ARE TWO ELEMENTS IN THE DEFINITION OF SYNTHETIC BIOLOGY:
(1) Redesigning and fabrication of EXISTING biological systems.
(2) DESIGN and FABRICATION of biological components and systems that DO NOT
EXIST IN NATURE.
Examples:
Program DNA in plants so that it grows into furniture.
Repopulate northern Europe with Woolly Rhinoceros
Genetic material which uses NON-ATCG nucleotides
Details:
On ‘APPLYING ENGINEERING PRINCIPLES to BIOLOGICAL SYSTEMS’:
This is a process of DESIGN, BUILD, TEST and LEARN.
g)
For instance, in a case of testing MULTIPLE PROMOTERS SIMULTANEOUSLY,
If we apply the view of engineering, this can be separated into 3 levels:
SYSTEMS: where promoter takes effect. THIS COULD BE CELL or CHASSIS.
DEVICES: This may include GENETIC CIRCULTS, LIGHT SENSORS, INVENTORS,
CELL SIGNALLING.
PARTS: PROMOTER, REPRESSOR, TERMINATOR, CDS, ADAPTOR.
Devices have to come together to become LARGER COHORTS.
-
The way they interact is ANALOGOUS TO CIRCULTS
-
SYSTEMS AND COMPUTATIONAL BIOLOGY ARE INTEGRAL TO DESIGN.
That is:
• Design and synthesis of biological entities:
– Parts
– Devices
– Systems (entire cells)
• Enabled by the development of parts that can be assembled into larger, functioning
devices
• Directly analogous to the design of integrated circuits
• Systems and computational biology are integrated into design
History:
General:
Watson and Crick: DNA DOUBLE HELIX STRUCTURE SOLVED 1953
First oligomer synthesis: 1955
Initially ENZYMES WERE USED TO SYNTHESIZE OLIGOMERS.
Improvement of OLIGOMER SYNTHESIS:
PHOSPHORAMIDITE OLIGOSYNTHESIS – 1985
Sequencing:
-SANGER: 1975
-Automated sequencing, complete genome sequence of s. cerevisiae and E. coli: 1990s.
-PCR: 1983
Proteins:
Arber discovers RESTRICTION ENZYMES: 1968
GFP: marker/reporter for devices (1978)
PCR: 1983
2003: THE NOTION OF BIO-BRICKS: DNA, PROMOTERS, etc. IN COLOUR CODED
ABSTRACTION
2005: CRISPR
2009: GIBSON ASSEMBLY:
ASSEMBLY USING NON-RESTRICTION ENZYME BASE/Homologous type of assembly.
Details:
-DNA sequencing.
This has greatly advanced since development of HIGH-THROUGHPUT, NEXTGENERATION SEQUENCING in the mid-2000s.
This means that we can:
(1) Check assemblies really fast
(2) Look at bigger contiques
(3) Discover new parts
(4) Discover NEW chassis to hold genetic constructs in
-Bioinformatics/Computational biology:
Throughout the 1980s and 1990s, computational biologists (bioinformaticians) developed
computational methods to handle DNA and protein data – mainly driven by reductionist
approaches
‘ABSTRACTION IN SILICON’
This is an example of OBJECT-ORIENTED PROGRAMMING
We can ‘assign a sequence’, ‘give sequence code’, ‘pull sequence from database’,
‘translation in silico into virtual protein models’.
-Systems biology:
Modelling systems.
Metabolic control analysis (MCA) is a mathematical framework used to quantify the
distribution of control over concentrations and fluxes in steady state metabolic pathways.
Although ultimately a metabolic system consists of an entire cell, or even an entire
organism, some degree of isolation is needed in order to analyse it.
That is:
Use constraint to answer questions of a SUBSET of a pathway.
Looking at complex systems by simply focusing on A FEW COMPONENTS IN THE
SYSTEM.
Details:
DNA SYNTHESIS:
ONE OF THE BOTTLENECKS IN SYNTHETIC BIOLOGY.
Traditionally, we use OLIGONUCLEOTIDE SYNTHESIS:
• Chemical synthesis 3’ to 5’
• practical limit is about 200 bp
• Cheap, accurate but low output
• PCR primers
This method also uses PHOSPHORAMIDITES AS ACTIVATION REAGENT.
The main problem with this is that 200bp is NOT SUFFICIENT TO ENCODE ENTIRE
GENES.
HOW TO SYNTHESIZE BIGGER SEQUENCES?
Traditionally, genes, promoters, etc. were synthesized AS SEPARATE PARTS before
BEING JOINED TOGETHER. Now we can directly determine the sequence in
COMPUTER and send fully-synthesized sequence to company that makes DNA.
Gene synthesis from oligonucleotides:
(1) Make 180-200bp oligos and make them OVERLAP.
(2) We do PROGRESSIVE ONE-POT OVERLAP EXTENSION ASSEMBLY.
(3) We will have some population of long DNA, which is amplified by PCR.
(4) Length: 5kb
That is:
• 0.3 – 5 kb genes cloned into a plasmid
• Oligonucleotide and enzyme based
• 9c per base (3.5kb = $USD 3150)
• Errors increase with the length of DNA
NOW: AFTER ‘PARTS’ ARE SYNTHESIZED, HOW TO BRING THEM TOGETHER?
i.e. DNA ASSEMBLY.
Preferred method: HOMOLOGY-BASED
ENZYME-BASED:
Disadvantage: leave scars, ‘randomness in cloning’
Use combinatory approach: assembly in automated way
Issue: SIZE LIMIT:
Example: E. coli: 50 kb plasmid is upper limit. (That is roughly 10 genes)
However: a pathway/screening may include hundreds of proteins
Thus: OTHER SYSTEMS:
YEAST:
Bit more complicated, eukaryote, ‘not perfect’
Example of application: CONSTRUCTION OF MYCOPLASMA GENITALIUM GENOME
IN YEAST:
Synthesized overlapping pieces of the genome through poly-PCR amplification, put pieces
into yeast, YEAST HAS HOMOLOGOUS RECOMBINATION PROCESS to reconstruct
genome.
Use ‘partial yeast chromosomes’ hosted in yeast
DNA AS PARTS:
(1) Choose plasmid base: BACTERIA or YEAST PLASMID, (backbone, has origin of
replication, can have MULTIPLE OR LOW COPIES)
(2) Pick a promoter: We don’t always aim for high expression. Sometimes we need low
expression for some parts of the pathway (throughput and flux). We may also try all
combinations.
(3) RIBOSOME BINDING SITE: different ribosome binding sites work better with different
combinations of promoters.
(4) CODING SEQUENCE: Depends of ‘what we are trying to make’
(5) Construct it together through synthesis (homologous region at either end, etc.)
Examples of Synthetic biology:
REPRESSILATOR:
First example of a BIOLOGICAL TOGGLE SWITCH SYSTEM.
‘We used three transcriptional repressor systems that are not part of any natural biological
clock to build an oscillating network.’
‘The network periodically induces the synthesis of green fluorescent protein as a readout
of its state in individual cells.’
[See 自虐的代理代理戦争]
Artemisin precursor pathway:
Recapitulated a plant pathway into an E. coli to make anti-malerial drug.
Light-sensing circuits, 2005
Creation of synthetic Mycoplasma genome, 2008 (as shown above, in yeast system)
Biofuel production using amino acid metabolism: 2008
Creation of a bacterial cell with a chemically synthesized genome, 2010
Engineering of synthetic yeast chromosome arms, 2011
Complete biosynthesis of opioids in yeast, 2015
Semi-synthetic organism that stores and retrieves increased genetic information, 2017
Full synthesis of E.coli genome 2019 (Mycoplasma has smaller genome than E. coli, thus
piecing together a larger genome is important).
WHAT IS COVERED WITHIN SYNTHETIC BIOLOGY:
1. Genome synthesis
ASSEMBLY OF LARGE AMOUNTSS OF DNA
Precursor of synthetic organisms.
•Minimal genomes (E.coli, B. subtilis)
WHAT IS THE MINIMAL AMOUNT OF GENES THAT CAN SUSTAIN AN
ORGANISM LIKE E. COLI?
This is to FORM A BASE CHASSIE.
•Whole-cell models
•Synthetic viruses (e.g.to kill bacteria)
BACTERIA WOULD NOT RECOGNISE SYNTHETIC EPITOPES
•Artificial chromosomes from S. cerevisae
Can we assemble large amounts of DNA
•Mammoth / Tasmanian Tiger
2. Gene regulatory circuits
ON/OFF
To do complex metabolic engineering, one need to turn genes on/off.
Natural: Promoters, regulators
TOGGLE SWITCH: ON AND OFF DEPENDS ON CERTAIN CONDITIONS
• DNA-based bio-circuits
• analog to electrical circuits
3. Protocells
• Synthetic colloidal system mimicking biological processes
• Giant lipid vesicles are often used for constructing cell-like systems
• Enzymes and other macromolecules are easily encapsulated inside giant vesicles,
allowing
the
construction
of
micro-compartmentalized
systems
capable
of
programmable behaviour
Example: Put a synthetic genome into a SYNTHETIC LIPID BILAYER
That is: make PROTO CELLS with DIFFERENT LIPID COMPOSITION.
Normally the system includes genes encoding proteins that make the lipids to maintain
lipid sequence.
Aim: recreate transmembrane electrical gradient, enable self-replication of proto cell.
4. Optogenetics
• From Greek optikós, meaning 'seen, visible’
• Is technique that involves the use of light to control cells in living tissue, typically
neurons
• Used for optical interrogation of neural activity
That is:
USE OF LIGHT TO CONTROL CELLS IN LIVING TISSUE, TYPICALLY NEURONS
Example: photosensitive version of GTPase RAC control cell movement,
5. Xenobiology
• Derives from the Greek word xenos, which means "stranger, alien". What is covered
within Synthetic biology? 31
• not (yet) familiar to science and is not found in nature
• biological systems differing from the canonical DNA, RNA, 20 AA system
• nucleic acid analogues (Xeno Nucleoic Acid) as information carriers
• expanded genetic code and the incorporation of non-proteinogenic amino acids into
proteins
Example: incorporate novel bases and amino acids into an organism.
XENO-ORGANISMS WILL NOT INTERACT WITH DNA-BASED LIFE BUT IS
ANALOGOUS TO LIFE.
6. Metabolic Engineering
• Attempts to optimise cellular processes to increase the production of a certain
substance
Making compounds like antimalarial on scale, such as biofuel and bio-diesels.
KEY CASES:
Repressilator
•
constructed from three repressor–promoter interactions (cI, LacIand TetR)
•
linked together to form a ring-shaped network, in which Tet Regulates a GFP-reporter
•
using time-lapse fluorescence microscopy, the circuit exhibits periodic oscillations in
GFP expression
(See above and 自虐的代理代理戦争)
If we set up this situation, we can get E.coli to ‘pulse’ regularly.
A GFO is cycling with one of the expressed proteins.
‘Continuous turnover of proteins’.
Toggle switch:
Two repressors, reporter linked to one repressor.
i.e. An on and off switch
Autoregulatory circuit
BEHAVIOUR OF GENE EXPRESSION WITH OR WITHOUT FEEDBACK.
[WIP]
Lecture 5 – Fluorescent proteins
(1) Background:
2017: In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact
mouse brain.
-
Cell layers in brain are monitored.
-
Magenta dye indicated blood vessels.
-
GFP (GREEN FLUORESCENT PROTEINS) show synapses.

When Calcium ions are released into inter-synaptic space, GFP lights up,
indicating that mouse is using this part of the brain.
(2) Major ways to label proteins other than fluorescent proteins:
1. Dyes:
Used
to
label
LARGE
CELLULAR
COMPONENTS
COMPARTMENTS.
Example:
DAPI – blue, stains nucleus (DNA)
Mitotracker Red CMKROS – red, stains mitochondria.
2. Labelled immunoglobulin (labelled Ig)
This is a part of IMMUNOHISTOCHEMISTRY.
Using labelled Ig, we can LOCATE SPECIFIC PROTEINS.
History:
1942:
Coons et al.
/
ORGANELLES
/

Determination of location of Ag of pneumococcal infection using Fluorescent
Ig.

Pneumococcal infection is caused by streptococcus.

At the time, it was believed that the bacterial agents can be found within the
lesion.

Also: at that time, it is known that Ig is part of the immune system and that it
can bind to and mark disease agents, but its specific nature was uncertain.

Coons used an Ig combined with dye, and visualised a lesion of a mouse
liver with pneumococcal infection by adding the Ig to the lesion. It reveals
that streptococcal Ag was indeed found within the lesion on the liver, proving
prior theories to be true.
Later:
Ig is found to be able to target any antigen on any material.
In 1974, Lazarides et al. visualised actin filaments in chicken non-muscle cells
using labelled Ig.
Later, all cytoskeletal materials were visualised with the same method.
Problem:
Dyes and labelled Ig can only be used in cell cultures in vitro. When it comes to
in vivo labelling and living organisms. They will not work.
‘You don’t get a mouse with labelled brain cells; you get a mouse with weird hair
colour.’
(3) Green Fluorescent Proteins (GFP)
(SEE NEXT PAGE)
Origin: Aequorea Victoria
Initial research done by Shimomura, who fragmented the jellyfish to retrieve the protein.
The initial protein discovered was a protein COMPLEX called AEQUORIN
AEQUORIN:
The basis of aequorin bioluminescence is ‘RESONANCE ENERGY TRANSFER’.
(A) Aequorin bioluminescence reaction:
Aequorin complex consists of an APOAEQUORIN and a COELENTERAZINE.
1. Calcium ion binds to the APOAEQUORIN.
2. This induces a conformation change in APOPEQUORIN, resulting in a
PEROXIDE (-O-O-) linking APOAEQUORIN to COELENTERAZINE
becoming unstable.
3. This results in the formation of the intermediate: COELENTERAZINE
PEROXIDE, which is still connected to APOAEQUORIN by one ionic
interaction.
4. This last interaction was broken, and COELENTERAZINE PEROXIDE
decomposes, forming COELENTERAZINE and CO2.
5. At the 4th step, COELENTERAZINE is EXCITED, and during its complete
dissociation from APOAEQUORIN, it produces BLUE LIGHT at
wavelength 470 nm.
6. COELENTERAZINE dissociates into COELENTERAMIDE.
(B) GFP- aequorin BRET reaction:
The Aequorin fluorescence reaction does not involve ‘resonance energy transfer’
in
any way. Only when GFP is bound to the aequorin complex can ‘resonance energy
transfer be discussed.
INITIALLY ONLY AEQUORIN IS FOUND. ONLY AFTER FINDING OF GFP WAS
THE SOURCE OF GREEN LIGHT FOUND!
The 6 steps in aequorin bioluminescence reaction still exists in GFP-aequorin
BRET reaction, with ONE EXTRA STAGE at the original 5th step:
…
5. At the 4th step, COELENTERAZINE is EXCITED, and during its complete
dissociation from APOAEQUORIN, but INSTEAD OF PRODUCING BLUE LIGHT
at wavelength 470 nm, it TRANSFERS THE ENERGY IN A NON-RADIOACTIVE
WAY TO GREEN FLUORESCENT PROTEIN, WHICH IS ATTACHED TO
AEQUORIN.
6. COELENTERAZINE dissociates into COELENTERAMIDE. IN THE MEAN
TIME:
GFP EMITS GREEN LIGHT AT 510nm as it is RETURNED TO GROUND STATE.
(Energy once used to produce blue light in COELENTERAZINE is used instead to
activate GFP. This is RESONANCE ENERGY TRANSFER. ‘Energy that is used
to produce light comes from light from other proteins’.)
In bioluminescent proteins, the energy comes from an enzymatic reaction.
Additional notes:
AEQUORIN is a HOLOPROTEIN:
An apoprotein (APOAEQUORIN) connected to a prosthetic group. It does nothing
itself, and is only used to promote energy transfer to COELENTERAZINE.
As COELENTERZINE becomes COELENTERAMIDE, its energy level returns to
ground state as extra energy is released in the form of light.
Details on RESONANCE ENERGY TRANSFER:
Wavelength is always associated with PHOTON ENRGY. The LONGER the
wavelength, the LOWER the energy.
For this case:
Photons at wavelength 475 nm (from COELENTERAZINE) is absorbed by GFP.
‘When the molecule absorbs the proton, an ELECTRON in the molecule’s outer
shell gets KICKED UP TO A HIGHER ENERGY LEVEL as it is excited by the
energy of the photon. (S0 state to S1 state).’
Within S1 there are SEVERAL VIBRATION LEVELS.
Electron goes to a high vibration level, and IMMEDIATELY DROPS BACK TO
GROUND STATE DUE TO INTERACTION WITH ENVIRONMENT.
This results in another proton being sent out.
As energy is inevitably lost during energy transfer, the new proton has LOWER
energy and hence LONGER WAVELENGTH.
HOWEVER: IN GFP- aequorin BRET reaction, NO BLUE LIGHT WAS
PRODUCED AT ALL.
In fact: RESONANCE ENERGY TRANSFER CAN TAKE ANOTHER FORM:
‘Energy transfer between two proteins that are close to each other.’
(1) COELENTERAZINE has electrons going into EXCITED STATE DUE TO
ENERGY FROM APOAEQUORIN DUE TO CALCIUM ION BINDING.
(2) rather than releasing energy in the form of a proton, it can transfer the energy
to the electron in a neighbouring fluorophore: in this case gfp.
(3) The energy is accepted by the new electron on GFP, which goes to excited
state, and then ENERGY IS RELEASE FROM THIS ELECTRON IN THE
FORM OF PROTON.
Due to TWO ENERGY TRANSFER EVENTS, MORE ENERGY IS LOST.
[How to test whether aequorin-GFP resonance transfer is valid?]
EVERY FLUORESCENT PROTEIN HAS A CERTAIN WAVELENGTH THAT IT
CAN BE EXCITED BY AND A CERTAIN WAVELENGTH IT GIVES LIGHT OFF
IN.
GFP:
Excitation
spectrum
has
TWO
PEAKS.
ENERGY
EMITTED
BY
COELENTERAZINE FALLS UNDER THE SECOND (SMALLER) PEAK, AT 400475 nm.
This smaller peak overlaps with output peak, but not exactly at 510 nm.
To find the gene that codes for aequorin, Douglas Prasher uses protein amino acid
sequence to infer DNA sequence of aequorin, and expressed aequorin in vitro and
in vivo.
The PROBLEM WITH AEQUORIN:
Aequorin is NOT A GOOD INDICATOR FOR DIAGNOSTICS!
Being a holoprotein, it is unstable and difficult to control.
Hence:
We don’t know how many cells can take up complete and functional aequorin.
Also:
EVERY CELL HAS CALCIUM ION OSCILLATIONS, which will INTERFERE WITH
VISUALISATION.
Hence:
GREEN FLUORESCENT PROTEINS are used individually.
Advantage: only need to match light with excitation spectrum peak to activate, needs
no cofactor to function. Stable.
However, at the time mainstream science does not believe that GFP can act alone,
and Prasher had no resources to conduct research. He did isolate and publish the
sequence of the GFP gene, but that was as far as he could go. Stranded in a marine
biology research institute, he suffered from depression and finally left the field and
became an uber driver. He was the only person among the major GFP researchers
that DID NOT GET A NOBEL PRIZE…
Eventually, Chalfie et al. (husband and wife, both researchers) took up Prasher’s paper
and EXPRESSED GFP IN E. COLI.
GFP as a reporter for gene expression and protein localisation
Initially, GFP was expressed in E. coli and C. Elegans, and later in Drosophila.
‘GFP fusion proteins in Drosophila melanogaster are fluorescent after formaldehyde
fixation.’
Further study:
1988: a gradient of bicoid protein in Drosophila embryos.
1994: Implications for bcd mRNA localisation from special distribution of exu protein
in Drosophila oogenesis.
Basic applications of GFP:
(1) Indication of activity of a promoter:
GFP replaces a protein after a promoter When the promoter is bound the level of
GFP expression corresponds to the expression level of the protein if it was in its
original locus.
(2) Indication of protein localisation
GFP and the original protein expressed as a fusion protein. Hence by illuminating
GFP with light with wavelength between 400-475 nm, we can find the localisation
of the protein. This has been used in the two embryonic studies on Drosophila
mentioned above.
Example: in bicoid pathway:
Bicoid pathway is the first ‘morphogen’ to be discovered. In a Drosophila embryo,
the embryo experiences segmentation, with each segment becoming different
body parts. However, the way segment differentiation is determined remained
unknown. By applying GFP to bicoid protein localisation, it is shown that there is
RNA deposited at anterior part of embryo. The RNA is translated into bicoid
proteins, which start to diffuse towards posterior end, forming a concentration
gradient. THE CONCENTRATION GRADIENT DETERMINES FATE OF
SEGMENT. High concentration: head, low concentration: tail.
Source of mRNA:
‘Mother delivers mRNA, embryo makes protein’.
Exu is required for formation of gradient. If exu is knocked out, the accumulation
of the bicoid does not work anymore. Thus: exu-GFP are expressed: exu localised
to dots in membrane that connect nurse cell to oocyte. mRNA transported with the
aid of exu to the anterior end of oocyte and gets deposited there.
Mutation in GFP (Tsien)
(1) P4-mutant, in which Tyr-66 is replaced by histidine. This mutant produced blue
fluorescence.
(2) Single point mutation:
As the 400-475 nm peak is not the highest excitation peak in GFP, we hope to have
the highest excitation peak located near the emission spectrum and within the visible
light range. If we excite at 350-425 nm, we need to USE LASERS: NOBLE GASES.
Noble gases are difficult to control. Also: filters good for emission bump and not good
for excitation spectrum.
Thus: optimise spectrum.
Ser-65, CHANGED SPECTRUM so that best excitation is located at 475nm.
Also: BRIGHT EMISSION, PERFECT FOR MICROSCOPY
(3) Thr-203:
This shifted the entire spectrum so it is emitting lights of longer wavelength (yellow
fluorescent protein)
With the publication of Thr-203, the CRYSTAL STRUCTURE of GFP is also revealed.
[FORMS A BARREL-LIKE STRUCTURE]:
Containing a couple of large beta-helices, a ROD STRUCTURE in the middle.
When folded this forms a BETA BARREL: beta helices folded tightly over the central rod.
In the centre of the barrel is the chromophore which absorbs photons.
Barrel:

PROTECT chromophores so that SOLVENTS DO NOT GET INTO THE CENTRE OF
THE BARREL AND QUENCH THE CHROMOPHORE.

POSITIONS THE CHROMOPHORE EXACTLY IN MIDDEL AT A CERAIN ANGLE. It
one changes the position slightly, it changes the spectrum.
The point mutations CHANGES POSITION OF CHROMOPHORE and thus changes the
spectrum.
-65 and 66 are IN THE MIDDLE OF THE ROD.
-203 is in A BETA-SHEET HOLDING THE CHROMOPHORE.
Also:
WE NEED A NARROE SPECTRUM.
-mechanically CHROMOPHORES ARE NOT SUPER-STABLE. Hence chromophores
may be activated by a BROAD SPECTRUM, including laser (POLARISED LIGHT).
The broad spectrum is BAD FOR MICROSCOPY, and STRAY LIGHT NEEDS TO BE
FILTERED.
By altering the residues on the beta-barrel structure, we may produce a database/family
tree of fluorophores.
Now: LIMITATION OF GFP: GFP variants can only produce light within the spectrum of
BLUE TO YELLOW.
Thus, to produce red light:
Initially, red fluorophore was collected from ANTHOZOA SPEICES.
It is a TERAMERIC PROTEIN with 4 barrel clustered together and takes LONG TO
MATURE.
After mutating the protein into a single barrel, THE MUTATION TIME IS SPED UP 10 times
greater AND THE BRIGHTNESS BECOMES 5 times GREATER.
From this protein the ENTIRE RED SPECTRUM WAS CREATED by Nathan Shaner.
Further discoveries:
Finding the brightest fluorophore:
4 homologues of GFP in Aequorea.
AsusFP1:
Found in Australia
Narrow excitation and emission spectra
Brightest fluorophore found today.
Lecture 8-Gene Drives
The basic biology of a gene drive:
BASE EXAMPLE: Eliminating mosquitoes
•Major disease vectors
•Malaria; Dengue, Zika, and Ross River Fever; Buruli Ulcer
•Mosquitoes have developed resistance to many insecticides
•Do we want to be using insecticides anyway?
•How can we apply our understanding of mosquito biology to reduce disease burden
DETAILS:
Major form of controlling mosquitoes is INSECTICIDE. But the problem is that mosquitoes
would develop resistance, and insecticides are TOXIC COMPOUNDS THAT CAN
DAMAGE ENVIRONMENT.
Hence: HOW TO APPLY UNDERSTANDING OF MOSQUITO BIOLOGY TO REDUCE
DISEASE BURDEN?
The possible answer is ENGINEERING MOSQUITOES.
Examples:
(1) Reduce female offspring
(2) Make females resistant to disease
(3) Put scorpion toxin into mosquito to KILL MALERIA PARASITE
However, the next question would be:
HOW TO GET GENETICALLY-MODIFIE MOSQUITOES INTO THE POPULATION?
Even if gene is very beneficial, gene introduction into a stable population is hard.
The HARDY WEINBERG EQUILIBRIUM
Assumes ideal circumstances:

No new mutations

No natural selection

Random mating

No GENETIC DRIFT

Genetic drift: change in frequency of an existing gene variant in a population
due to RANDOM CHANCE.

Large population size

No GENE FLOW:

GENE FLOW: Transfer of genetic material with another population

If gene flow is fast, two populations may also be seen as ‘ONE
POPULATION’.

One single population
UNDER IDEAL CIRCUMSTANCES, ALLELE FREQUENCIES IN A POPULATION
REMAINS STABLE.
This remains true in REAL CIRCUMSTANCES, where the above conditions may not be
true. Thus, INTRODUCED MUTATION MAY SIMPLY DISAPPEAR IN A POPULATION
DUE TO CHANCE…
[ONLY WHEN MUTATION IS ADVANTAGEOUS CAN IT REMAIN IN POPULATION IN
HIGH NUMBERS. AND EVEN FOR ADVANTAGEOUS VARIANTS, RANDOMNESS MAY
STILL CAUSE ITS DISAPPEARANCE IF POPULATION IS TOO SMALL…
In gene drives, MOST MUTATIONS INTRODUCED RE DELATERIOUS OR AT MOST
NEUTRAL.
THUS:
[CAN WE CHANGE INHERITANCE PATTERNS TO OVERCOME THE TENDENCY OF
NATURAL POPULATIONS TO REMAIN GENETICALLY STABLE?]
Thus, enter gene drives.
GENE DRIVES: UNNATURAL SELECTION.
The problem – uncontrolled vector-borne disease
The goal – change the vector population so disease doesn’t spread
• Idea of a gene drive
• Introduce a neutral or deleterious gene into a population and have it spread to fixation
(present in the whole population)
• Their mathematical models showed how to do it
• Shift inheritance patterns so the new gene is inherited by most offspring regardless of
“fitness”
• Few biological methods were available
That is:
WE STOP THE SPREAD BY DECREASING NUMBER OF VECTORS, OR INDIVIDUALS
THAT CAN SPREAD THE DISEASE. WE DO THAT THROUGH GENETIC MEANS:
INTRODUCE A NEUTRAL/DELETERIOUS GENE INTO POPULATION AND HAVE IT
SPREAD TO FIXATION (that is: make sure that said neutral/deleterious gene is present in
entire population).
IF IT IS POSSBLE TO SHIFT INHERITANCE PATTERNS:
We hope that offspring can carry the gene regardless of whether it is deleterious or not.
Natural examples: TRANSPOSON
“Jumping genes”
AUTONOMOUS DNA ELEMENTS
CARRY ALL NECESSARY MACHINERY TO:
(1) REPLICATE THEMSELVES
(2) CUT A STRAND OF DNA
(3) INSERT ITSELF INTO THAT SITE THAT HAS BEEN CUT
Example: Corn.
When crossing corn, if one parent has a transposon, THE TRANSPOSON WILL
PRODUCE PROTEINS THAT CLEAVES CORRESPONDING LOCI IN THE OTHER
ALLELE, AND INSERT REPLICAS OF ITSELF INTO CUT REGIONS. Thus, BOTH
ALLELES WILL HAVE THE TRANSPOSON!
Another more notable case: FRUIT FLIES.
• Wild caught fruit flies
• Before 1950 – no P-type transposons
• After 1990 – all have P-type transposons
That is: The P-type transposon completely took over the worldwide fruit fly population in
30 years!
THE P-TRANSPOSONS WORK IN GERMLINE:
(1) They REPLICATE DURING MATING, JUMPING INTO NEW POSITIONS IN
GENOME.
(2) MOST OFFSPRING CARRY AT LEAST ONE COPY.
The P-transposon is not even a neutral or positive gene. IT ACTUALLY CAUSES
DEVELOPMENTAL DEFECTS LIKE VESTIGIAL WINGS.
Even so, P-transposon has spread worldwide, just because it could shift its inheritance.
FLIES EVENTUALLY ‘SHUT DOWN’ THE EFFECT OF P-TYPE TRANSPOSON.
Now: CAN WE MAKE GENE DRIVES USING TRANSPOSONS?
Unlikely to succeed. THIS IS NOT A GREAT SYSTEM:
(1) THEY DO NOT ALWAYS WORK WHENYOU INTEGRATE THEM FROM ONE
SPECIES TO ANOTHER.
(2) They INTEGRATE RANDOMLY, and may lead to unexpected adverse effects that may
lead to individuals carrying such a gene drive being selected against.
(3) THERE IS NO WAY TO ENSURE THAT THE PAYLOAD GENE IS ALWAYS
CARRIED WITHIN THE TRANSPOSON. AS LONG AS THE MINIMAI MACHINERY
IS PRESENT, TRANSPOSONS MAY REPLICATE ON WITHOUT PAYLOAD.
(4) ONLY ONE CHANCE. (MAIN PROBLEM)
Example: Drosophila can now shut off effects of P-transposon. If we use gene drive
based on P-transposon, the payload gene would not be expressed anyway…
ENTER CRISPR: a DESIGNER TRANSPOSON.
This is a key stage in gene drive studies.
We need the following:
(1) Cas9
(2) Guide RNA
(3) Payload
THE GENE IS INSERTED INTO THE FLANKS OF THE DESIRED LOCI OF INSERTION.
When Cas9 cuts target, guide RNA/cargo cassette gets replicated in genome.
That is: CLEAVED DNA GETS REPAIRED USING TRANSGENE AS TEMPLATE.
Hence: ALL OFFSPRING WILL CARRY INTRODUCED GENE.
Effect/application:
YEAST
Example: Yeast mutation ‘ADE2’ interrupt purine metabolism.
If one grow yeast on ADENINE (PURINE) – FREE MEDIA: YEAST TURN PINK.
If the yeast contains ADE2, they stay white.
Lecture 9: Gene Therapy
Basics:
Cell contains genetic information that can be inherited.
Genetic diseases: mutations I DNA that leads to defects.
[GENERALLY, THERE IS NO CURE FOR GENETIC DISEASES, BUT GENE
THERAPY IS A PROMISING SOLUTION.]
History:
Initial proposal: 1972, Theodore Friedmann and Richard Robin
‘Incorporating functional DNA into patient cells to treat genetic disorder’.
‘Introducing a healthy copy of gene to replace function of mutated gene’.
However: BOTTLENECKS:
(1) Limited
understanding
of
GENE
REGULATION
AND
GENETIC
RECOMBINATION in human cells.
(2) Limited understanding of RELATION BETWEEN MOLECULAR DEFECT AND
DISEASE.
(3) NO INFORMATION of SHORT- AND LONG-TERM SIDE EFFECTS.
1990: first approved gene therapy trial
Target: SEVERE COMBINED IMMUNODEFICIENCY (SCID).
Symptoms of the disease:
Lack of ADENOSINE DEAMINASE (ADA)
Hence: Defective T and B cell function and recurrent infections
Also called ‘Bubble-boy disease’.
Trials:
RETROVIRAL VECTOR carrying ADA gene targeting T-cells.
10 infusions over 2 years.
Result: RESTORED IMMUNE FUNCTION.
1999: Major setback
Target: ORNITHINE TRANSCARBAMYLASE DEFICIENCY (OTCD)
Mutation in GENE ENCODING ORNITHINE TRANSCARBAMYLASE
Thus: INABILITY FOR LIVEER TO BREAK DOWN AMMONIA
Hence: LETHAL AMOUNTS OF AMMONIA IN BLOOD
[BABIES WITH OTCD USUALLY DIE OR FALL INTO COMMA SOON AFTER BIRTH.]
In this case patient has a mild variant of the disease and has adopter a LOW PROTEIN
DIET and 50 PILLS PER DAY.
Trial:
ADENOVIRAL VECTOR WITH A HEALTHY COPY OF OTC GENE INTO LIVER.
18th person in trial
However:
Death due to IMMUNE REACTION.
FDA suspend + investigate 69 other gene therapy trials
2002: More setback
Target: X-linked SCID.
Trial:
Gammaretroviral vector to restore IL2RG gene and target hematopoietic stem cells
9/10 cures, but 4/9 SUFFERED FROM T CELL LEUKAEMIA WITHIN 5 YEARS AND 1
DIED.
Cause of the T cell leukaemia:
There is no control where the healthy copy of the gene is inserted. The vector inserted
the IL2RG gene into known PROTO-ONCOGENES:
LMO2
BMI1
CCND2
This eventually results in abnormal karyotypes.
Thus:
Where are we now?
Currently:
(1) Better vectors, more safety.
(2) More forms of gene therapy approved.
(3) Many targets, for instance SCID.
(4) More therapies had been approved since 2012, the first of these being LUXTURNA.
GOALS FOR GENE THERAPY:
1. HIGH EFFICACY
2. SAFETY
Details:
EFFICACY:
DEPENDS ON VECTOR AND ROUTE OF DELIVERY.
We need ‘Efficient vector for gene delivery’.
Now there are a series of MOLECULAR TRICKS to enhance gene expression and
protein stability to achieve long-lasting effects.
SAFETY:
PRECISION IS KEY.
As seen in the X-linked SCID case (2002), there is serious concern of off-target effects.
OFF TARGET GENE EDITING AND DELIVERY TO NON_TARGET CELLS MAY
CAUSE SERIOUS ADVERSE EFFECTS INCLUDING CANCER.
We must also AVOID EXCESSIVE IMMUNE RESPONSE AS SHOWN IN
ORNITHINE TRANSCARBAMYLASE DEFICIENCY case (1999).
Hence:
Considerations:

Types of gene therapy?

Somatic vs germline?

Nuclear vs mitochondrial?

Method of delivery?
Details:
Types of gene therapy:
2 FORMS:
(1) GENE AUGEMNTATION
Introducing a healthy copy of the gene into cell.
The functional copy will replace the mutant copy in delivering the normal effects.
DEFECTIVE COPY IS STILL AROUND.
Problem: WILL NOT WORK WELL FOR ‘DOMINANT NEGATIVE’ GENES.
(2) GENE EDITING
Correct genomic mutation in the DNA:
THERE WILL BE NO MORE DEFECTIVE COPIES IN CELL.
Somatic vs germline?
Somatic gene therapy:
GENOME MODIFICATION IN SOMATIC CELLS.
In vivo: gene is transferred into cells INSIDE THE PATIENT’S BODY.
Ex vivo: gene therapy on PATIENT’S CELL OUTSIDE THE BODY, BEFORE CELL IS
TRANSPLANTED BACK INTO THE PATIENT.
[IMPORTANTLY: SOMATIC GENE THERAPY WILL NOT BE CARRIED DOWN TO
THE NEXT GENERATION.]
Germline gene therapy:
GENE MODIFICATION IN GERM CELLS.
WILL BE CARRIED TO NEXT GENERATION.
Example: CRISPR BABIES, MITOCHONDRIAL REPLACEMENT THERAPY.
Ethical considerations with manipulating gene pool.
Nuclear vs mitochondrial genomes as target:
Nuclear:
23 chromosomes, 46000 nuclear genes.
Mitochondria:
37 mitochondrial DNA genes, multiple copies.
Example: TRANSFER OF MITOCHONDRIA ALONG HUMAN OPTIC NERVES.
Gene delivery method: VIRAL vs NON-VIRAL.
A large number of viral vectors may be used:
Example:
ADENO-ASSOCIATED VIRUS.
RETROVIRUS and LENTIVIRUS.
[DIFFERENT CELLS HAVE DIFFERENT TRANSDUCTION EFFICIENCIES FOR
DIFFERENT VIRUSES.]
This is related to chromosomes.
Example:
ADENO-ASSOCIATED VIRUS TARGET DIVIDING AND NON-DIVIDING CELLS.
RETROVIRUS AND LENTIVIRUSTARGET ONLY DIVIDING CELLS.
The other aspect is GENOME INTEGRATION.
That is, WILL THE VIRUS INTEGRATE INTO GENOME OF TRANSDUCED CELLS?
ADENO-ASSOCIATED VIRUS: NON-INTEGRATING.
RETROVIRUS AND LENTIVIRUS: INTEGRATING.
Advantages and disadvantages of integration:
Advantage:
LONG-LASTING EXPRESSION.
Disadvantage:
IF INTEGRATED GENE IS NEXT TO PROTO-ONCOGENE, IT MAY LEAD TO CANCER.
In this way, it seems that Adeno-associated viruses are safer.
However, THEY DO HAVE ONE MAJOR DRAWBACK: PACKING CAPACITY.
That is, HOW MUCH GENETIC MATERIAL CAN BE PACKED INTO THE VECTOR for
delivery.
ADENO-ASSOCIATED VIRUS has small packing capacity: 4.5 kb.
In contrast, retrovirus and lentivirus have packing capacity of 8-10 kb.
IMPROVING VIRAL VECTOR DESIGN:
Enhancing expression:
PROMOTERS:
-Ubiquitous expression
(That is, expression in all cell types)
-Cell-specific:
(That is, limit expression to a single type of cell to enhance safety and ensure less target
effects)
-Improve protein expression stability:
Kosak sequence
Other DNA-regulating elements such as WPRE
Viral tropism:
-Viral capsid can be MODIFIES TO IMPROVE SPECIFICITY TO CERTAIN TARGET
CELLS.
Example: ADENO-ASSOCIATED VIRUS:
Different serotypes of AAV targets different cell types with different efficiency, get into
different retinal cells with different degrees of efficiency.
That is, we need to come up with a serotype that has greater cell specificity.
CASE STUDIES:
[INHERITED RETINAL DEGENERATIVE DISEASES]
Overview:
Light travels through CORNEA, hits back of eye (RETINA)
Retina is a MULTILAYERED structure which includes PHOTORECEPTORS (light-sensory
cells). It PICKS UP LIGHT AND CONVERTS IT INTO ELECTRICAL SIGNALS.
The electric signal gets passed on into subsequent cells: BIPOLAR CELLS to GANGLIAL
CELLS (optic nerve).
GANGLIAN CELLS transfers signal to brain and brain interprets signal as image.
WHY EYE IS A GOOD SYSTEM FOR GENE THERAPY:
-External, easy to access.
-Advanced, non-invasive imaging technology can visualise what happens after treatment
is implemented.
-The eye is ‘contained’.
Anything injected into the eye is CONTAINED IN THE EYE.
This is thanks to the BLOOD-RETINA BARRIER, which prevents many substances from
entering circulation and being transported to other organs (less off-target effects, effects
limited to eye).
-Retina is PART OF CENTRAL NERVOUS SYSTEM. This means that it is immuneprivileged: THERE IS LESS IMMUNE REJECTION AND EFFECTS ARE EASY TO
MANAGE.
Inherited retinal degenerative diseases include:

Leber’s hereditary optic neuropathy (LHON)

Retinitis pigmentosa, Leber’s cogential amaurosis
Details:
CASE 1: Leber’s hereditary optic neuropathy (LHON):
Characterised by degeneration of optic nerve cells and resulting essential vision loss.
Happens between teens and early-twenties:
Patient loses vision in one eye, before losing vision in the other eye after a few weeks.
This is the most common MITOCHONDRIAL DNA DISEASE, AFFECTING 1 in 30000
INDIVIDUALS, PREDOMINANTLY YOUNG MALES.
All Leber’s cases are caused by:
MUTATION IN MITOCHONDRIAL DNA ENCODING FOR MITOCHONDRIAL COMPLEX
I SUBUNITS.
Precise mechanisms of how RGC (retinal ganglion cells) die is unknown:
The mutation of mitochondria happens in all cell types. But somehow, due to unknown
property of retinal ganglion cells, ONLY RETINAL GANGLIAL CELLS/OPTIC NERVE
CELLS DIE!
There is no clinically-relevant animal model, no long-term patients, and only one
preliminary treatment which only shows limited effectiveness in large-scale clinical trials.
Thus: WHAT CAN BE DONE:
(1) ‘We really need to study the MECHANISM OF OPTIC NERVE CELL DEGENERATION’
Thus, we apply IPS cell technology, or cell reprogramming:
We take SKIN CELLS from patient, and BY IPS, WE GENERATE INDUCED
PLURIPOTENT PATIENT-SPECIFIC STEM CELLS.
We then differentiate this into optic cells.
These optic cells may be used for DRUG SCREENING AND TEST FOR GENETIC
CORRECTION.
When the defect is corrected, we may even TRANSPLANT OPTIC NERVE CELL BACK
INTO PATIENT AS A CELL THERAPY.
i.e. Gene therapy in vitro.
Current results based on this approach:
IPS cells are generated using FIBROBLASTS FROM HEALTHY CONTROLS AND LHON
PATIENTS.
Outcome: The cells look THE SAME AT STEM CELL LEVEL. All IPS expressed the
induced pluripotent stem cell marker OCT4. IN TERMS OF GENOTYPE, the LHON
MUTATION IS ONLY SEEN IN PATIENT CELLS AND NOT IN CONTROLS.
A 4–5-day protocol was developed, involving the use of media, supplements, growth
factors and extracellular matrix, to DIFFERENTIATE THE IPS INTO OPTIC NERVE
CELLS. The characteristic long axon was observed, and electrophysiological data
indicated that the cells are functional.
When we look at optic nerve cells of LHON patients, they exhibit HIGH LEVEL OF
APOPTOSIS, which corresponds to clinical observations.
Conclusion: IPS cells can be used as stem cell model to study clinical phenotype.
(2) CAN WE CORRECT OR REPLACE MITOCHONDRIAL MUTATION IN CELLS?
The technology used in this case was called ‘Cybrid’.
‘For LHON, the cybrid technology can be used to replace mutated mitochondria with
healthy donor mitochondria’
We took a cell containing mutant mitochondria:
1. Use R6G to DEPLETE MITOCHONDRIA. What is left is a cell with only nucleus.
2. Take healthy donor cell: the donor cell is a KARYOTYPE, NOT A FIBROBLAST.
3. Enucleation of donor cell removes donor cell nucleus and leaves it with only
mitochondria.
4. We FUSE THE TWO CELLS TOGETHER. The fused cell is a CYBRID, or
CYTOPLASMIC HYBRID.
Result:
CYBRID APOPTOSIS RATE DROPS DOWN TO SAME AS NORMAL CELL.
We create the cybrid at IPS stage before differentiating them into optic nerve cells.
What really supports the above therapy for LHON is an analogous mitochondrial therapy:
the MITOCHONDRIAL REPLACEMENT THERAPY.
Mitochondrial replacement therapy comes in two forms:
Method 1:
PRONUCLEAR TRANSFER.
Replacing mitochondria at FERTILISED EGG STAGE AND FUSE CELLS TOGETHER.
Method 2:
MATERNAL SPINDLE TRANSFER.
Eliminate unhealthy mitochondria AT STAGE OF UNFERTILISED EGG.
Remove the nucleus of a mother’s egg, and FUSE WITH ENUCLEATED DONOR EGG
containing HEALTHY MITOCHONDRIA. THIS METHOD IS ANALOGOUS TO CYBRID.
The resulting infant may be called a ‘THREE PARENT BABY’.
3 parent IVF:
2015: UK become first to legalise mitochondrial replacement therapy
2016: first 3 parent baby born in Mexico to treat carry Leigh syndrome
2017: 3 parent baby born in Ukraine to treat infertility
2019: 3 parent baby born in Greece to treat infertility
2022: Mitochondrial donation legalised and took effect on 1 Oct 2022
Notably, all mitochondrial replacement therapies are GERMLINE: DOES NOT CURE
DISEASE IN HOST, BUT PREVENT DISEASE BEING PASSED DOWN TO NEXT
GENERATION.
Meanwhile, LHON TREATMENT IS SOMATIC.
It is a ‘GENE AUGMENTATION’ therapy: A HEALTHY COPY OF THE ND4 GENE (most
common LHON mutation) IS DELIVERED. It does not alter the genetic material of the
original mitochondria. It simply disposes of them.
Current clinical trials for LHON:
• Neuropath
– NR082: AAV2 vector carrying ND4 gene
– Chinese clinical trial 9 patients with 7 years follow up (Yuan 2020 Ophthalmology)
– Safe and vision improvement in some patients
• USA phase I clinical trials
– AAV2 vector carrying ND4 gene
– Phase I with 14 patients, 12 month follow up: Safe and vision improvement in some
patients (Guy et al 2017 Ophthalmology)
– Phase I with 28 patients, 24 month follow up: Good safety profile, limited efficacy (Lam
et al. Am J Ophthalmol
• 2x GenSight Biologics (France) Phase III clinical trials with 90 patients:
– GS010: AAV2 vector carrying ND4 gene
– Multi centre, randomized, double blind, placebo controlled trial
– bilateral visual improvement in patients with unilateral gene therapy (Newman 2021
Ophthalmology, Yu Wai Man 2020 Sci Transl Med)
This trial seems to indicate that the therapy is EFFECTIVE AGAINST CERTAIN
MUTATIONS.
CASE 2: Leber’s cogential amaurosis (LCA)
• One of the most common causes of blindness in children, affecting ~1 in 40000
• Severe visual loss at birth
• Other eye related conditions such as abnormal sensitivity to light, roving eye movements
CAUSES DEGENERATION OF PHOTORECEPTORS.
Key mutation:
RPE65 MUTATION.
This causes INABILITY TO PROCESS VITAMIN A and DEGENERATION OF
PHOTORECEPTORS.
Other mutations include: CEP290, CRB1, GUCY2D
For RPE65, a therapy has already been developed, LUXTURNA.
It is also the first FDA-APPROVED GENE THERAPY IN THE EYE.
It is an ADENO-ASSOCIATED VIRAL VECTOR (AAV) that carries a HEALTHY COPY OF
THE RPE65 GENE INTO THE EYE.
Thus, it is a GENE AUGMENTATION THERAPY (the original mutant is still there and not
corrected.)
THE DOWNSIDE OF THE THERAPY IS THAT: IT IS TOO EXPENSIVE! $850000USD!
• 2020: approved by TGA in Australia
• 2022: Cost subsidize jointly by federal + state government in Australia
Ethical considerations for gene therapy
Regulation:
Therapeutic Goods Act 1989 (TGA), Gene Technology Act 2000 (Gene Technology
Regulator)
These NEEDS TO BE UPDATED.
Ethical side:
(1) Patient’s consent.
(2) Patients must understand the RISK of gene therapy.
(3) In terms of germline modification:
Is this a Eugenics method?
In many countries this is illegal, especially if it does not have a legitimate reason for doing
so.
(4) This could be applied to HUMAN ENHANCEMENT AND MODIFICATION.
Future approach:
APPLICATION OF NEXT GENERATION GENE EDITING.
Problem: For gene augmentation: SOME GENES ARE TOO BIG TO BE PACKED INTO
A VIRAL VECTOR.
Example: ABCA4 gene for Stargardt’s disease, which causes DEGENERATION OF
RETINAL EPITHELIAL CELLS.
Solution: We must EDIT AND CORRECT THE MUTATION WITHIN THE CELL.
This may be used by zinc finger, TALENS or CRISPR (see earlier pages).
Advantage of CRISPR:
Easy and efficient. It not only corrects DNA, but can now also correct RNA.
Furthermore, it can also be used for gene repression and molecular splicing.
(1) 1st generation of CRISPR
-‘Cutting DNA’ and ‘causing homology-directed repair BASED ON TEMPLATE’
(2) New technology
-
Editing SINGLE BASE without cleavage
(3) RNA EDITING
Challenges:
(1) Mutations in ~280 genes are known to cause inherited retinal diseases
(2) Multiple mutations have been identified in each disease causing gene
If we want to fix all, it would cost too much time and money.
Possible solution: USE ONE SINGLE GENE THERAPY TO TARGET MULTIPLE
MUTATIONS AT ONCE.
Future:
(1) Optogenetic gene therapy to restore vision?
OPTOGENETIC GENE THERAPY:
Normally photoreceptors are LIGHT-SENSING.
Optogenetics: Introducing a ‘light-sensing protein to activate neurons’.
Retrosense/Allegan
is
CHANNELRHODOPSIN
developing
to
make
a
bipolar
AAV
gene
cells
therapy
light-sensitive
to
introduce
and
BYPASS
PHOTORECEPTORS.
i.e. Turn OTHER CELLS INTO LIGHT-SENSING CELLS.
‘Engineer new function to existing cells to treat disease’.
This may be the cure for MULTIPLE DISEASE, REGARDLESS OF MUTATION.
(2) Can we use gene therapy to simulate regeneration?
Use cell reprogramming to stimulate regeneration of retina with INTRODUCTION OF
TRANSCRIPTION FACTORS.
Key advantage:
AT LATE STAGE OF DEGENERATION.THERE MAY NOT BE SUFFICIENT CELLS LEFT
FOR PHARMACOLOGICAL OR GENE THERAPY.
Thus we reprogram cell identity by controlling genes in a cell.
For example: From skin cells to IPS cells.
This may also be applied to other organs (heart, lungs, liver, brain, spinal cord)
Example:
We inject reprogramming gene targeting MULLER GLIAL CELLS (STEM CELLS) WITHIN
RETINA.
Then we switch on a set of genes to REGENERATE THEM INTO PHOTORECEPTORS.